KR20030036139A - HLA Binding peptides and their uses - Google Patents

Abstract

The present invention provides means and methods for selecting immunogenic peptides and immunogenic peptide compositions capable of specifically binding to glycoproteins encoded by HLA alleles and capable of inducing T cell activation in T cells restricted by such alleles. To provide The peptides are useful for eliciting an immune response to the desired antigen.

Description

HLA Binding peptides and their uses

Reference to related application

This application is a partial consecutive application of USSN O8 / 205,713, filed March 4, 1994. This application also discloses USSN O9 / 017,735, USSN O8 / 753,622, USSN O8 / 822,382, USSN 60 / 013,980, USSN O8 / 589,108, USSN O8 / 454,033, USSN O8 / 349,177, USSN O8 / 073,205 and USSN O8 / 027,146. It is about. The present application also discloses USSN O9 / 017,524, USSN O8 / 821,739, USSN 60 / 013,833, USSN O8 / 758,409, USSN O8 / 589,107, USSN O8 / 451,913, USSN O8 / 347,610, USSN O8 / 186,266, USSN O8 / 159,339, USSN O9 / 116,061, USSN O8 / 103,396, USSN O8 / 027,746 and USSN O7 / 926,666. The present application also relates to USSN O9 / 017,743; USSN O8 / 753,615; USSN O8 / 590,298; USSN O8 / 452,843; USSN O9 / 115,400; USSN O8 / 344,824; And USSN O8 / 278,634. The present application also relates to USSN O8 / 197,484 and USSN O8 / 815,396. All of these applications are incorporated herein by reference.

The present invention relates to compositions and methods for preventing, treating or diagnosing a number of 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 class I or class II molecules. Class II MHC molecules are mainly expressed in cells involved in initiating and sustaining an immune response, such as T lymphocytes, B lymphocytes, macrophage, and the like. Class II MHC molecules are recognized by helper T lymphocytes, inducing proliferation of helper T lymphocytes and inducing amplification of the immune response to the particular immunogenic peptides indicated. Class I MHC molecules are expressed on almost all nucleated cells and recognized by cytotoxic T lymphocytes (CTLs), destroying antigen-bearing cells. CTLs are particularly important in tumor rejection and also in combating viral infections.

CTLs recognize antigens in the form of peptide fragments bound to MHC class I molecules, rather than the original foreign antigen itself. The antigen normally must be synthesized intrinsically by the cell, and certain portions of this protein antigen are broken down into small peptide fragments in the cytoplasm. Some of these small peptides are translocated into the pre-Golgi compartment to interact with class I heavy chains to promote proper folding and binding with subunit β2 microglobulin. The peptide-MHC class I complex is then delivered to the cell surface for potential recognition and expression by specific CTLs.

Studies on the crystal structure of HLA-A2.1, a human MHC class I molecule, have shown that folding of the α1 and α2 domains of class I heavy chains produces peptide binding grooves (Bjorkman et al. , Nature 329: 506 (1987). However, even in these studies, the identity of the peptide bound to the groove was not found.

Buus et al., Science 242: 1065 (1988) describe for the first time a method of acid eluting bound peptides from MHC. Subsequently, Rammensee and his co-workers developed an approach to identify the properties of naturally processed peptides bound to class I molecules. Falk et al., Nature 351: 290 (1991). Other researchers have found that Class I molecules of type A2.1 (Hunt et al., Science 225: 1261 (1992)) and type B (jardetzky et al., Nature 353: 326 (1991)) by mass spectrometry. Peptides eluted from were successfully analyzed for amino acid sequences of more abundant peptides in various HPLC fractions through conventional automated sequencing. A review of the characterization of naturally processed peptides in MHC class I is described in Rotzschke and Falk, Immunol. Today 12: 447 (1991).

See Sette et al., Proc. Natl. Acad. Sci. USA 86: 3296 (1989) states that MHC allele specific motifs could be used to predict MHC binding capacity. See Schaeffer et al., Proc. Natl. Acad. Sci. USA 86: 4649 (1989) reports that MHC binding is associated with immunogenicity. Some authors have reviewed De Bruijn et al., Eur. J. Immunol., 21: 2963-2970 (1991); Pamer et al., 991 Nature 353: 852-955 (1991), provide preliminary evidence that Class I binding motifs can be applied to identify potential immunogenic peptides in animal models. No Class I motifs specific for a number of human alleles of a given Class I isotype have yet been reported. It is desired that the sum of the incidences of these different alleles be high enough to cover a significant or perhaps majority of the human crossbred population.

Despite the research and development in the art, the prior art did not provide useful human peptide-using vaccines or therapeutics based on these studies. 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 to an HLA-A2.1 molecule. Immunogenic peptides that are associated with suitable MHC alleles are preferably 9 to 10 residues in length and include residues conserved at specific positions, such as positions 2 and 9. Furthermore, the peptide may be at other positions in the case of a peptide of 9 amino acids in length, for example at positions 1, 3, 6 and / or 7, position 1, in the case of a peptide of 10 amino acids in length. It does not include negative binding moieties as defined herein at 3, 4, 5, 7, 8 and / or 9. The present invention defines a location within the motif that allows the selection of peptides that will effectively bind HLA A2.1.

The motif of the present invention has a first conserved residue at a second position from the N-terminus selected from the group consisting of I, V, A and T; Included are peptides of nine amino acids having a second conserved residue in the C-terminal position, selected from the group consisting of V, L, I, A and M. In addition, the peptide has a first conserved residue at a second position from the N-terminus selected from the group consisting of L, M, I, V, A and T; It may have a second conserved residue at the C-terminal position selected from the group consisting of A and M. If the peptide is ten residues, it has a first conserved residue at the second position from the N-terminus selected from the group consisting of L, M, I, V, A and T; There will be a second conserved residue at the C-terminal position, selected from the group consisting of V, I, L, A and M, wherein the first conserved residue and the second conserved residue are spaced seven residues apart.

The peptides of the invention can be used to identify epitopes on numerous immunogenic target proteins. Examples of suitable antigens include prostate cancer specific antigen (PSA), prostate specific membrane antigen (PSM), hepatitis B core and surface antigen (HBVc, HBVs), hepatitis C antigen, Epstein-Barr Viral antigen, human immunodeficiency type 1 virus (HIV1), Kaposi's sarcoma herpes virus (KSHV), human papilloma virus (HPV) antigen, Lassa virus, Mycobacterium tuberculosis (MT), p53 and Murine p53 (mp53), CEA, tripanosome surface antigen (TSA), members of the tyrosinase related protein (TRP) family, and Her2 / neu. Thus, the peptides are useful in pharmaceutical compositions for both in vivo and ex vivo therapeutic and diagnostic applications.

The invention also provides compositions comprising immunogenic peptides having binding motifs for MHC class I molecules. Such immunogenic peptides typically contain about 8 to about 11 residues and comprise conserved residues associated with binding the protein encoded by the appropriate MHC allele. Numerous allele specific motifs have been identified.

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

The motif for HLA-A1 includes a first conserved residue of T, S or M, a second conserved residue of D or E, and a third conserved residue of Y, from the N-terminus to the C-terminus. The other second conserved residue is A, S or T. These first and second conserved residues are contiguous and are preferably spaced apart from the third conserved residue by six to seven residues. The second motif consists of a first conserved residue of E or D and a second conserved residue of Y, wherein the first conserved residue and the second conserved residue are spaced 5 to 6 residues apart.

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

The motif for HLA-A24.1 includes a first conserved residue of Y, F or W, and a C-terminal conserved residue of F, I, W, M or L at position 2 from the N-terminus to the C-terminus do. This first conserved residue and the second conserved residue are preferably separated by 6 to 7 residue intervals.

In this way, epitopes on a number of potential target proteins can be identified. Examples of suitable antigens include prostate cancer specific antigen (PSA), prostate specific membrane antigen (PSM), hepatitis B core and surface antigens (HBVc, HBVs), hepatitis C antigen, malignant melanoma antigen (MAGE-1) , Epstein-Barr virus antigen, human immunodeficiency type-1 virus (HIV1), papilloma virus antigen, Lhasa virus, Mycobacterium tuberculosis (MT), p53 and p53 (mp53) in rats, CEA, Her2 / neu, and Members of the tyrosinase related protein (TRP) family. Thus, the peptides are useful in pharmaceutical compositions for both in vivo and ex vivo therapeutic and diagnostic applications.

The invention also provides compositions comprising immunogenic peptides having binding motifs for non-A HLA alleles. Such immunogenic peptides are preferably about 9 to 10 residues in length and contain conserved residues at specific positions, for example, include proline at position 2 and aromatic residues at the carboxy terminus (eg, Y, W, F) or hydrophobic residues (eg, L, I, V, M or A). In particular, an advantage of the peptides according to the invention is that they have the ability to bind two or more different HLA alleles.

In this way, epitopes on a number of potential target proteins can be identified. Examples of suitable antigens include prostate cancer specific antigens (PSA), hepatitis B core and surface antigens (HBVc, HBVs), hepatitis C antigens, malignant melanoma antigens (MAGE-1), Epstein-Barr virus antigens, humans Immunodeficiency type 1 virus (HIV1), papilloma virus antigens, Lhasa virus, Mycobacterium tuberculosis (MT), p53, CEA and Her2 / neu. Thus, the peptides are useful in pharmaceutical compositions for both in vivo and ex vivo therapeutic and diagnostic applications.

Justice

The term "peptide" is typically interchangeable with "oligopeptide" herein to refer to a series of residues, typically L-amino acids, linked together by peptide bonds between the alpha-amino and carbonyl groups of adjacent amino acids. Used as Oligopeptides of the invention are less than about 15 residues in length and typically consist of about 8 to about 11 residues, preferably 9 or 10 residues.

An “immunogenic peptide” is a peptide that contains an allele-specific motif to bind to an MHC molecule and induce a CTL response. The immunogenic peptides of the present invention can bind to appropriate HLA-A2.1 molecules and can induce cytotoxic T cell responses against antigens that induce immunogenic peptides.

Immunogenic peptides are conveniently identified using the algorithm of the present invention. This algorithm is a mathematical process that represents a score that allows the selection of immunogenic peptides. Typically, algorithmic scores are used that represent “limit of binding” that allow for the selection of peptides that are likely to bind with a particular affinity and eventually are immunogenic. This algorithm is based on the effect of specific amino acids on MHC binding at specific positions of peptides, or on the binding of specific substituents on motif-containing peptides.

A "conservation residue" is an amino acid that is present at a significantly higher frequency than would be expected by random distribution at specific locations within the peptide. Typically, conserved residues are those in which the MHC structure can provide a point of contact with an immunogenic peptide. At least one to three or more, preferably two conserved residues within the peptide of the defined length represent motifs for the immunogenic peptide. These residues are typically in close contact with the peptide binding grooves whose side chains are buried in the specific pockets of the grooves themselves. Typically, an immunogenic peptide will comprise no more than three, more typically two conserved residues.

As used herein, a “negative binding moiety”, when present at a specific position (eg, positions 1, 3 and / or 7 of the hexamer), is not bound at all or binds to a weak level, resulting in non-immunogenicity. That is, amino acids that produce peptides that do not induce a CTL response.

The term “motif” refers to a pattern of residues within a peptide of defined length, typically about 8 to about 11 amino acids, recognized by a particular MHC allele. Such peptide motifs typically differ for each human MHC allele and differ in the pattern of highly conserved residues and negative residues.

Binding motifs for certain alleles can be defined with increasing precision. In one case, all conserved residues are present at the correct position in the peptide and no negative residues are present at positions 1, 3 and / or 7 .

