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Publication numberUS20080305122 A1
Publication typeApplication
Application numberUS 11/708,107
Publication dateDec 11, 2008
Filing dateFeb 16, 2007
Priority dateSep 24, 2002
Also published asDE60332645D1, US7179645, US20040058881
Publication number11708107, 708107, US 2008/0305122 A1, US 2008/305122 A1, US 20080305122 A1, US 20080305122A1, US 2008305122 A1, US 2008305122A1, US-A1-20080305122, US-A1-2008305122, US2008/0305122A1, US2008/305122A1, US20080305122 A1, US20080305122A1, US2008305122 A1, US2008305122A1
InventorsRobert E. Humphreys, Minzhen Xu
Original AssigneeAntigen Express, Inc.
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Ii-Key/antigenic epitope hybrid peptide vaccines
US 20080305122 A1
Abstract
Disclosed is a nucleic acid molecule comprising a first expressible sequence encoding a protein of interest or polypeptide of interest which contains an MHC Class II-presented epitope. In addition, the nucleic acid molecule comprises a second expressible nucleic acid sequence encoding an antigen presentation enhancing hybrid polypeptide. The antigen presentation enhancing hybrid polypeptide includes the following elements: i) an N-terminal element consisting essentially of 4-16 residues of the mammalian Ii-Key peptide LRMKLPKPPKPVSKMR (SEQ ID NO: 1) and non-N-terminal deletion modifications thereof that retain antigen presentation enhancing activity; ii) a C-terminal element comprising an MHC Class II-presented epitope in the form of a polypeptide or peptidomimetic structure which binds to the antigenic peptide binding site of an MHC class II molecule, the MHC Class II-presented epitope being contained in the protein of interest of step a); and iii) an intervening peptidyl structure linking the N-terminal and C-terminal elements of the hybrid, the peptidyl structure having a length of about 20 amino acids or less.
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Claims(10)
1. A non-naturally occurring protein or polypeptide modified by recombinant DNA techniques, comprising:
a) a C-terminal element comprising an MHC Class II-presented epitope;
b) an N-terminal element comprising an Ii-key motif; and
c) an intervening element comprising a sequence from about 4 to about 11 amino acid residues;
the modification by recombinant DNA techniques taking place within elements b) and c).
2. The protein or polypeptide of claim 1 wherein the Ii-Key motif comprises a segment of 5 contiguous amino acid residues containing at least two amino acids selected from the group consisting of LIVFM (SEQ ID NO: 790) and at least one selected from the group consisting of HKR.
3. The method of claim 1 wherein the MHC Class II-presented epitope is a molecular feature in a therapeutically significant protein.
4. An expressible nucleic acid sequence encoding a non-naturally occurring protein or polypeptide, the protein or polypeptide comprising:
a) a C-terminal element comprising an MHC Class II-presented epitope;
b) an N-terminal element comprising an Ii-key motif, the Ii-Key motif comprising a segment of 5 contiguous amino acid residues containing at least two amino acids selected from the group consisting of LIVFM (SEQ ID NO: 790) and at least one selected from the group consisting of HKR; and
c) an intervening element comprising a sequence from about 4 to about 11 amino acid residues.
5. A method for suppressing an immune response directed toward an MHC Class II-presented epitope of interest, the method comprising:
a) providing a nucleic acid sequence encoding the MHC Class II-presented epitope of interest, the nucleic acid sequence encoding an Ii-Key motif located 4-11 amino acids upstream from the N-terminal residue of the MHC Class II-presented epitope of interest; and
b) modifying the Ii-Key motif to decrease its conformance to the archetypal Ii-Key regulatory motif, the archetype Ii-Key regulatory motif comprising a segment of 5 contiguous amino acids comprising at least two amino acids selected from the group consisting of leucine, isoleucine, valine, phenylalanine and methionine, and at lease one amino acid selected from the group consisting of histidine, lysine and arginine.