"Isolated" or "biologically pure" refers to a substance that, as found in its original state, typically contains little or essentially no accompanying components. Thus, the peptides of the present invention typically do not contain substances associated with their in situ environment, eg, MHC I molecules on antigen presenting cells. Even when a particular protein is separated into homologous or dominant bands, trace contaminants are present within the range of 5-10% of the original protein, which is co-purified with the desired protein. Isolated peptides of the present invention do not contain such co-purified endogenous proteins.

The term "residue" refers to an amino acid or amino acid mimetic introduced into an oligopeptide by an amide bond or an amide bond mimetic.

I. HLA-A2.1 motif

The present invention relates to the determination of allele-specific peptide motifs for human class I MHC (often referred to as HLA) allele subtypes, particularly peptide motifs recognized by the HLA-A2.1 allele. These motifs are then used to define T cell epitopes from all antigens of interest, particularly antigens associated with human viral diseases, cancers or autoimmune diseases, for which the amino acid sequences of potential antigens or autoantigen targets are known. .

In this way, epitopes on a number of potential target proteins can be identified. Examples of suitable antigens include prostate cancer specific antigens (PSA), hepatitis B core and surface antigens (HBVc, HBVs), hepatitis C antigens, Epstein-Barr virus antigens, melanoma antigens (eg MAGE-1), Human immunodeficiency virus (HIV) antigens, human papilloma virus (HPV) antigens, Lhasa virus, mycobacterium tuberculosis (MT), p53, CEA, tripanosome surface antigen (TSA) and Her2 / neu.

Peptides comprising epitopes from these antigens are synthesized and subjected to, for example, immunofluorescent staining and flow rate microfluorescence, peptide-dependent Class I assembly assays, and inhibition of CTL recognition by peptide competition. , Their ability to bind appropriate MHC molecules in assays using cells expressing purified Class I molecules and radioiodinated peptides and / or empty Class I molecules. Peptides that bind class I molecules, as well as their ability to act as targets for CTLs derived from infected or immunized individuals, as well as CTL populations that can react with tumor cells or virally infected target cells as potential therapeutic agents. Further assess their ability to induce a first in vitro or in vivo CTL response that can cause.

MHC class I antigens are encoded by HLA-A, B and C locus. HLA-A and B antigens are expressed at the cell surface at approximately the same density, while the expression of HLA-C is significantly lower (possibly 10-fold lower). Each of these loci has a number of alleles. Peptide binding motifs of the invention are relatively specific for each allele subtype.

In the case of peptide-using vaccines, the peptides of the invention preferably comprise motifs recognized by MHC I molecules which are widely distributed in the human population. Since MHC alleles exist at different frequencies within different racial groups and races, the choice of target MHC allele can depend on the target population. Table 1 shows the frequency of occurrence of various alleles in the HLA-A locus product between different species. For example, the majority Caucasus population may be covered by peptides that bind four HLA-A allele subtypes, specifically HLA-A2.1, A1, A3.2 and A24.1. Similarly, the majority of Asian populations are encompassed by the addition of peptides that bind the fifth allele HLA-A11.2.

This table is described in B. DuPont, Immunobiology of HLA, Vol. I, Histocompatibility Testing 1987, Springer-Verlag, New York 1989].

* N = black; A = Asian; C = Caucasian. The numbers in parentheses indicate the number of individuals involved in the analysis.

The nomenclature used to describe peptide compounds is in accordance with the conventional practice of displaying amino groups to the left (N-terminus) of each amino acid residue and carboxyl groups to the right (C-terminus). In the formulas illustrating certain selective embodiments of the present invention, although the amino and carboxyl-terminal groups are not specifically indicated, unless otherwise stated, they are present in forms that can be taken at physiological pH values. In amino acid structural formulas, each residue is generally represented by a standard three letter or single letter name. L-form amino acid residues are represented by a single letter in uppercase or a three letter code in which the first letter is in uppercase, and a D-type amino acid is represented by a single letter in lowercase or a three letter code in lowercase. Glycine is simply referred to as "Gly" or G because it lacks an asymmetric carbon atom.

The procedure used to identify the peptides of the present invention is generally described in Falk et al., Nature 351: 290 (1991); Incorporated herein by reference. Briefly, the method involves the large scale separation of MHC class I molecules from suitable cells or cell lines, typically by immunoprecipitation or affinity chromatography. Examples of other separation methods of the MHC molecules of interest, which are also well known to those skilled in the art, include ion exchange chromatography, lectin chromatography, size exclusion, high performance ligand chromatography, and combinations of all these techniques.

In typical cases, immunoprecipitation is used to isolate the desired allele. Depending on the specificity of the antibody used, many protocols can be 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 are available for isolating HLA-A molecules. Monoclonal BB7.2 is suitable for isolating HLA-A2 molecules. Affinity columns made with these mAbs were successfully used to purify each HLA-A allele product using standard techniques.

In addition to allele-specific mAbs, broadly reactive anti-HLA-A, B, C mAbs such as W6 / 32 and B9.12.1, and one anti-HLA-B, C mAb, B1.23.2 Can be used in another affinity purification protocol as described in the previous application specification.

Peptides bound to the peptide binding grooves of the isolated MHC molecules are typically eluted by acid treatment. Peptides may also be dissociated from Class I molecules by various standard denaturation means, such as heat, pH, detergents, salts, chaotropic agents, or combinations thereof.

Peptide fractions are further separated from MHC molecules by reverse phase high performance liquid chromatography (HPLC) and then sequenced. Peptides can be separated by various other standard means well known to those skilled in the art, including filtration, ultrafiltration, electrophoresis, size chromatography, precipitation with specific antibodies, ion exchange chromatography, isoelectric focusing, and the like.

Sequence analysis on isolated peptides can be performed according to standard techniques such as Edman digestion. Hunkapiller, MW, et al., Methods Enzymol. 91, 399 (1983). Other methods suitable for sequencing include methods for sequencing individual peptides by mass spectroscopy, as mentioned previously. Hunt et al., Science 225: 1261 (1992); Which is incorporated herein by reference]. Amino acid sequence analysis of bulky heterologous peptides (eg, collected HPLC fractions) from different class I molecules typically shows characteristic sequence motifs for each class I allele.

By defining motifs specific for different class I alleles, it is possible to identify potential peptide epitopes from antigenic proteins with known amino acid sequences. Typically, identification of potential peptide epitopes is initially performed using a computer that scans the amino acid sequence of the desired antigen for the presence of the motif. Subsequently, epitope sequences are synthesized. The ability to bind MHC class molecules is measured in a variety of different ways. One method is a class I molecular binding assay as described in the related application mentioned above. Other alternative methods described in the literature include methods of inhibiting antigen presentation [Sette et al., J. Immunol. 141: 3893 (1991)], in vitro assembly assays (Townsend et al., Cell 62: 285 (1990)) and FACS-using assays using mutated cells such as RMA.S [Melief et al., Eur. J. Immunol. 21: 2963 (1991).

Peptides tested as positive in the MHC Class I binding assays are then assayed for the ability of the peptide to induce specific CTL responses in vitro. For example, antigen-presenting cells incubated with peptides can be assayed for their ability to induce CTL responses in responder cell populations. Antigen-presenting cells can be normal cells, eg, peripheral blood mononuclear cells or dendritic cells. Inaba et al., J. Exp. Med. 166: 182 (1987); Boog, Eur. J. Immunol. 18: 219 (1988).

On the other hand, mutant mammalian cell lines lacking the ability to add internally processed peptides to Class I molecules, such as the mouse cell line RMA-S. Karre et al., Nature 319: 675 (1986). ; Ljunggren et al., Eur. J. Immunol. 21: 2963-2970 (1991)] and human celiac T cell hybrids, T-2 (Cerundolo et al., Nature 345: 449-452 (1990)), and mutants transfected with appropriate human class I genes. Mammalian cell lines are used for convenience in testing the ability of peptides to be added to them to induce primary CTL responses in vitro. Other eukaryotic cell lines that can be used include various insect cell lines, such as mosquito larvae (ATCC cell lines CCL 125, 126, 1660, 1591, 6585, 6586), silkworms (ATCC CRL 8851), armyworms (ATCC CRL). 1711), moths (ATCC CCL 80) and drosophila cell lines, such as Schneider cell lines (Schneider J. Embryol. Exp. Morphol. 27: 353-365 (1927).

Peripheral blood lymphocytes are usually isolated after normal donor or patient leukocytosis, and then used as a responder cell source of CTL precursors. In one embodiment, the appropriate antigen-presenting cells are incubated with 10-100 μM peptides in serum-free medium for 4 hours under suitable culture conditions. The peptide-added antigen-presenting cells are then incubated with the responder cell population in vitro for 7-10 days under optimized culture conditions. The cultures were assayed for the presence of CTLs that both killed specific peptide-pulsed target radiolabeled target cells as well as target cells expressing intrinsically processed forms of the relevant virus or tumor antigen from which the peptide sequence was derived. Positive CTL activation can then be determined.

By testing for different peptide target cells expressing appropriate or inappropriate human MHC class I, the specificity and MHC restriction of CTLs are determined. Peptides that are tested to be positive in the MHC binding assay and elicit specific CTL responses are referred to herein as immunogenic peptides.

Immunogenic peptides can be prepared synthetically or by recombinant DNA techniques or from natural sources such as complete viruses or tumors. Although it is preferred that the peptide contains few other naturally occurring host cell proteins and fragments thereof, in some embodiments, the peptide can be conjugated synthetically with the original fragment or particle.

The polypeptide or peptide may be of various lengths in neutral (no charge) form or in salt form and does not include or include modifications such as glycosylation, side chain oxidation or phosphorylation, but such modifications are herein It is assumed that it does not destroy the biological activity of the polypeptide as described.

Desirably, the peptide will be as small as possible while maintaining substantially all of the biological activity of the large peptide. Where possible, it may be desirable to optimize the peptides of the invention to 9 or 10 amino acid residues in length, consistent in size with the intrinsically processed viral peptides or tumor cell peptides bound to the MHC class I molecules on the cell surface. .

Peptides having the desired activity can be modified as needed to provide certain desired properties, such as improved pharmacological properties, but unmodified peptides that bind to the desired MHC molecule and activate appropriate T cells. All biological activity of must substantially increase or at least maintain it. For example, these peptides can be subject to various changes, for example, conservative or non-conservative substitutions, which alter certain advantages in their use, such as improving MHC binding. You can also provide Conservative substitutions mean replacing one amino acid residue with another biologically and / or chemically similar amino acid residue, for example, replacing one hydrophobic residue with another hydrophobic residue or another polar residue. It is replaced with another polar residue. Such substitutions include Gly, Ala; Val, Ile, Leu, Met; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; And combinations of Phe, Tyr and the like. The effect of single amino acid substitutions can also be probed using D-amino acids. Such modifications are described, for example, in Merrifield, Science 232: 341-347 (1986), Barany and Merrifield, The Peptides , Gross and Meienhofer, eds. (NY, Academic Press) , pp. 1-284 (1979); and Stewart and Young, Solid Phase Peptide Synthesis , (Rockford, I11., Pierce), 2d Ed. (1984), using well known peptide synthesis procedures.

Peptides may also be modified by increasing or decreasing the amino acid sequence of the compound, for example, by adding or deleting amino acids. Peptides or homologues of the invention may also be modified by changing the order or composition of certain residues, wherein certain amino acid residues essential for biological activity, such as residues or conserved residues at critical contact sites, generally It is readily recognized that it cannot be changed without adverse effects. Non-deterministic amino acids need not be limited to natural amino acids in the protein, such as L-α-amino acids or D-isomers thereof, but also include non-natural amino acids such as β-γ-δ- Many derivatives of L-α-amino acids as well as amino acids can be included.

Typically, a series of peptides representing single amino acid substitutions are used to determine the effects of electrostatic charge, hydrophobicity, and the like on binding. For example, a series of positively charged amino acid substitutions (e.g. Lys or Arg) or negatively charged amino acid substitutions (e.g., depending on length of peptides exhibiting different sensitivity patterns for various MHC molecules and T cell receptors) Glu). It is also possible to use multiple substituents using relatively neutral small residues such as Ala, Gly, Pro or similar residues. Such substituents may be homo-oligomers or hetero-oligomers. The number and type of residues that are substituted or added depend on the specific functional properties sought (eg hydrophobic versus hydrophilic) and the required distance between the required contact points. Binding affinity for MHC molecules or T cell receptors, increased in comparison to the affinity of the parent peptide, may also be achieved by such substitutions. In any case, such substitutions should, for example, utilize amino acid residues or other molecular fragments selected to avoid steric and charge disturbances that may break the bond.

Amino acid substitutions typically consist of a single residue. Substitutions, deletions, insertions or any combination thereof can be combined to reach the final peptide. Substitution variants are those in which one or more residues of a particular peptide have been removed with different residues in place. Such substitutions are generally made in accordance with the following Table 2 if desired to control the characteristics of the peptide in question.

Substantial changes in function (e.g., changes in affinity for MHC molecules or T cell receptors) can be achieved by selecting less conservative substitutions from Table 2, namely (a) the structure of the peptide backbone in the substitutions. Select residues with significantly different influences on maintaining the structure, for example in sheet or helical form, (b) maintaining the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the size of the side chains. By doing so. In general, substitutions that are expected to bring about the largest change in peptide properties include (a) hydrophilic residues (e.g. seryl) to hydrophobic residues (e.g. Louisyl, isoleucyl, phenylalanyl, valelyl or alanyl) Replace; (b) replace residues with positively charged side chains (such as lysyl, arginyl or histidyl) with residues having negatively charged side chains (such as glutamyl or aspartyl), or (c) replace large side chains. A residue having a residue (eg phenylalanine) by a residue having no such side chain (eg glycine).

The peptide may also include isotopes of two or more residues in an immunogenic peptide. Isotopes as defined herein are two or more residue sequences that can be replaced with such a second sequence because the conformation of the first sequence fits the binding site specific to the second sequence. Such terms specifically include peptide backbone modifications that are well known to those skilled in the art. Such modifications include amide nitrogen, α-carbons, modifications of amide carbonyls, complete replacements of amide bonds, extensions, deletions or backbone crosslinks. Spatola, Chemistry and Biochemistry of Amino Acids, peptides and Proteins, Vol. VII (Weinstein ed., 1983).

Modification of peptides with various amino acid mimetics or non-natural amino acids is particularly useful for increasing the stability of peptides in vivo. Stability can be tested in a number of ways. For example, stability has been tested using peptidase and various biological media such as human plasma and serum. Verhoef et al., Eur. J. Drug Metab. Pharmacokin. 11: 291-302 (1986). The half life of the peptides according to the invention is conveniently determined using a 25% human serum (v / v) assay. These protocols are generally as follows. Collected human serum (Type AB, non-heat activated) is degelatinized by centrifugation prior to use. The serum is then diluted to 25% using RPMI tissue culture medium and then used to test peptide stability. Small amounts of reaction solution are removed at predetermined time intervals and added to 6% aqueous trichloroacetic acid or ethanol. The turbid reaction sample is cooled for 15 minutes (4 ° C.) and then spun to pellet the precipitated serum protein. The presence of the peptide is then determined by reversed phase HPLC using stability-specific chromatography conditions.

The peptides of the invention or their analogs with CTL stimulatory activity can be modified to provide desired properties other than improved serum half-life. For example, the peptide's ability to induce CTL activity can be enhanced by linking to sequences containing one or more epitopes capable of inducing T helper cell responses. Particularly preferred immunogenic peptide / T helper conjugates are linked by spacer molecules. Such spacers typically consist of relatively small neutral molecules such as amino acids or amino acid mimetics that are substantially free of charge under physiological conditions. The spacer is typically selected from, for example, Ala, Gly, or other neutral spacers of nonpolar or neutral polar amino acids. It will be appreciated that the spacer present optionally need not consist of the same moiety, so it may be a hetero- or homo-oligomer. If present, the spacer will typically be one or two or more residues, more typically three to six residues. Alternatively, CTL peptides can be linked to T helper peptides without spacers.

The immunogenic peptide can be linked to the T helper peptide directly or through a spacer at the amino or carboxy terminus of the CTL peptide. The amino terminus of an immunogenic peptide or T helper peptide can be acylated. Exemplary T helper peptides include tetanus toxoid 830-843, influenza 307-319, malaria circumsporozoite 382-398 and 378-389.

In some embodiments, it may be desirable to include one or more ingredients that prime CTLs in the pharmaceutical compositions of the present invention. Lipids have been identified as agents capable of priming CTLs in vivo against viral antigens. For example, palmitic acid residues are attached to the alpha and epsilon amino groups of the Lys residues and then immunogenicized, for example, via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, etc. Can be linked to the peptide. This lipidated peptide can then be injected directly in micelle form, incorporated into liposomes, or emulsified in an adjuvant such as imcomplete Freund's adjuvant. In a preferred embodiment, particularly effective immunogens include palmitic acid attached to the alpha and epsilon amino groups of Lys, which are attached to the amino terminus of the immunogenic peptide via a linkage such as Ser-Ser.

As another example of a lipid that primes a CTL reaction, E. coli lipoproteins such as trippalmitoyl-S-glycerylcysteineriseryl-serine (P ₃ CSS) can be used to prime virus specific CTLs when covalently attached to appropriate peptides. Deres et al., Nature 342: 561-564 (1989); Incorporated herein by reference. The peptides of the invention are coupled to, for example, P ₃ CCS, and these lipopeptides are administered to the individual to specifically prime the CTL response to the target antigen. In addition, since the induction of neutralizing antibodies can be primed using P ₃ CCS conjugated to a peptide displaying the appropriate epitope, the two compositions can be combined to provide more humoral and cell-mediated responses to infection. It can be induced effectively.

In addition, additional amino acids can be added to the ends of certain peptides to easily link the peptides together, to couple with a carrier support or larger peptide, or 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- or N-terminus of the peptide or oligopeptide. In some cases, due to modification at the C terminus, the binding characteristics of the peptide may change. In addition, the peptide or oligopeptide sequence may comprise terminal-NH ₂ acylation, for example alkanoyl (C ₁ -C ₂₀ ) or thioglycolyl acetylation, terminal-carboxyl amidation, for example ammonia, methylamine And may be different from the native sequence by modification. In some cases, these modifications may provide a site for linking to a support or other molecule.

Peptides of the invention can be prepared by a wide variety of methods. Because the peptides are relatively short in size, they can be synthesized in solution or on a solid support according to conventional techniques. Various automated synthesizers are commercially available and can be used according to well known protocols. See, for example, Stewart and Young, Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co. (1984) supra.

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

Coding sequences for peptides of length contemplated herein can be found in chemical techniques such as, for example, Matteucci et al., J. Am. Chem. Soc. 103: 3185 (1981), such a coding sequence can be modified simply by substituting the base encoding the original peptide sequence with the appropriate base (s). The linker is then provided with an appropriate linker, which is linked into an expression vector commonly available in the art, and the vector is used to transform a host suitable for producing the desired fusion protein. Many such vectors and host systems suitable therefor are currently available. To express the fusion protein, the coding sequence is provided with operably linked start and stop codons, promoter and terminator regions and typically replication systems to provide expression vectors for expression in the desired cellular host. will be. For example, promoter sequences corresponding to bacterial hosts are provided in plasmids containing restriction sites that are convenient 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 may be employed using suitable vectors and regulatory sequences.

The peptides of the invention, and pharmaceutical and vaccine compositions thereof, are useful for administration to mammals, especially humans, for the treatment and / or prevention of viral infections and cancers. Examples of diseases that can be treated with the immunogenic peptides of the invention include prostate cancer, hepatitis B, hepatitis C, AIDS, kidney carcinoma, cervical carcinoma, lymphoma, CMV and condlyloma acuminatum.

For pharmaceutical compositions, an immunogenic peptide of the invention is administered to an individual with cancer or infected with the virus of interest. Individuals in the latent or acute phase of infection may optionally be treated with the immunogenic peptides of the present invention separately from other therapies, or the immunogenic peptides of the present invention may be used with these therapies. In therapeutic applications, a composition is administered to a patient in an amount sufficient to induce an effective CTL response to a viral or tumor antigen, and also sufficient to treat or at least partially inhibit symptoms and / or complications. A suitable amount to do this is defined as "therapeutically effective dose." The amount effective for this use will depend, for example, on the composition of the peptide, the mode of administration, the condition and severity of the disease being treated, the weight and health of the patient, and the judgment of the attending physician, but generally in the case of early immunity (ie Peptide dosage ranges from about 1.0 to about 5000 μg for 70 kg patients, followed by measuring specific CTL activity in the patient's blood, depending on the patient's response and condition. Boosting administration of from about 1.0 to about 1000 μg of the peptide is done over a few weeks to months according to the boosting technique. It should be borne in mind that the peptides and compositions of the present invention can generally be used under severe disease conditions, i.e. fatal or potentially fatal. In such cases, based on the minimization of foreign bodies and the relative nontoxicity of the peptides, it may be considered possible and desirable for the attending physician to administer a significant excess of the peptide composition.

For therapeutic use, dosing should begin the first time signs of viral infection appear, or after tumor detection or surgical removal, or immediately after diagnosis in the case of an acute infection. Subsequently, at least the symptoms are significantly alleviated and some boosting administration thereafter. In the case of chronic infection, a boosting dose may be required following the loading dose.

Treatment of an infected individual with a composition of the present invention can quickly diminish the symptoms of infection of an acutely infected individual. For individuals who are susceptible to developing (or susceptible to) chronic infections, they are particularly useful in methods that prevent the composition from progressing from acute infection to chronic infection. For example, as described herein, where such susceptible individuals are identified prior to or during infection, the composition can be targeted to these individuals, thus minimizing the need to administer to more populations.

The peptide composition can also be used to treat chronic infections and stimulate the immune system to remove virus-infected cells in a carrier. Within formulations and dosage forms, it is important to provide sufficient amounts of immune-enhancing peptides to effectively stimulate cytotoxic T cell responses. Thus, to treat chronic infections, a representative dose range for a 70 kg patient per batch is about 1.0 to about 5000 μg, preferably about 5 to 1000 μg. Boosting doses may be required at appropriate intervals following the immunization dose, for example, for one to four weeks, possibly for a long period of time necessary to effectively immunize an individual. In the case of chronic infections, administration should be continued for at least some time thereafter, at least until clinical symptoms or laboratory tests indicate that the viral infection has disappeared or has been significantly alleviated.

Pharmaceutical compositions for therapeutic purposes are for parenteral, topical, oral or topical administration. Preferably, the pharmaceutical composition is administered parenterally, eg, intravenously, subcutaneously, intradermal or intramuscularly. Accordingly, the present invention provides compositions for parenteral administration comprising a solution of an immunogenic peptide dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. Various aqueous carriers such as water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like can be used. These compositions may be sterilized by conventional and well known sterilization techniques or may be sterile filtered. The resulting aqueous solution can be packaged or lyophilized for use as such, such a lyophilized formulation is combined with a sterile solution prior to administration. The composition may optionally contain pharmaceutically acceptable auxiliary substances such as pH adjusters and buffers, tonicity adjusters, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, Calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like.