6. A method for enhancing an immune response directed toward an MHC Class II-presented epitope of interest, the method comprising:
a) providing a nucleic acid sequence encoding the MHC Class II-presented epitope of interest, the nucleic acid sequence lacking an Ii-key motif located 4-11 amino acids upstream from the N-terminal residue of the MHC Class II-presented epitope of interest; and
b) modifying the nucleic acid sequence to introduce an Ii-key motif appropriately spaced from the MHC Class II-presented epitope.
7. A method for immunizing a mammal, thereby altering immunological sensitivity toward a predetermined epitope or determinant, the method comprising:
a) providing a first nucleic acid sequence comprising a first expressible sequence encoding a protein of interest, the protein of interest corresponding to a protein encoded by an infectious pathogen;
b) providing a second nucleic acid sequence comprising a second expressible sequence encoding an antigen presentation enhancing hybrid polypeptide comprising:
i) an N-terminal element consisting essentially of 4-16 residues of the mammalian Ii-Key peptide LRMKLPKPPKPVSKMR (SEQ ID NO: 1) and non-N-terminal deletion modifications thereof that retain antigen presentation enhancing activity;
ii) a C-terminal element comprising an MHC Class II-presented epitope in the form of a polypeptide or peptidomimetic structure which binds to the antigenic peptide binding site of an MHC class II molecule, the MHC Class II-presented epitope being contained in the protein of interest of step a); and
iii) an intervening peptidyl structure linking the N-terminal and C-terminal elements of the hybrid, the peptidyl structure having a length of about 20 amino acids or less; and
c) administering the nucleic acid sequence of step b) to a patient in an amount sufficient to stimulate an immune response; and
d) following T cell expansion in response to the administration of step c), administering the first nucleic acid sequence of step a) to the patient in an amount sufficient to stimulate an immune response.
8. A method for immunizing a mammal, thereby altering immunological sensitivity toward a predetermined epitope or determinant, the method comprising:
a) providing the genome of a vaccine pathogen of interest;
b) incorporating into the genome of step a) an expressible nucleic acid sequence encoding an antigen presentation enhancing hybrid polypeptide comprising:
i) an N-terminal element consisting essentially of 4-16 residues of the mammalian Ii-Key peptide LRMKLPKPPKPVSKMR (SEQ ID NO: 1 and non-N-terminal deletion modifications thereof that retain antigen presentation enhancing activity;
ii) a C-terminal element comprising an MHC Class II-presented epitope in the form of a polypeptide or peptidomimetic structure which binds to the antigenic peptide binding site of an MHC class II molecule, the MHC Class II-presented epitope being encoded by the genome of step a); and
iii) an intervening peptidyl structure linking the N-terminal and C-terminal elements of the hybrid, the peptidyl structure having a length of about 20 amino acids or less;
c) administering the nucleic acid sequence of step b) to a patient in an amount sufficient to stimulate an immune response; and
d) following T cell expansion in response to the administration of step c), administering the first nucleic acid sequence of step a) to the patient in an amount sufficient to stimulate an immune response.
9. A method for displaying a virus-specific antigenic epitope of interest on the surface of an MHC Class II molecule-positive cell in which Ii protein expression is suppressed, the method comprising:
a) providing an MHC Class II-positive cell; and
b) introducing into the cell of step a) a recombinant virus comprising:
i) an expressible nucleic acid sequence encoding the virus-specific antigenic epitope of interest; and
ii) an expressible reverse gene construct which encodes an RNA molecule which is complementary to an mRNA molecule which encodes human Ii protein, the RNA molecule having the ability to hybridize with the mRNA molecule thereby inhibiting translation of the mRNA molecule.