The concentration of CTL stimulatory peptides of the present invention in pharmaceutical formulations may vary, i.e., less than about 0.1% by weight, typically from about 2% by weight to 20 to 50% by weight, depending on the particular mode of administration chosen. Fluid volume, viscosity and the like.

The peptides of the present invention also provide liposomes that act to target such peptides to specific tissues, such as lymphoid tissues, or to selectively target infected cells as well as increase the half-life of the peptide composition. It can be administered through. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, thin layers and the like. In these formulations, the monoclonal antibody which incorporates the peptide to be delivered alone as part of a liposome, or for example binds a molecule which binds to a receptor widely distributed in lymphoid cells, for example the CD45 antigen. In combination with the other therapeutic or immunogenic composition. Thus, liposomes filled or decorated with the peptides of interest of the invention can be directed into the lymphoid cell site, where the liposomes deliver the selected therapeutic / immunogenic peptide composition. Liposomes for use in the present invention are formed from standard vesicle-forming lipids, which lipids generally include neutral and negatively charged phospholipids and sterols such as cholesterol. The choice of lipids is generally made taking into account, for example, liposome size, acid instability and stability of liposomes in the bloodstream. See, eg, Szoka et al., Ann. Rev. Biophys. Bioeng. 9: 467 (1980). U.S. Patents 4,235,871, 4,501,728, 4,837,028, and 5,019,369; As incorporated herein by reference, various methods of preparing liposomes are available.

To target immune cells, the ligands to be incorporated into liposomes can include, for example, antibodies or fragments thereof that are specific for the cell surface determinants of the desired immune system cells. Liposomal suspensions containing specific peptides may be administered intravenously, topically, topically, and the like, in varying dosages, particularly depending on the mode of administration, the peptide to be delivered and the condition of the disease to be treated.

For solid compositions, conventional non-toxic solid carriers can be used, including, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate and the like. have. For oral administration, pharmaceutically acceptable non-toxic compositions may contain from 10 to 95%, more preferably from 25 to 10 excipients commonly employed, such as the carriers and active ingredients listed above, ie one or more peptides of the invention. It is formed by incorporating 75%.

In the case of aerosol administration, the immunogenic peptide is preferably supplied in finely divided form together with a surfactant and a propellant. Typical proportions of peptides are 0.01 to 20% by weight, preferably 1 to 10% by weight. The surfactant should of course be nontoxic and preferably soluble in the propellant. Representative examples of such agents include fatty acids having 6 to 22 carbon atoms (e.g. caproic acid, octanoic acid, lauric acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, olesteric acid and oleic acid) and aliphatic polyhydric alcohols or cyclics thereof. Esters or partial esters with anhydrides. Mixed esters such as mixed or natural glycerides may also be used. The surfactant may comprise 0.1 to 20%, preferably 0.25 to 5%, based on the weight of the composition. The remainder of the composition is usually a propellant. The carrier may optionally be included with, for example, lecithin for intranasal delivery.

In another aspect, the present invention relates to a vaccine containing, as an active ingredient, an immunogenic effective amount of an immunogenic peptide as described herein. The peptide (s) may be linked to their carrier or introduced into the host (including humans) as a homopolymer or heteropolymer of active peptide units. Such polymers have the advantage of increasing immunological responses and, when using different peptides to construct such polymers, the additional ability to induce CTLs and / or antibodies to react with different antigenic determinants of viral or tumor cells. It has the advantage of having. Useful carriers are well known in the art and include, for example, tyroglobulin, albumin (e.g. human serum albumin), tetanus toxoid, polyamino acids (e.g. poly (lysine: glutamic acid)), influenza, hepatitis B Viral core proteins, hepatitis B virus recombinant vaccines, and the like. The vaccine may contain physiologically acceptable diluents such as water, phosphate buffered saline, or saline, and further typically comprise an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide or alum are well known in the art. As mentioned above, CTL reactions can be primed by conjugating peptides of the invention to lipids (eg, P ₃ CCS). When immunized with a peptide composition as described herein, via injection, aerosol, oral, transdermal or other routes, the host's immune system responds to the vaccine by producing large amounts of CTLs specific for the antigen of interest, and the host Is at least partially immune to later infections or resistant to ongoing chronic infections.

Vaccine compositions containing the peptides of the present invention are administered to a patient susceptible to or at risk of viral infection or cancer, thereby enhancing the patient's own immune response ability by eliciting an immune response to the antigen. This amount is defined as "immunologically effective dose." The exact amount for this use depends on the patient's state of health and body weight, mode of administration, type of dosage form, etc., but generally ranges from about 1.0 to about 5000 μg for a 70 kg patient, more typically from about 10 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 vaccines that induce neutralizing antibody responses against the virus of interest, in particular viral envelope antigens.

For therapeutic or immunological purposes, nucleic acids encoding one or more peptides of the invention may be administered to a patient. Numerous methods are conveniently used to deliver the nucleic acid to a patient. For example, nucleic acids can be delivered directly as "naked DNA." This approach is described, for example, in US Pat. Nos. 5,580,859 and 5,589,466, as well as in Wolff et al., Science 247: 1465-1468 (1990). The nucleic acid may also be administered using ballistic delivery, as described, for example, in US Pat. No. 5,204,253. Particles consisting solely of DNA can be administered. Alternatively, DNA can be attached to particles, such as golden particles. Nucleic acids can also be delivered in complex with cationic compounds, such as cationic lipids. Lipid-mediated gene delivery methods are described, for example, in WO 96/18372; WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques 6 (7): 682-691; Rose USPN 5,279,833; WO 91/06309; and Felgner et al., (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7414. Peptides of the invention can also be expressed by an attenuated viral host, such as vaccinia or fowlpox virus. This approach involves using vaccinia virus as a vector for expressing a nucleotide sequence encoding a peptide of the invention. Upon introduction into an acute or chronically infected host or into an uninfected host, recombinant vaccinia virus induces a host CTL response by expressing the immunogenic peptide. Vaccinia vectors and methods useful in immune protocols are described, for example, in US Pat. No. 4,722,848, incorporated herein by reference. Another vector is BCG (Bacille Galmette Guerin). BCG vectors are described in Stover et al., Nature 351: 456-460 (1991), which is incorporated herein by reference. A wide variety of other vectors useful for therapeutic administration or immunization of peptides according to the invention, such as Salmonella typhi vectors, etc. will be apparent to those skilled in the art from this specification.

Preferred methods for administering nucleic acids encoding peptides of the invention utilize minigene constructs encoding the multiple epitopes of the invention. To generate DNA sequences encoding CTL epitopes (minigenes) selected for expression in human cells, the amino acid sequences of these epitopes are reversed. The human codon utilization table is used to guide the codon selection for each amino acid. These epitope-encoding DNA sequences are directly joined to form contiguous polypeptide sequences. In order to optimize expression and / or immunogenicity, additional elements may be incorporated into the minigene construct. Examples of amino acid sequences that may be reverse detoxified and included in the minigene sequence include: helper T lymphocyte epitopes, leader (signal) sequences, and endoplasmic reticulum retention signals. In addition, MHC presentation of CTL epitopes can be improved by including synthetic (eg, poly-alanine) or natural flanking sequences adjacent to the CTL epitopes.

Minigene sequences are converted to DNA by combining oligonucleotides encoding the plus and minus chains of the minigene. Overlapping oligonucleotides (30-100 bases in length) are synthesized, phosphorylated, purified, and annealed under appropriate conditions using well known techniques. The ends of these oligonucleotides are linked using T4 DNA ligase. The synthetic minigene encoding the CTL epitope polypeptide can then be cloned into the desired expression vector.

Standard regulatory sequences well known to those skilled in the art are included in the vector to ensure expression in target cells. Several vector elements are required: a promoter with a bottom cloning site for minigene insertion; Polyadenylation signals for efficient transcription termination; this. E. coli replication origin; And this. E. coli selectable markers such as ampicillin or kanamycin resistance. Numerous promoters can be used for this purpose, such as the human cytomegalovirus (hCMV) promoter. For other suitable promoter sequences, see US Pat. Nos. 5,580,859 and 5,589,466.

Additional vector modifications may be desired to optimize minigene expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or natural introns could be introduced into the transcription region of the minigene. For the purpose of increasing minigene expression, the introduction of mRNA stabilizing sequences may also be considered. It has recently been reported that immunostimulatory sequences (ISS or CpG) play a specific role in the immunogenicity of DNA vaccines. These sequences can be included in the vector beyond the minigene coding sequence if they are found to enhance immunogenicity.

In some embodiments, biointronic expression vectors that allow for the generation of minigene-encoded epitopes, and second proteins included to enhance or decrease immunogenicity, can be used. Examples of proteins or polypeptides that can advantageously enhance an immune response when co-expressed include cytokines (eg, IL2, IL12, GM-CSF), cytokine-induced molecules (eg, LeIF) or costimulatory molecules do. Helper (HTL) epitopes are bound to intracellular targeting signals and expressed separately from CTL epitopes. This will direct HTL epitopes to different cell compartments than for CTL epitopes. In some cases, this may enhance CTL induction by allowing HTL epitopes to be introduced more efficiently into the MHC class II pathway. In contrast to CTL induction, specifically lowering the immune response by co-expressing an immunosuppressive molecule (eg TGF-β) may be beneficial in certain diseases.

Once the expression vector is selected, the minigene is cloned into the bottom of the polylinker region of the promoter. Make this plasmid a suitable tooth. Transform into E. coli strains and prepare DNA using standard techniques. The orientation of the minigene and the DNA sequence as well as other elements contained in the vector are confirmed through restriction mapping and DNA sequence analysis. Bacterial cells bearing the correct plasmid can be stored as master cell banks and working cell banks.

Therapeutic amount of plasmid DNA is E. coli. It is produced by fermentation in E. coli and then purification. Aliquots from working cell banks are used to inoculate fermentation medium (eg TerrificBroth) and grown saturated in shake flasks or bioreactors according to well known techniques. The plasmid DNA can be purified using standard bioseparation techniques such as solid phase anion-exchange resin (Quiagen). If desired, ultra-helix DNA can be separated from open circular and linear forms using gel electrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using various formulations. The simplest method is to reconstitute lyophilized DNA in sterile phosphate buffered saline (PBS). Various methods have been reported and new techniques are available. As previously recognized, nucleic acids are conveniently formulated with cationic lipids. In addition, glycolipids, fusion-inducing liposomes, peptides and compounds, collectively referred to as protective, interactive, non-condensable (PINC), also complex with purified plasmid DNA to provide stability, intramuscular dispersion, or specific Parameters such as trafficking to organs or cell types can be affected.

Target cell sensitization can be used as a functional assay for MHC class I presentation and expression of minigene-encoded CTL epitopes. Plasmid DNA is introduced into a suitable mammalian cell line as a target for a standard CTL chromium release assay. The transfection method used will depend on the final formulation. Electroporation can be used for "extracted" DNA, while cationic lipids can direct in vitro transfection. Plasmids expressing green fluorescent protein (GFP) can be co-transfected to enhance transfected cells using fluorescence activated cell sorting (FACS). These cells are then labeled with chromium-51 and epitopes Used as target cells for specific CTL lines. Cytolysis detected by 51Cr release indicates that MHC presentation of minigene-encoded CTL epitopes was made.

In vivo immunogenicity is the second approach to functionally test minigenic DNA formulations. Transgenic mice expressing suitable human MHC molecules are immunized with the DNA product. Dosage and route of administration are formulation dependent (eg IM for DNA in PBS and IP for DNA complexed with lipids). 21 days after immunization, splenocytes are harvested and re-stimulated for 1 week in the presence of peptides encoding each epitope tested. These effector cells (CTLs) are subjected to assays for cytolysis of peptide-added, chromium-51 labeled target cells using standard techniques. Lysing of sensitized target cells by adding peptides corresponding to minigene-encoded epitopes to MHC demonstrates DNA vaccine function for CTL induction in vivo.

Antigenic peptides can also be used to elicit CTL ex vivo. The CTLs thus generated can be used to treat chronic infections (viruses or bacteria) or tumors in patients who will not respond to other conventional forms of therapy or will not respond to peptide vaccine treatment approaches. In vitro CTL responses to specific pathogens (infectious agents or tumor antigens) are induced by incubating a patient's CTL precursor cells (CTLp) in tissue culture with a source of antigen-presenting cells (APCs) and appropriate immunogenic peptides. Let's do it. After a suitable incubation period (typically 1 to 4 weeks) of activating CTLp, maturing it and then augmenting it with effector CTL, the cells are re-injected into the patient, and they are treated with their specific target cells (infected cells or Tumor cells).

The peptide may have use as a diagnostic reagent. For example, using the peptides of the present invention, by determining the specific individual's susceptibility to treatment methods using such peptides or related peptides, it may be helpful to alter existing treatment protocols or determine prognosis for infected individuals. Can be. In addition, the peptides can be used to predict whether chronic infections that are ongoing in an individual are at significant risk.

The following examples are illustrated but are not limited to this.

Example 1

Class I antigen separation was performed as described in the above-mentioned related application. The naturally processed peptide was then isolated and sequenced as described in the related application above. Allele-specific motifs and algorithms were determined and quantitative binding assays were performed.

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

The above examples are provided for purposes of illustration and are not intended to limit the scope of the invention. Other variants 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.

II. Non-HLA-A2 Motif

The present invention relates to the determination of allele-specific peptide motifs for human class I MHC (often referred to as HLA) allele subtypes. These motifs are then used to define T cell epitopes from all antigens of interest, particularly antigens associated with human viral diseases, cancers or autoimmune diseases, for which the amino acid sequences of potential antigens or autoantigen targets are known. .

In this manner, epitopes on a number of potential target proteins can be identified. Examples of suitable antigens include prostate cancer specific antigens (PSA), hepatitis B core and surface antigens (HBVc, HBVs), hepatitis C antigens, Epstein-Barr virus antigens, melanoma antigens (eg MAGE-1), Human immunodeficiency virus (HIV) antigens, human papilloma virus (HPV) antigens, Lhasa virus, Mycobacterium tuberculosis bacillus (MT), p53, CEA and Her2 / neu.

Peptides comprising epitopes from these antigens are synthesized and subjected to, for example, immunofluorescent staining and flow rate microfluorescence, peptide-dependent Class I assembly assays, and inhibition of CTL recognition by peptide competition. , Their ability to bind appropriate MHC molecules in assays using cells expressing purified Class I molecules and radioiodinated peptides and / or empty Class I molecules. Peptides that bind class I molecules, as well as their ability to act as targets for CTLs derived from infected or immunized individuals, as well as CTL populations that can react with tumor cells or virally infected target cells as potential therapeutic agents. Further assess their ability to induce a first in vitro or in vivo CTL response that can cause.

MHC class I antigens are encoded by HLA-A, B and C loci. HLA-A and B antigens are expressed at the cell surface at approximately the same density, while the expression of HLA-C is significantly lower (possibly 10-fold lower). Each of these loci has a number of alleles. Peptide binding motifs of the invention are relatively specific for each allele subtype.

In the case of peptide-using vaccines, the peptides of the invention preferably comprise motifs recognized by MHC I molecules which are widely distributed in the human population. Since MHC alleles exist at different frequencies within different racial groups and races, the choice of target MHC allele can depend on the target population. Table 4 shows the frequency of occurrence of various alleles in the HLA-A locus product between different species. For example, the majority Caucasus population may be covered by peptides that bind four HLA-A allele subtypes, specifically HLA-A2.1, A1, A3.2 and A24.1. Similarly, the majority of Asian populations are encompassed by the addition of peptides that bind the fifth allele HLA-A11.2.

This table is described in B. DuPont, Immunobiology of HLA, Vol. I, Histocompatibility Testing 1987, Springer-Verlag, New York 1989].

* N = black; A = Asian; C = Caucasian. The numbers in parentheses indicate the number of individuals involved in the analysis.

The nomenclature used to describe peptide compounds is in accordance with the conventional practice of displaying amino groups to the left (N-terminus) of each amino acid residue and carboxyl groups to the right (C-terminus). In the formulas illustrating certain selective embodiments of the present invention, although the amino and carboxyl-terminal groups are not specifically indicated, unless otherwise stated, they are present in forms that can be taken at physiological pH values. In amino acid structural formulas, each residue is generally represented by a standard three letter or single letter name. L-form amino acid residues are represented by a single letter in uppercase or a three letter code in which the first letter is in uppercase, and a D-type amino acid is represented by a single letter in lowercase or a three letter code in lowercase. Glycine is simply referred to as "Gly" or G because it lacks an asymmetric carbon atom.

The procedure used to identify the peptides of the present invention is generally described in Falk et al., Nature 351: 290 (1991); Incorporated herein by reference. Briefly, the method involves the large scale separation of MHC class I molecules from suitable cells or cell lines, typically by immunoprecipitation or affinity chromatography. Examples of other separation methods of the MHC molecules of interest, which are also well known to those skilled in the art, include ion exchange chromatography, lectin chromatography, size exclusion, high performance ligand chromatography, and combinations of all these techniques.

Numerous cells with defined MHC molecules, in particular MHC class I molecules, are known and readily available. For example, human EBV-transformed B cell lines have been found to be good sources for preliminary separation of class I and class II MHC molecules. Well-established cell lines are primate and commercial sources, for example, the American Type Culture Collection ("Catalogue of Cell Lines and Hybridomas", 6th edition (1988) Rockville, Maryland, U.S.A.); National Institute of General Medical Sciences 1990/1991 Catalog of Cell Lines (NIGMS) Human Genetic Mutant Cell Repository, Camden, NJ; and ASHI Repository, Bingham and Wowen's Hospital, 75 Francis Street, Boston, MA 02115. Table 5 lists some B cell lines suitable for use as a source for the HLA-A allele. All of these cell lines grow 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 that many other cell sources are available. Similar EBV B cell lines homozygous for HLA-B and HLA-C can be used as sources for HLA-B and HLA-C alleles, respectively.

In typical cases, immunoprecipitation is used to isolate the desired allele. Depending on the specificity of the antibody used, many protocols can be 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 are available for isolating HLA-A molecules (Table 6). For each targeted HLA-A allele, reagents are available that can be used to directly separate HLA-A molecules. Affinity columns made with these mAbs were successfully used to purify each HLA-A allele product using standard techniques.

In addition to allele-specific mAbs, broadly reactive anti-HLA-A, B, C mAbs such as W6 / 32 and B9.12.1, and one anti-HLA-B, C mAb, B1.23.2 Can be used in another affinity purification protocol as described in the following examples.

Peptides bound to the peptide binding grooves of the isolated MHC molecules are typically eluted by acid treatment. Peptides may also be dissociated from Class I molecules by various standard denaturation means, such as heat, pH, detergents, salts, chaotropic agents, or combinations thereof.

Peptide fractions are further separated from MHC molecules by reverse phase high performance liquid chromatography (HPLC) and then sequenced. Peptides can be separated by various other standard means well known to those skilled in the art, including filtration, ultrafiltration, electrophoresis, size chromatography, precipitation with specific antibodies, ion exchange chromatography, isoelectric focusing, and the like.

Sequence analysis on isolated peptides can be performed according to standard techniques such as Edman digestion. Hunkapiller, MW, et al., Methods Enzymol. 91, 399 (1983). Other methods suitable for sequencing include methods for sequencing individual peptides by mass spectroscopy, as mentioned previously. Hunt et al., Science 225: 1261 (1992); Which is incorporated herein by reference]. Amino acid sequence analysis of bulky heterologous peptides (eg, collected HPLC fractions) from different class I molecules typically shows characteristic sequence motifs for each class I allele.

By defining motifs specific for different class I alleles, it is possible to identify potential peptide epitopes from antigenic proteins with known amino acid sequences. Typically, identification of potential peptide epitopes is initially performed using a computer that scans the amino acid sequence of the desired antigen for the presence of the motif. Subsequently, epitope sequences are synthesized. The ability to bind MHC class molecules is measured in a variety of different ways. One method is a class I molecular binding assay as described in the related application mentioned above. Other alternative methods described in the literature include methods of inhibiting antigen presentation [Sette et al., J. Immunol. 141: 3893 (1991)], in vitro assembly assays (Townsend et al., Cell 62: 285 (1990)) and FACS-using assays using mutated cells such as RMA.S [Melief et al., Eur. J. Immunol. 21: 2963 (1991).

Peptides tested as positive in the MHC Class I binding assays are then assayed for the ability of the peptide to induce specific CTL responses in vitro. For example, antigen-presenting cells incubated with peptides can be assayed for their ability to induce CTL responses in responder cell populations. Antigen-presenting cells can be normal cells, eg, peripheral blood mononuclear cells or dendritic cells. Inaba et al., J. Exp. Med. 166: 182 (1987); Boog, Eur. J. Immunol. 18: 219 (1988).

On the other hand, mutant mammalian cell lines lacking the ability to add internally processed peptides to Class I molecules, such as the mouse cell line RMA-S. Karre et al., Nature 319: 675 (1986). ; Ljunggren et al., Eur. J. Immunol. 21: 2963-2970 (1991)] and human celiac T cell hybrids, T-2 (Cerundolo et al., Nature 345: 449-452 (1990)), and mutants transfected with appropriate human class I genes. Mammalian cell lines are used for convenience in testing the ability of peptides to be added to them to induce primary CTL responses in vitro. Other eukaryotic cell lines that can be used include various insect cell lines, such as mosquito larvae (ATCC cell lines CCL 125, 126, 1660, 1591, 6585, 6586), silkworms (ATCC CRL 8851), armyworms (ATCC CRL). 1711), moths (ATCC CCL 80) and drosophila cell lines, such as Schneider cell lines (Schneider J. Embryol. Exp. Morphol. 27: 353-365 (1927).

Peripheral blood lymphocytes are usually isolated after normal donor or patient leukocytosis, and then used as a responder cell source of CTL precursors. In one embodiment, the appropriate antigen-presenting cells are incubated with 10-100 μM peptides in serum-free medium for 4 hours under suitable culture conditions. The peptide-added antigen-presenting cells are then incubated with the responder cell population in vitro for 7-10 days under optimized culture conditions. Both cultures were assayed for the presence of CTLs that killed specific peptide-pulsed target radiolabeled target cells as well as target cells expressing intrinsically processed forms of the relevant virus or tumor antigen from which the peptide sequence was derived. Positive CTL activation can then be determined.

By testing for different peptide target cells expressing appropriate or inappropriate human MHC class I, the specificity and MHC restriction of CTLs are determined. Peptides that are tested to be positive in the MHC binding assay and elicit specific CTL responses are referred to herein as immunogenic peptides.

Immunogenic peptides can be prepared synthetically or by recombinant DNA techniques or from natural sources such as complete viruses or tumors. Although it is preferred that the peptide contains few other naturally occurring host cell proteins and fragments thereof, in some embodiments, the peptide can be conjugated synthetically with the original fragment or particle.