10. A method of claim 9 wherein the recombinant virus is a vaccinia virus.
Description
    BACKGROUND OF THE INVENTION
  • [0001]
    The immune system responds to foreign pathogens, to tumor cells, to autoimmune disease-inducing processes, to allergens, to grafts, through the recognition of the ‘foreign’ or ‘abnormal’ structures, as antigens. Most of those antigens are proteins, which are synthesized either by cells of the host, or by a pathogen. Such antigens are processed (proteolytically digested) into peptide fragments which come to be presented to the responding lymphocytes of the immune system, in a peptide-presenting structure on the surface of the antigen presenting cell. Those peptide presenting structures are called major histocompatibility complex (MHC) molecules. They obtained that name since they were first recognized as products of polymorphic, allelic genes in the MHC locus, which genes control graft rejection among inbred strains of mice.
  • [0002]
    The immune response to a specific antigen is mediated by T lymphocytes which recognize peptide fragments of those antigens in the MHC molecules. Within an antigen presenting cell (APC), peptide fragments of a proteolytically processed antigen become bound into the antigenic peptide binding site of major histocompatibility complex (MHC) molecules. These peptide-MHC complexes are then transported to the cell surface for recognition (of both the foreign peptide and the adjacent surface of the presenting MHC molecule) by T cell receptors on responding T lymphocytes. Those T lymphocytes can have either immunoregulatory functions (to help or suppress an immune response) or effector functions (to clear the pathogen or tumor, for example, through a cytotoxic immune response). The antigen-specific recognition event initiates the immune response cascade which leads to a protective immune response, or in the case of autoimmune processes, a deleterious immune response.
  • [0003]
    Two classes of MHC molecules function as immune system presenters of antigenic peptides to T cells. MHC class I molecules receive peptides from endogenously synthesized proteins, such as an infectious virus, in the endoplasmic reticulum about the time of synthesis of the MHC class I molecules. The MHC class I-bound antigenic peptides are presented at the cell surface to CD8-positive cytotoxic T lymphocytes, which then become activated and can directly kill the virus-expressing cells. In contrast, MHC class II molecules are synthesized in the endoplasmic reticulum with their antigenic peptide binding sites blocked by the invariant chain protein (Ii). These complexes of MHC class II molecules and Ii protein are transported from the endoplasmic reticulum to a post-Golgi compartment where Ii is released by proteolysis and a specific antigenic peptide becomes bound to the MHC class II molecule (Blum et al., Proc. Natl. Acad. Sci. USA 85: 3975 (1988); Riberdy et al., Nature 360: 474 (1992); Daibata et al., Mol. Immunol. 31: 255 (1994); Xu et al., Mol. Immunol. 31: 723 (1994); Xu et al., Antigen Processing and Presentation, Academic Press, NY p 227 (1994); Kropshofer et al., Science 270: 1357 (1995); and Urban et al., J. Exp. Med. 180: 751 (1994)).
  • [0004]
    R. Humphreys (1996) U.S. Pat. No. 5,559,028, and Humphreys et al. (1999) U.S. Pat. No. 5,919,639 revealed the mechanisms by which Ii protein is cleaved, releasing fragments in the course of cleavage to regulate the binding and locking in of antigenic peptides within the antigenic peptide binding site of MHC class II molecules (Adams et al., Eur. J. Immunol. 25: 1693 (1995); Adams et al., Arzneim. Forsch./Drug Research 47: 1069 (1997); and Xu et al., Arzneim. Forsch./Drug Research in press (1999)). One segment of the Ii protein, Ii(77-92), was found to act at an allosteric site outside the antigenic peptide binding site near the end of that site holding the N-terminus of the antigenic peptide. The referenced patents, furthermore, disclosed novel therapeutic compounds and methods to control this initial regulatory, antigenic peptide recognizing event of the immune response by three classes of mechanisms. In the first mechanism, antigenic peptides are spilled from cell surface MHC class II molecules by the action of compounds of the invention.
  • [0005]
    In the second, the charging of the antigenic peptide binding site on those molecules is promoted with compounds of the invention for binding of other, synthetic peptides. Such inserted peptide sequences can be either antigenic epitopes or nonantigenic peptide sequences which nevertheless bind tightly to block the antigenic peptide binding site. The third mechanism involves altering the rates of association/dissociation of antigenic peptides from those complexes and the nature of the interaction of components of the trimolecular MHC molecule/antigenic peptide/T cell receptor complex, and furthermore the interaction of that trimolecular complex with auxiliary cell-to-cell interaction molecules, in a manner to regulate differentiation and function of the responding T lymphocytes.
  • [0006]
    The identification of the mechanisms referred to above opens new avenues of therapeutic intervention. New methods and compositions based on these discoveries offer the promise of epitope-specific therapies.
  • SUMMARY OF THE INVENTION
  • [0007]
    In one aspect, the present invention relates to a nucleic acid molecule comprising a first expressible sequence encoding a protein of interest or polypeptide of interest which contains an MHC Class II-presented epitope. In addition, the nucleic acid molecule comprises a second expressible nucleic acid sequence encoding an antigen presentation enhancing hybrid polypeptide. The antigen presentation enhancing hybrid polypeptide includes the following elements: i) an N-terminal element consisting essentially of 4-16 residues of the mammalian Ii-Key peptide LRMKLPKPPKPVSKMR (SEQ ID NO: 1) and non-N-terminal deletion modifications thereof that retain antigen presentation enhancing activity; ii) a C-terminal element comprising an MHC Class II-presented epitope in the form of a polypeptide or peptidomimetic structure which binds to the antigenic peptide binding site of an MHC class II molecule, the MHC Class II-presented epitope being contained in the protein of interest of step a); and iii) an intervening peptidyl structure linking the N-terminal and C-terminal elements of the hybrid, the peptidyl structure having a length of about 20 amino acids or less.
  • [0008]
    In preferred embodiments, the modifications of the I-Key peptide include deletion of amino acids from the C-terminus; N-terminal extensions; and amino acid substitutions. In other embodiments, the C-terminal element further comprises an MHC Class I-presented epitope, or a portion thereof, the amino acid residues comprising the MHC Class I-presented epitope or portion thereof being constituent residues of the MHC Class II-presented epitope.
  • [0009]
    Disclosed embodiments also include embodiments wherein the C-terminal element further comprises an antibody-recognized determinant, or a portion thereof. The amino acid residues comprising the antibody-recognized determinant, or a portion thereof, are preferably constituent residues of the MHC Class II-presented epitope. Embodiments are disclosed in which the infectious pathogen is selected from the group consisting of anthrax, EBOLA, HIV and influenza. In addition to the disclosed compositions, methods of use are also described.
  • DETAILED DESCRIPTION OF THE INVENTION
  • [0010]
    As discussed in the Background of the Invention section of the present disclosure, U.S. application Ser. No. 09/396,813 (now U.S. Pat. No. 6,432,409) discloses hybrid peptides useful in connection with modulation of the immune system (referred to herein as “the '813 enhancing hybrid peptide”). The disclosure was based on the discovery that an MHC Class II-restricted antigenic epitope which is covalently linked to a mammalian Ii key peptide by an appropriate intervening chemical structure, to form a hybrid polypeptide, is presented to T lymphocytes by antigen presenting cells with significantly higher efficacy than is the precursor antigenic epitope. The disclosure of U.S. Pat. No. 6,432,409 is incorporated herein by reference.
  • [0011]
    The hybrid polypeptide disclosed was referred to as an “MHC Class II antigen presentation enhancing hybrid polypeptide”, or more simply as an “enhancing hybrid”. In this disclosure, such peptides have also been referred to as “Ii-Key/antigenic epitope hybrids” or “hybrid peptides”. Alternatively, short-hand designations based on functional elements may be used, particularly in the Exemplification section. For example, Ii-Key/MHC Class II-presented antigenic epitope hybrids, Ii-Key/MHC Class II-presented antigenic epitope/MHC Class I-presented antigenic epitope hybrids, Ii-Key/MHC Class II-presented antigenic epitope/antibody-recognized determinant (ARD) hybrids. The preceding listing of alternative terminology may not be comprehensive, but reference to such enhancing hybrids will be clear in context.