The polypeptide or peptide may be of various lengths in neutral (no charge) form or in salt form and does not include or include modifications such as glycosylation, side chain oxidation or phosphorylation, but such modifications are herein It is assumed that it does not destroy the biological activity of the polypeptide as described.

Desirably, the peptide will be as small as possible while maintaining substantially all of the biological activity of the large peptide. Where possible, it may be desirable to optimize the peptides of the invention to 9 or 10 amino acid residues in length, consistent in size with the intrinsically processed viral peptides or tumor cell peptides bound to the MHC class I molecules on the cell surface. .

Peptides having the desired activity can be modified as needed to provide certain desired properties, such as improved pharmacological properties, but unmodified peptides that bind to the desired MHC molecule and activate appropriate T cells. All biological activity of must substantially increase or at least maintain it. For example, these peptides can be subject to various changes, for example, conservative or non-conservative substitutions, which alter certain advantages in their use, such as improving MHC binding. You can also provide Conservative substitutions mean replacing one amino acid residue with another biologically and / or chemically similar amino acid residue, for example, replacing one hydrophobic residue with another hydrophobic residue or another polar residue. It is replaced with another polar residue. Such substitutions include Gly, Ala; Val, Ile, Leu, Met; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; And combinations of Phe, Tyr and the like. The effect of single amino acid substitutions can also be probed using D-amino acids. Such modifications are described, for example, in Merrifield, Science 232: 341-347 (1986), Barany and Merrifield, The Peptides , Gross and Meienhofer, eds. (NY, Academic Press) , pp. 1-284 (1979); and Stewart and Young, Solid Phase Peptide Synthesis , (Rockford, I11., Pierce), 2d Ed. (1984), using well known peptide synthesis procedures.

Peptides may also be modified by increasing or decreasing the amino acid sequence of the compound, for example, by adding or deleting amino acids. Peptides or homologues of the invention may also be modified by changing the order or composition of certain residues, wherein certain amino acid residues essential for biological activity, such as residues or conserved residues at critical contact sites, generally It is readily recognized that it cannot be changed without adverse effects. Non-deterministic amino acids need not be limited to natural amino acids in the protein, such as L-α-amino acids or D-isomers thereof, but also include non-natural amino acids such as β-γ-δ- Many derivatives of L-α-amino acids as well as amino acids can be included.

Typically, a series of peptides representing single amino acid substitutions are used to determine the effects of electrostatic charge, hydrophobicity, and the like on binding. For example, a series of positively charged amino acid substitutions (e.g. Lys or Arg) or negatively charged amino acid substitutions (e.g. Glu). It is also possible to use multiple substituents using relatively neutral small residues such as Ala, Gly, Pro or similar residues. Such substituents may be homo-oligomers or hetero-oligomers. The number and type of residues that are substituted or added depend on the specific functional properties sought (eg hydrophobic versus hydrophilic) and the required distance between the required contact points. Binding affinity for MHC molecules or T cell receptors, increased in comparison to the affinity of the parent peptide, may also be achieved by such substitutions. In any case, such substitutions should, for example, utilize amino acid residues or other molecular fragments selected to avoid steric and charge disturbances that may break the bond.

Amino acid substitutions typically consist of a single residue. Substitutions, deletions, insertions or any combination thereof can be combined to reach the final peptide. Substitution variants are those in which one or more residues of a particular peptide have been removed with different residues in place. Such substitutions are generally made in accordance with the following Table 2 if desired to control the characteristics of the peptide in question.

TABLE 2

Substantial changes in function (e.g., changes in affinity for MHC molecules or T cell receptors) can be achieved by selecting less conservative substitutions from Table 2, namely (a) the structure of the peptide backbone in the substitutions. Select residues with significantly different influences on maintaining the structure, for example in sheet or helical form, (b) maintaining the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the size of the side chains. By doing so. In general, substitutions that are expected to bring about the largest change in peptide properties include (a) hydrophilic residues (e.g. seryl) to hydrophobic residues (e.g. Louisyl, isoleucyl, phenylalanyl, valelyl or alanyl) Replace; (b) replace residues with positively charged side chains (such as lysyl, arginyl or histidyl) with residues having negatively charged side chains (such as glutamyl or aspartyl), or (c) replace large side chains. A residue having a residue (eg phenylalanine) by a residue having no such side chain (eg glycine).

The peptide may also include isotopes of two or more residues in an immunogenic peptide. Isotopes as defined herein are two or more residue sequences that can be replaced with such a second sequence because the conformation of the first sequence fits the binding site specific to the second sequence. Such terms specifically include peptide backbone modifications that are well known to those skilled in the art. Such modifications include amide nitrogen, α-carbons, modifications of amide carbonyls, complete replacements of amide bonds, extensions, deletions or backbone crosslinks. Spatola, Chemistry and Biochemistry of Amino Acids, peptides and Proteins, Vol. VII (Weinstein ed., 1983).

Modification of peptides with various amino acid mimetics or non-natural amino acids is particularly useful for increasing the stability of peptides in vivo. Stability can be tested in a number of ways. For example, stability has been tested using peptidase and various biological media such as human plasma and serum. See Verhoef et al., Eur. J. Drug Metab. Pharmacokin. 11: 291-302 (1986). The half life of the peptides according to the invention is conveniently determined using a 25% human serum (v / v) assay. These protocols are generally as follows. Collected human serum (Type AB, non-heat activated) is degelatinized by centrifugation prior to use. The serum is then diluted to 25% using RPMI tissue culture medium and then used to test peptide stability. Small amounts of reaction solution are removed at predetermined time intervals and added to 6% aqueous trichloroacetic acid or ethanol. The turbid reaction sample is cooled for 15 minutes (4 ° C.) and then spun to pellet the precipitated serum protein. The presence of the peptide is then determined by reversed phase HPLC using stability-specific chromatography conditions.

The peptides of the invention or their analogs with CTL stimulatory activity can be modified to provide desired properties other than improved serum half-life. For example, the peptide's ability to induce CTL activity can be enhanced by linking to sequences containing one or more epitopes capable of inducing T helper cell responses. Particularly preferred immunogenic peptide / T helper conjugates are linked by spacer molecules. Such spacers typically consist of relatively small neutral molecules such as amino acids or amino acid mimetics that are substantially free of charge under physiological conditions. The spacer is typically selected from, for example, Ala, Gly, or other neutral spacers of nonpolar or neutral polar amino acids. It will be appreciated that the spacer present optionally need not consist of the same moiety, so it may be a hetero- or homo-oligomer. If present, the spacer will typically be one or two or more residues, more typically three to six residues. Alternatively, CTL peptides can be linked to T helper peptides without spacers.

The immunogenic peptide can be linked to the T helper peptide directly or through a spacer at the amino or carboxy terminus of the CTL peptide. The amino terminus of an immunogenic peptide or T helper peptide can be acylated. Exemplary T helper peptides include tetanus toxoid 830-843, influenza 307-319, malaria circumsporozoite 382-398 and 378-389.

In some embodiments, it may be desirable to include one or more ingredients that prime CTLs in the pharmaceutical compositions of the present invention. Lipids have been identified as agents capable of priming CTLs in vivo against viral antigens. For example, palmitic acid residues are attached to the alpha and epsilon amino groups of the Lys residues and then immunogenicized, for example, via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, etc. Can be linked to the peptide. This lipidated peptide can then be injected directly in micelle form, incorporated into liposomes, or emulsified in an adjuvant such as imcomplete Freund's adjuvant. In a preferred embodiment, particularly effective immunogens include palmitic acid attached to the alpha and epsilon amino groups of Lys, which are attached to the amino terminus of the immunogenic peptide via a linkage such as Ser-Ser.

As another example of a lipid that primes a CTL reaction, E. coli lipoproteins such as trippalmitoyl-S-glycerylcysteineriseryl-serine (P ₃ CSS) can be used to prime virus specific CTLs when covalently attached to appropriate peptides. Deres et al., Nature 342: 561-564 (1989); Incorporated herein by reference. The peptides of the invention are coupled to, for example, P ₃ CCS, and these lipopeptides are administered to the individual to specifically prime the CTL response to the target antigen. In addition, since the induction of neutralizing antibodies can be primed using P ₃ CCS conjugated to a peptide displaying the appropriate epitope, the two compositions can be combined to provide more humoral and cell-mediated responses to infection. It can be induced effectively.

In addition, additional amino acids can be added to the ends of certain peptides to easily link the peptides together, to couple with a carrier support or larger peptide, or 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- or N-terminus of the peptide or oligopeptide. In some cases, due to modification at the C terminus, the binding characteristics of the peptide may change. In addition, the peptide or oligopeptide sequence may comprise terminal-NH ₂ acylation, for example alkanoyl (C ₁ -C ₂₀ ) or thioglycolyl acetylation, terminal-carboxyl amidation, for example ammonia, methylamine And may be different from the native sequence by modification. In some cases, these modifications may provide a site for linking to a support or other molecule.

Peptides of the invention can be prepared by a wide variety of methods. Because the peptides are relatively short in size, they can be synthesized in solution or on a solid support according to conventional techniques. Various automated synthesizers are commercially available and can be used according to well known protocols. See, for example, Stewart and Young, Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co. (1984) supra.

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

Coding sequences for peptides of length contemplated herein can be found in chemical techniques such as, for example, Matteucci et al., J. Am. Chem. Soc. 103: 3185 (1981), such a coding sequence can be modified simply by substituting the base encoding the original peptide sequence with the appropriate base (s). The linker is then provided with an appropriate linker, which is linked into an expression vector commonly available in the art, and the vector is used to transform a host suitable for producing the desired fusion protein. Many such vectors and host systems suitable therefor are currently available. To express the fusion protein, the coding sequence is provided with operably linked start and stop codons, promoter and terminator regions and typically replication systems to provide expression vectors for expression in the desired cellular host. will be. For example, promoter sequences corresponding to bacterial hosts are provided in plasmids containing restriction sites that are convenient 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 may be employed using suitable vectors and regulatory sequences.

The peptides of the invention, and pharmaceutical and vaccine compositions thereof, are useful for administration to mammals, especially humans, for the treatment and / or prevention of viral infections and cancers. Examples of diseases that can be treated with the immunogenic peptides of the invention include prostate cancer, hepatitis B, hepatitis C, AIDS, kidney carcinoma, cervical carcinoma, lymphoma, CMV and condlyloma acuminatum.

For pharmaceutical compositions, an immunogenic peptide of the invention is administered to an individual with cancer or infected with the virus of interest. Individuals in the latent or acute phase of infection may optionally be treated with the immunogenic peptides of the present invention separately from other therapies, or the immunogenic peptides of the present invention may be used with these therapies. In therapeutic applications, a composition is administered to a patient in an amount sufficient to induce an effective CTL response to a viral or tumor antigen, and also sufficient to treat or at least partially inhibit symptoms and / or complications. A suitable amount to do this is defined as "therapeutically effective dose." The amount effective for this use will depend, for example, on the composition of the peptide, the mode of administration, the condition and severity of the disease being treated, the weight and health of the patient, and the judgment of the attending physician, but in general for early immunity Peptide dosage ranges from about 1.0 to about 5000 μg for 70 kg patients, followed by measuring specific CTL activity in the patient's blood, depending on the patient's response and condition. Boosting administration of from about 1.0 to about 1000 μg of the peptide is done over a few weeks to months according to the boosting technique. It should be borne in mind that the peptides and compositions of the present invention can generally be used under severe disease conditions, i.e. fatal or potentially fatal. In such cases, based on the minimization of foreign bodies and the relative nontoxicity of the peptides, it may be considered possible and desirable for the attending physician to administer a significant excess of the peptide composition.

For therapeutic use, dosing should begin the first time signs of viral infection appear, or after tumor detection or surgical removal, or immediately after diagnosis in the case of an acute infection. Subsequently, at least the symptoms are significantly alleviated and some boosting administration thereafter. In the case of chronic infection, a boosting dose may be required following the loading dose.

Treatment of an infected individual with a composition of the present invention can quickly diminish the symptoms of infection of an acutely infected individual. For individuals who are susceptible to developing (or susceptible to) chronic infections, they are particularly useful in methods that prevent the composition from progressing from acute infection to chronic infection. For example, as described herein, where such susceptible individuals are identified prior to or during infection, the composition can be targeted to these individuals, thereby minimizing the need to administer to more populations.

The peptide composition can also be used to treat chronic infections and stimulate the immune system to remove virus-infected cells in a carrier. Within formulations and dosage forms, it is important to provide sufficient amounts of immune-enhancing peptides to effectively stimulate cytotoxic T cell responses. Thus, to treat chronic infections, a representative dose range for a 70 kg patient per batch is about 1.0 to about 5000 μg, preferably about 5 to 1000 μg. Boosting doses may be required at appropriate intervals following the immunization dose, for example, for one to four weeks, possibly for a long period of time necessary to effectively immunize an individual. In the case of chronic infections, administration should be continued for at least some time thereafter, at least until clinical symptoms or laboratory tests indicate that the viral infection has disappeared or has been significantly alleviated.

Pharmaceutical compositions for therapeutic purposes are for parenteral, topical, oral or topical administration. Preferably, the pharmaceutical composition is administered parenterally, eg, intravenously, subcutaneously, intradermal or intramuscularly. Accordingly, the present invention provides compositions for parenteral administration comprising a solution of an immunogenic peptide dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. Various aqueous carriers such as water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like can be used. These compositions may be sterilized by conventional and well known sterilization techniques or may be sterile filtered. The resulting aqueous solution can be packaged or lyophilized for use as such, such a lyophilized formulation is combined with a sterile solution prior to administration. The composition may optionally contain pharmaceutically acceptable auxiliary substances such as pH adjusters and buffers, tonicity adjusters, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, Calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like.

The concentration of CTL stimulatory peptides of the present invention in pharmaceutical formulations may vary, i.e., less than about 0.1% by weight, typically from about 2% by weight to 20 to 50% by weight, depending on the particular mode of administration chosen. Fluid volume, viscosity and the like.

The peptides of the present invention also provide liposomes that act to target such peptides to specific tissues, such as lymphoid tissues, or to selectively target infected cells as well as increase the half-life of the peptide composition. It can be administered through. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, thin layers and the like. In these formulations, the monoclonal antibody which incorporates the peptide to be delivered alone as part of a liposome, or for example binds a molecule which binds to a receptor widely distributed in lymphoid cells, for example the CD45 antigen. In combination with the other therapeutic or immunogenic composition. Thus, liposomes filled or decorated with the peptides of interest of the invention can be directed into the lymphoid cell site, where the liposomes deliver the selected therapeutic / immunogenic peptide composition. Liposomes for use in the present invention are formed from standard vesicle-forming lipids, which lipids generally include neutral and negatively charged phospholipids and sterols such as cholesterol. The choice of lipids is generally made taking into account, for example, liposome size, acid instability and stability of liposomes in the bloodstream. See, eg, Szoka et al., Ann. Rev. Biophys. Bioeng. 9: 467 (1980). U.S. Patents 4,235,871, 4,501,728, 4,837,028, and 5,019,369; As incorporated herein by reference, various methods of preparing liposomes are available.

To target immune cells, the ligands to be incorporated into liposomes can include, for example, antibodies or fragments thereof that are specific for the cell surface determinants of the desired immune system cells. Liposomal suspensions containing specific peptides may be administered intravenously, topically, topically, and the like, in varying dosages, particularly depending on the mode of administration, the peptide to be delivered and the condition of the disease to be treated.

For solid compositions, conventional non-toxic solid carriers can be used, including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate and the like. have. For oral administration, pharmaceutically acceptable non-toxic compositions may comprise from 10 to 95%, more preferably from 25 to 10 excipients commonly employed, such as the carriers and active ingredients listed above, ie one or more peptides of the invention. It is formed by incorporating 75%.

In the case of aerosol administration, the immunogenic peptide is preferably supplied in finely divided form together with a surfactant and a propellant. Typical proportions of peptides are 0.01 to 20% by weight, preferably 1 to 10% by weight. The surfactant should of course be nontoxic and preferably soluble in the propellant. Representative examples of such agents include fatty acids having 6 to 22 carbon atoms (e.g. caproic acid, octanoic acid, lauric acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, olesteric acid and oleic acid) and aliphatic polyhydric alcohols or cyclics thereof. Esters or partial esters with anhydrides. Mixed esters such as mixed or natural glycerides may also be used. The surfactant may comprise 0.1 to 20%, preferably 0.25 to 5%, based on the weight of the composition. The remainder of the composition is usually a propellant. The carrier may optionally be included with, for example, lecithin for intranasal delivery.

In another aspect, the present invention relates to a vaccine containing, as an active ingredient, an immunogenic effective amount of an immunogenic peptide as described herein. The peptide (s) may be linked to their carrier or introduced into the host (including humans) as a homopolymer or heteropolymer of active peptide units. Such polymers have the advantage of increasing immunological responses and, when using different peptides to construct such polymers, the additional ability to induce CTLs and / or antibodies to react with different antigenic determinants of viral or tumor cells. It has the advantage of having. Useful carriers are well known in the art and include, for example, tyroglobulin, albumin (e.g. human serum albumin), tetanus toxoid, polyamino acids (e.g. poly (lysine: glutamic acid)), influenza, hepatitis B Viral core proteins, hepatitis B virus recombinant vaccines, and the like. The vaccine may contain physiologically acceptable diluents such as water, phosphate buffered saline, or saline, and further typically comprise an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide or alum are well known in the art. As mentioned above, CTL reactions can be primed by conjugating peptides of the invention to lipids (eg, P ₃ CCS). When immunized with a peptide composition as described herein, via injection, aerosol, oral, transdermal or other routes, the host's immune system responds to the vaccine by producing large amounts of CTLs specific for the antigen of interest, and the host Is at least partially immune to later infections or resistant to ongoing chronic infections.

Vaccine compositions containing the peptides of the present invention are administered to a patient susceptible to or at risk of viral infection or cancer, thereby enhancing the patient's own immune response ability by eliciting an immune response to the antigen. This amount is defined as "immunologically effective dose." The exact amount for this use depends on the patient's state of health and body weight, mode of administration, type of dosage form, etc., but generally ranges from about 1.0 to about 5000 μg for a 70 kg patient, more typically from about 10 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 vaccines that induce neutralizing antibody responses against the virus of interest, in particular viral envelope antigens.

For therapeutic or immunological purposes, nucleic acids encoding one or more peptides of the invention may be administered to a patient. Numerous methods are conveniently used to deliver the nucleic acid to a patient. For example, nucleic acids can be delivered directly as "naked DNA." This approach is described, for example, in US Pat. Nos. 5,580,859 and 5,589,466, as well as in Wolff et al., Science 247: 1465-1468 (1990). The nucleic acid may also be administered using ballistic delivery, as described, for example, in US Pat. No. 5,204,253. Particles consisting solely of DNA can be administered. Alternatively, DNA can be attached to particles, such as golden particles. Nucleic acids can also be delivered in complex with cationic compounds, such as cationic lipids. Lipid-mediated gene delivery methods are described, for example, in WO 96/18372; WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques 6 (7): 682-691; Rose USPN 5,279,833; WO 91/06309; and Felgner et al., (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7414. Peptides of the invention can also be expressed by an attenuated viral host, such as vaccinia or fowlpox virus. This approach involves using vaccinia virus as a vector for expressing a nucleotide sequence encoding a peptide of the invention. Upon introduction into an acute or chronically infected host or into an uninfected host, the recombinant vaccinia virus induces a host CTL response by expressing the immunogenic peptide. Vaccinia vectors and methods useful in immune protocols are described, for example, in US Pat. No. 4,722,848, incorporated herein by reference. Another vector is BCG (Bacille Galmette Guerin). BCG vectors are described in Stover et al., Nature 351: 456-460 (1991), which is incorporated herein by reference. A wide variety of other vectors useful for therapeutic administration or immunization of peptides according to the invention, such as Salmonella typhi vectors, etc. will be apparent to those skilled in the art from this specification.

Preferred methods for administering nucleic acids encoding peptides of the invention utilize minigene constructs encoding the multiple epitopes of the invention. To generate DNA sequences encoding CTL epitopes (minigenes) selected for expression in human cells, the amino acid sequences of these epitopes are reversed. The human codon utilization table is used to guide the codon selection for each amino acid. These epitope-encoding DNA sequences are directly joined to form contiguous polypeptide sequences. In order to optimize expression and / or immunogenicity, additional elements may be incorporated into the minigene construct. Examples of amino acid sequences that may be reverse detoxified and included in the minigene sequence include: helper T lymphocyte epitopes, leader (signal) sequences, and endoplasmic reticulum retention signals. In addition, MHC presentation of CTL epitopes can be improved by including synthetic (eg poly-alanine) or natural flanking sequences adjacent to the CTL epitopes.

Minigene sequences are converted to DNA by combining oligonucleotides encoding the plus and minus chains of the minigene. Overlapping oligonucleotides (30-100 bases in length) are synthesized, phosphorylated, purified, and annealed under appropriate conditions using well known techniques. The ends of these oligonucleotides are linked using T4 DNA ligase. The synthetic minigene encoding the CTL epitope polypeptide can then be cloned into the desired expression vector.

Standard regulatory sequences well known to those skilled in the art are included in the vector to ensure expression in target cells. Several vector elements are required: a promoter with a bottom cloning site for minigene insertion; Polyadenylation signals for efficient transcription termination; this. E. coli replication origin; And this. E. coli selectable markers such as ampicillin or kanamycin resistance. Numerous promoters can be used for this purpose, such as the human cytomegalovirus (hCMV) promoter. For other suitable promoter sequences, see US Pat. Nos. 5,580,859 and 5,589,466.

Additional vector modifications may be desired to optimize minigene expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or natural introns could be introduced into the transcription region of the minigene. For the purpose of increasing minigene expression, the introduction of mRNA stabilizing sequences may also be considered. It has recently been reported that immunostimulatory sequences (ISS or CpG) play a specific role in the immunogenicity of DNA vaccines. These sequences can be included in the vector beyond the minigene coding sequence if they are found to enhance immunogenicity.

In some embodiments, biointronic expression vectors that allow for the generation of minigene-encoded epitopes, and second proteins included to enhance or decrease immunogenicity, can be used. Examples of proteins or polypeptides that can advantageously enhance an immune response when co-expressed include cytokines (eg, IL2, IL12, GM-CSF), cytokine-induced molecules (eg, LeIF) or costimulatory molecules do. Helper (HTL) epitopes are bound to intracellular targeting signals and expressed separately from CTL epitopes. This will direct HTL epitopes to different cell compartments than for CTL epitopes. In some cases, this may enhance CTL induction by allowing HTL epitopes to be introduced more efficiently into the MHC class II pathway. In contrast to CTL induction, specifically lowering the immune response by co-expressing an immunosuppressive molecule (eg TGF-β) may be beneficial in certain diseases.

Once the expression vector is selected, the minigene is cloned into the bottom of the polylinker region of the promoter. Make this plasmid a suitable tooth. Transform into E. coli strains and prepare DNA using standard techniques. The orientation of the minigene and the DNA sequence as well as other elements contained in the vector are confirmed through restriction mapping and DNA sequence analysis. Bacterial cells bearing the correct plasmid can be stored as master cell banks and working cell banks.