  • [0012]
    The '813 enhancing hybrid has an N-terminus comprised of a mammalian Ii-Key peptide, or a modification thereof, which retains antigen presentation enhancing activity. Covalently, but indirectly, linked to the Ii-Key peptide is the specific MHC Class II antigenic epitope to be presented. Between the Ii-Key peptide and the antigenic epitope is an intervening chemical structure which covalently links the other two components. This intervening chemical structure was referred to simply as a “spacer”. Necessary parameters of the spacer were described in detail.
  • [0013]
    The present disclosure specifically contemplates enhancing hybrid peptides containing antigenic epitopes/determinants in addition to the MHC Class II antigenic epitope disclosed in connection with the '813 enhancing hybrid. For example, the enhancing hybrids of the present invention may contain multiple MHC Class II epitopes. The inclusion of multiple MHC Class II epitopes enables a greater fraction of the human population to be immunized because the multiple epitopes are frequently presented by different alleles. In addition to a plurality of MHC Class II epitopes, the present invention also contemplates the inclusion of one or more MHC Class I epitopes and/or one or more ARDs (Antibody Recognized Determinants). The expressions “epitopes” and “determinants” are considered as synonyms by many skilled in the art. The use of the expression “epitope/determinant”, as used herein, is intended to encompass MHC Class II epitopes, MHC Class I epitopes and ARDs.
  • [0014]
    The Exemplification section which follows provides numerous specific examples of experimentally-determined or predicted MHC Class II epitopes, MHC Class I epitopes and ARDs, which can be incorporated in enhancing hybrid peptides. The experimentally determined epitopes are preferred over algorithm-predicted epitopes for preclinical trials in animal models for human disease, in part, because a significant percentage of algorithm-predicted epitopes are not found to be biologically functional. Nevertheless, the “significant percentage” is sufficiently small that such epitopes are a source of sequences for the development of enhancing hybrids. In the context of a focus on a particular disease or condition, reference is made to the compounds and methods of use described in the corresponding Exemplification section which follows.
  • [0015]
    As will be discussed below, the use of the '813 enhancing hybrid peptide to enhance or augment an MHC Class II-mediated immune response, created an untapped immune reservoir. As will be discussed in greater detail below, the interaction of the '813 enhancing peptide with cells of the immune system greatly amplified a number of responsive cell types. Molecular input for a subset of these responsive cell types, in the form of the MHC Class II epitope component of the enhancing hybrid, were provided. However, large numbers of primed and responsive immune cell types were stimulated by the '813 peptide, but no provision for appropriate molecular inputs was provided. Such additional molecule inputs, in the form of MHC Class I epitopes and ARDS, is provided herein.
  • [0016]
    More specifically, the enhancement of the T helper cell stimulation mediated by the Class II epitope of the '813 peptide is substantially augmented (i.e., about 250 times) by the effect of the Ii-Key moiety. The clonal expansion of an immunoregulatory cell type, such as an activated T cell, has a cascading effect through the immune system. As discussed above, this can create an excess of immune capacity which has not been addressed in the prior art.
  • [0017]
    Ultimately, an MHC Class II-presented antigen which is an element of the hybrid peptide (either an enhancing hybrid peptide of the present invention or an '813 enhancing hybrid peptide), exerts its influence through presentation by an MHC Class II molecule on the surface of an antigen presenting cell. Two particularly important classes of antigen presenting cells are dendritic cells and macrophages. These antigen presenting cells have on their respective surfaces two types of special molecules that function in antigen presentation. These two types of molecules are MHC Class I and MHC Class II molecules. Antigenic peptides (e.g., MHC Class I or MHC Class II epitopes) are noncovalently bound to MHC Class I or MHC Class II molecules for subsequent presentation to antigen-specific receptors on T cells.
  • [0018]
    While not wishing to be bound by theory, it is thought that peptides containing MHC Class I and/or MHC Class II epitopes may be displayed on the surface of an antigen presenting cell in association with the cognate display molecule (i.e., MHC Class I molecules or MHC Class II molecules) through at least two mechanisms. For example, following contact with an antigen presenting cell, such peptides may be internalized by the antigen presenting cell and processed through classical channels. Alternatively, the MHC Class I or MHC Class II-presented antigen portion of such a peptide may bind directly to an MHC Class I or MHC Class II molecule on the surface of an antigen presenting cell. Thus, in both cases, the MHC Class I or MHC Class II-presented epitope of the peptide is displayed on the surface of an antigen presenting cell in association with its cognate MHC Class I or MHC Class II molecule.
  • [0019]
    Such an MHC Class II-associated display triggers a cascade of immune-mediated effects including the induction of T cells and the subsequent expansion of this induced population. T helper cells, stimulated in this manner, respond in a variety of ways. For example, stimulated T helper cells function by releasing cytokines that provide various activation signals for B cells. B cells produce a surface immunoglobulin which can recognize and specifically bind to an ARD element which is present, for example, on a protein or peptide which contacts the cell surface. The protein or peptide is then internalized and any processed MHC Class I or MHC Class II-presented epitopes present are subsequently displayed on the B cell surface in association with MHC Class I or MHC Class II molecules, respectively.
  • [0020]
    The example of an ARD-containing molecule provided in the preceding paragraph was a protein or peptide. In connection with the present invention, the ARD is provided as an element of an enhancing hybrid peptide. As was the case in the previous example, the enhancing hybrid peptide is internalized by the B cell and any MHC Class II epitopes present as an element of the enhancing hybrid are processed for display on the surface of the B cell in association with MHC Class II molecules. Such presentation further stimulates the helper T cell population resulting in proliferation and maturation of B lymphocytes to plasma cells which produce the antibody specific to the ARD.
  • [0021]
    The enhancing hybrid polypeptide of the present invention is comprised of 3 elements, as was the '813 enhancing hybrid. The 3 elements are: 1) an N-terminal element consisting essentially of 4-16 residues of the mammalian Ii-Key peptide LRMKLPKPPKPVSKMR (SEQ ID NO: 1 and non-N-terminal deletion modifications thereof that retain antigen presentation enhancing activity; 2) a C-terminal element comprising an MHC Class II-presented epitope in the form of a polypeptide or peptidomimetic structure which binds to the antigenic peptide binding site of an MHC Class II molecule; and 3) an intervening chemical structure covalently linking the N-terminal and C-terminal elements of the hybrid, the chemical structure being a covalently joined group of atoms which when arranged in a linear fashion forms a flexible chain which extends up to the length of 20 amino acids likewise arranged in a linear fashion.
  • [0022]
    The included additional epitope(s) or determinant(s) which distinguish the enhancing hybrid of the present invention from the '813 enhancing hybrid are preferably located within the C-terminal element or the linker element. Additionally, an epitope or determinant may overlap the C-terminal element and the linker element. In some circumstances it may be possible for an additional epitope or determinant to overlap between the linker element and the N-terminal Ii-Key moiety.
  • [0023]
    Generally speaking, MHC Class I and MHC Class II epitopes are comprised of from about 8 to about 12 amino acid residues. ARD elements are typically have a size range somewhat broader than MHC Class I and MHC Class II epitopes. A commonly cited size range for ARDs is from about 6 to about 16 amino acid residues. ARDs are recognized based on their 3-dimensional structure whereas MHC Class I and MHC Class II epitopes are recognized on the basis of their linear, primary amino acid structure.