Therapeutic amount of plasmid DNA is E. coli. It is produced by fermentation in E. coli and then purification. Aliquots from working cell banks are used to inoculate fermentation medium (eg, Terrific Broth) and grown saturated in shake flasks or bioreactors according to well known techniques. The plasmid DNA can be purified using standard bioseparation techniques such as solid phase anion-exchange resin (Quiagen). If desired, ultra-helix DNA can be separated from open circular and linear forms using gel electrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using various formulations. The simplest method is to reconstitute lyophilized DNA in sterile phosphate buffered saline (PBS). Various methods have been reported and new techniques are available. As previously recognized, nucleic acids are conveniently formulated with cationic lipids. In addition, glycolipids, fusion-inducing liposomes, peptides and compounds, collectively referred to as protective, interactive, non-condensable (PINC), also complex with purified plasmid DNA to provide stability, intramuscular dispersion, or specific Parameters such as trafficking to organs or cell types can be affected.

Target cell sensitization can be used as a functional assay for MHC class I presentation and expression of minigene-encoded CTL epitopes. Plasmid DNA is introduced into a suitable mammalian cell line as a target for a standard CTL chromium release assay. The transfection method used will depend on the final formulation. Electroporation can be used for "extracted" DNA, while cationic lipids can direct in vitro transfection. Plasmids expressing green fluorescent protein (GFP) can be co-transfected to enhance transfected cells using fluorescence activated cell sorting (FACS). These cells are then labeled with chromium-51 and used as target cells for epitope-specific CTL lines. Cytolysis detected by 51Cr release indicates that MHC presentation of minigene-encoded CTL epitopes was made.

In vivo immunogenicity is the second approach to functionally test minigenic DNA formulations. Transgenic mice expressing suitable human MHC molecules are immunized with the DNA product. Dosage and route of administration are formulation dependent (eg IM for DNA in PBS and IP for DNA complexed with lipids). 21 days after immunization, splenocytes are harvested and re-stimulated for 1 week in the presence of peptides encoding each epitope tested. These effector cells (CTLs) are subjected to assays for cytolysis of peptide-added, chromium-51 labeled target cells using standard techniques. Lysing of sensitized target cells by adding peptides corresponding to minigene-encoded epitopes to MHC demonstrates DNA vaccine function for CTL induction in vivo.

Antigenic peptides can also be used to elicit CTL ex vivo. The CTLs thus generated can be used to treat chronic infections (viruses or bacteria) or tumors in patients who will not respond to other conventional forms of therapy or will not respond to peptide vaccine treatment approaches. In vitro CTL responses to specific pathogens (infectious agents or tumor antigens) are induced by incubating the patient's CTL precursor cells (CTLp) in tissue culture with a source of antigen-presenting cells (APCs) and appropriate immunogenic peptides. Let it be. After a suitable incubation period (typically 1 to 4 weeks) of activating CTLp, maturing it and then augmenting it with effector CTL, the cells are re-injected into the patient, which causes their specific target cells (infected cells or Tumor cells). To optimize the in vitro conditions for specific cytotoxic T cell development, stimulator cell cultures are maintained in a suitable serum-free medium.

Prior to incubation with a cell to be activated, eg, a precursor CD8 + cell, a sufficient amount of antigenic peptide is added to the human class I molecule to be expressed on the surface of the stimulator cell. Stimulator is added to the cell culture. In the present invention, a sufficient amount of peptide is an amount such that about 200, preferably at least 200, human class I MHC molecules to which the peptide is added can be expressed on the surface of each stimulator cell. Preferably, these stimulator cells are incubated with> 20 μg / ml peptide.

Resting or precursor CD8 + cells are then incubated in culture with appropriate stimulator cells for a time sufficient to activate these CD8 + cells. Preferably, CD8 + cells are activated in an antigen-specific manner. The ratio of resting or precursor CD8 + (effector) cells to stimulator cells may vary from person to person, which furthermore depends on the individual's lymphocyte amenability to culture conditions, and the disease state or existing treatment modality. It may depend on variables such as the nature and severity of the other disease state used. Preferably, however, the lymphocyte: stimulator cell ratio is in the range of about 30: 1 to 300: 1. Effector / stimulator cultures can be maintained for as long as possible as needed to stimulate a therapeutically available or effective number of CD8 + cells.

To induce CTLs in vitro, one must specifically recognize peptides that bind to allele specific MHC class I molecules on APC. The number of specific MHC / peptide complexes per APC stimulates CTLs, particularly critical for the primary immune response. Although a small amount of peptide / MHC complex per cell is sufficient to make the cells susceptible to lysis by CTL or to stimulate the secondary CTL response, in order to successfully activate the CTL precursor (pCTL) during the primary reaction, A significant number of MHC / peptide complexes are needed. By adding peptides to empty major histocompatibility complex molecules on cells, primary cytotoxic T lymphocyte responses can be induced. By adding peptides to empty major histocompatibility complex molecules on cells, primary cytotoxic T lymphocyte responses can be induced.

Since the mutant cell line is not present for every human MHC allele, it is a technique to remove the intrinsic MHC-associated peptide from the APC surface and then add the immunogenic peptide of interest to the resulting empty MHC molecule. It is advantageous. As APC, the use of non-transformed (non-tumorigenic), non-infected cells, preferably autologous cells, of a patient may be used in a CTL induction protocol aimed at developing in vitro CTL therapy. It is hoped for to devise. The present application describes a method for removing endogenous MHC-associated peptides from the surface of APC and then adding the desired peptides.

Stable MHC class I molecules are trimeric complexes formed of the following elements: 1) a peptide of typically 8 to 10 residues, 2) a transmembrane polymorphic protein heavy chain having peptide-binding sites in its α1 and α2 domains And 3) non-covalently bound non-polymorphic light chain, β ₂ microglobulin. The bound peptide is removed and / or dissociated β ₂ microglobulin from the complex, making the MHC class I molecule inactive and unstable, thereby rapidly breaking it down. All MHC class I molecules isolated from PBMCs have endogenous peptides bound to them. Thus, the first step is to remove all endogenous peptides bound to the MHC class I molecules on the APC without breaking them down, and then add the exogenous peptides to them.

Two possible methods for removing bound peptides from MHC class I molecules include destabilizing β ₂ microglobulin by lowering the culture temperature from 37 ° C. to 26 ° C. overnight, and inducing cells from cells using mild acid treatment. Included are methods for removing reproductive peptides. These methods can release existing bound peptides into the extracellular environment, allowing new endogenous peptides to bind to empty Class I molecules. Although the cold-warm incubation method allows exogenous peptides to efficiently bind to the MHC complex, this requires incubation overnight at 26 ° C., which can slow down the metabolism of cells. In addition, cells that do not actively synthesize MHC molecules (eg, resting PBMCs) are thought to fail to produce large quantities of empty surface MHC molecules by cold and hot processes.

Rough acid stripping involves extracting the peptide with trifluoroacetic acid (pH 2) or acid denaturing the immunoaffinity purified class I-peptide complex. These methods cannot induce CTLs because it is important to remove endogenous peptides while maintaining optimal metabolic state and APC viability in antigen presentation. Endogenous peptides were identified using a mild acid solution at pH 3, such as glycine or citrate-phosphate buffer, and tumor associated T cell epitopes were identified. This treatment is particularly effective in that other surface antigens, including MHC class II molecules, remain intact, whereas only MHC class I molecules destabilize (and release related peptides). Most importantly, treating the cells with a mild acid solution has no effect on cell viability or metabolic status. Mild acid treatment is rapid because removal of the endogenous peptide occurs 2 minutes at 4 ° C., and after addition of the appropriate peptide, APC is ready to perform its function. The technique is utilized herein to make peptide-specific APCs for generating primary antigen-specific CTLs. The resulting APCs are efficient at inducing peptide-specific CD8 + CTLs.

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 or CD8 + cells (or fragments thereof) added on such stimulator cells can be used to bind their appropriate complementary ligands. The antibody-tagged molecules can then be extracted from the stimulator-effector cell mixture via any suitable method, such as well known immunoprecipitation or immunoassay.

Effective cytotoxic amounts of activated CD8 + cells may vary depending on whether they are intended for use in vitro or in vivo, as well as the type and amount of cells that are ultimate targets of these killer cells. The amount will also vary depending on the condition of the patient, which should be determined by the practitioner taking into account all suitable factors. However, in the case of adult humans, preferably from about 1 X 10 ⁶ to about 1 X 10 ¹² , more preferably from about 5 X 10 ⁶ to about 5 X 10 ⁷ cells for mice 1 × 10 ⁸ to about 1 × 10 ¹¹ , even more preferably from about 1 × 10 ⁹ to about 1 × 10 ¹⁰ activated CD8 + cells.

Preferably, as discussed above, after activated CD8 + cells are harvested from the cell culture, such CD8 + cells are administered to the individual to be treated. However, it is important to recognize that, unlike other previously proposed treatment modalities, the methods of the present invention utilize cell culture systems that are not tumorigenic. Thus, without complete separation of the stimulator cells from the activated CD8 + cells, there is no inherent risk known to be associated with administration of a few stimulator cells, whereas administration of mammalian tumor-promoting cells It can be extremely dangerous.

Methods of reintroducing cellular components are known in the art and include procedures as illustrated in US Pat. No. 4,844,893 (Honsik et al.) And US Pat. No. 4,690,915 (Rosenberg). For example, it is appropriate to administer activated CD8 + cells via intravenous infusion.

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

The peptide may have use as a diagnostic reagent. For example, using the peptides of the present invention, by determining the specific individual's susceptibility to treatment methods using such peptides or related peptides, it may be helpful to alter existing treatment protocols or determine prognosis for infected individuals. Can be. In addition, the peptides can be used to predict whether chronic infections that are ongoing in an individual are at significant risk.

To identify peptides of the present invention, class I antigen isolation, and isolation and sequencing of naturally processed peptides are 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 as described above. The motifs described in the following Tables 8-11 are defined from collected sequencing data of naturally processed peptides, as described in the related application.

Example 2

Identification of Immunogenic Peptides

Using the above identified motifs for various MHC class I alleles, amino acid sequences from various pathogens and tumor-associated proteins were analyzed for the presence of these motifs. Sequence analysis was performed as described in the related application. Table 12 provides the antigenic findings.

Example 3

Identification of Immunogenic Peptides

Using the B7-like supermotifs identified in the above-mentioned related applications, sequences from various pathogens and tumor-associated proteins were analyzed for the presence of these motifs. Sequence analysis was performed as described in the related application. Table 13 provides the results of antigenic investigations.

The foregoing description is provided for purposes of illustration, which does not limit the scope of the invention. Other variants 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.

Claims (6)

A composition having an HLA-A2.1 binding motif and comprising an immunogenic peptide selected from the group consisting of: Cytotoxic T against a antigen pre-selected in a patient expressing HLA-A2.1 MHC product, comprising contacting a cytotoxic T cell from the patient with a composition comprising an immunogenic peptide selected from the group consisting of How to Induce Cellular Responses. A composition comprising an immunogenic peptide selected from the group consisting of: Contacting a cytotoxic T cell from a patient with a composition comprising an immunogenic peptide selected from the group consisting of: a method of inducing a cytotoxic T cell response to a preselected antigen in said patient. A composition comprising an immunogenic peptide selected from the group consisting of peptides listed in Table 13. Contacting cytotoxic T cells from the patient with a composition comprising an immunogenic peptide selected from the group of peptides listed in Table 13 for cytotoxic T cell responses against antigens preselected in said patient. How to induce.