  • [0024]
    To provide specificity to the options outlined in the preceding paragraph, it is necessary to discuss the anatomy of the enhancing peptide of the present invention in greater detail. The linker sequence has been described as an intervening chemical structure covalently linking the N-terminal and C-terminal elements of the hybrid, the chemical structure being a covalently joined group of atoms which when arranged in a linear fashion forms a flexible chain which extends up to the length of 20 amino acids likewise arranged in a linear fashion. Thus, to the extent that the linker sequence is comprised of amino acids (which is not a requirement), the disclosure of the present invention provides an additional functionality to the amino acid residues of the linker, above and beyond their required role as space occupiers.
  • [0025]
    The specified linker length (up to 20 amino acids arranged in a linear fashion) is long enough to contain a second complete MHC Class II epitope, a first complete MHC Class I epitope, or a first complete ARD or segments of such additional epitopes. Additionally, such a sequence length can accommodate a plurality of non-overlapping epitopes selected from the group consisting of MHC Class I epitopes, MHC Class II epitopes and ARDs.
  • [0026]
    It is known in the art that functional MHC Class I epitopes, MHC Class II epitopes and ARDs may be arranged in an overlapping manner while retaining full functionality of all represented epitopes. The respective functions of each epitope within a hybrid are not co-expressed at one point in time on a per peptide basis, because such peptides must be bound into MHC Class I or MHC Class II molecules and recognized in a folded structure by an antibody. Nevertheless, given a population of injected peptides with respective processing and/or binding to cell surface MHC molecules, all three classes of epitopes within any one Ii-Key enhancing hybrid can be effective immunogens within an immunized animal.
  • [0027]
    Minimum sequences are preferred for several reasons. These include simplicity and cost of synthesis, less opportunity for proteolytic degradation, less opportunity for metabolic change leading to clearance or adsorption. Thus, the linker element may contain a plurality of epitopes which overlap one another (i.e., an individual amino acid residues may be a components of more than one epitope). Similarly, the C-terminal element which includes an MHC Class II-presented epitope may also contain additional epitopes (MHC Class I, MHC Class II or ARD) in an overlapping or non-overlapping arrangement.
  • [0028]
    It is noted that the boundaries between the various elements of the enhancing hybrid peptide of the present invention are, within certain stated limits, somewhat arbitrary. Epitopes spanning the junctions between the various elements are encompassed within the scope of the present invention. Thus, for example, where a claim specifies that a portion of an epitope is contained within one of the enhancing hybrid peptide elements or domains (e.g., the linker region), this necessarily implies that the remaining portion is found in a contiguous portion of a flanking portion or domain. Partial (i.e., non-functional epitopes are of no utility in connection with the present invention).
  • [0029]
    Early work in this area demonstrated that the mammalian Ii key peptide LRMKLPKPPKPVSKMR (SEQ ID NO: 1), and a modified mammalian Ii-key peptide, YRMKLPKPPKPVSKMR (SEQ ID NO: 2), have the ability to alter presentation of certain MHC Class II-restricted, antigenic peptides to T lymphocyte-hybridomas which recognize those respective antigenic peptides (U.S. Pat. No. 5,559,028; U.S. Pat. No. 5,919,639, the disclosures of which are incorporated herein by reference). Previous experimentation with modified versions of the Ii-key peptide have indicated that a wide variety of modifications can be made to this polypeptide without detriment to activity. Indeed, modifications often enhanced antigen presentation activity of the polypeptide.
  • [0030]
    Results detailed in the Exemplification section of U.S. application Ser. No. 09/396,813, now U.S. Pat. No. 6,432,409, indicate that all modified Ii key peptides which retain antigen presentation enhancing activity will function in the enhancing hybrid of the present invention when appropriately incorporated. Modifications of the Ii key peptide include deletion of one or more amino acids from the C-terminus, protection of the N-terminus, amino acid substitutions, and introduction of cyclical peptides. Deletions of the Ii key peptide which retain at least 4 contiguous amino acids of the original sequence, or a substituted version thereof, exhibit functional activity. Various natural or non-natural amino acids may be substituted at respective residue positions. Some examples of molecules which may be substituted are peptidomimetic structures, D-isomer amino acids, N-methyl amino acids, L-isomer amino acids, modified L-isomer amino acids, and cyclized derivatives. In addition, procedures of medicinal chemistry may be applied by one skilled in the art using routine experimental methods to obtain additional modifications of the N-terminal segment of hybrids. Examples of such procedures are methods of rational drug design, molecular modeling based on structural information from X-ray diffraction data, nuclear magnetic resonance data, and other computational methods, and screening of products of combinatorial chemical syntheses, and isolations of natural products. Examples of modified versions of Ii key peptide which are known to retain high activity are LRMK (SEQ ID NO: 3), LRMKLPK (SEQ ID NO: 4), LRMKLPKS (SEQ ID NO: 5), LRMKLPKSAKP (SEQ ID NO: 6), and LRMKLPKSAKPVSK (SEQ ID NO: 7). Other modifications and modified versions of the Ii-key peptide are described in U.S. Pat. No. 5,919,639, and U.S. Pat. No. 5,559,028. A modified version of the Ii-key peptide (YRMKLPKPPKPVSKMR, SEQ ID NO: 2) which is known to retain activity is referred to herein as an ‘Ii-key homolog’. The term Ii key homolog as used herein is inclusive of the Ii key peptide itself.
  • [0031]
    Such Ii-Key peptides were demonstrated by several experimental methods to bind to an allosteric site at the end of the antigenic peptide binding site of MHC Class II molecules holding the N-terminal end of an antigenic peptide. That process of binding to the allosteric site, facilitated the release and exchange of endogenously bound antigenic peptide with cell surface MHC Class II molecules.
  • [0032]
    Peptide homologs of the Ii-Key peptide act on murine or human MHC Class II molecules to promote the release of bound antigenic peptides and their replacement with synthetic peptides (Adams S. Arneimittelforschung. 1997 47:1069-1077; Xu M. Arneimittelforschung. 1999 49:791-9). Hybrid constructs of the Ii-Key peptide linked to an antigenic epitope peptide through either a simple polymethylene linker or the extended, natural sequence of the Ii protein, have 500 to 2000 times the potency of presentation versus the antigenic peptides (Humphreys R E. Vaccine. 2000 18:2693-2697). This property has great clinical utility in diagnosis, treatment monitoring and therapy of various diseases and conditions, as presented herein. This activity of the Ii-Key moiety within Ii-Key/antigenic epitope hybrids is found either in vitro or in vivo. This activity can be ascribed to interaction with cell surface MHC Class II molecules because the Ii-Key compounds were active in vitro with either living or paraformaldehyde-fixed antigen presenting cells (Adams S. Eur J Immunol. 1995 25:1693-1702). However, since the compounds are potent in vivo, they may also be taken up by the pathway which processes exogenous antigens and bind to MHC Class II molecule sin the post-Golgi, antigen charging compartment.
  • [0033]
    The MHC Class I epitopes, MHC Class II epitopes and ARDs of the enhancing hybrid of the present invention have been discussed above. Such epitopes/determinants selected for use in the generation of an enhancing hybrid of the present invention may be further modified for use. That is to say, polypeptides of natural or modified sequence, peptidomimetic structures, and also chemical structures which are not natural or modified amino acids may be included in the epitope/determinant elements of the enhancing hybrids disclosed herein. In addition, various chemical modifications may be made to the antigenic epitope/determinant element of the enhancing hybrid. For example, the addition, in whole or in part, of non-natural amino acids, or of other backbone or side chain moieties, wherein the modifications preserve binding specificities of the antigenic epitope/determinant. Such chemical structures might bear moderate, little, or no apparent structural resemblance to any antigenic peptide which is derived from a natural protein sequence. Such modifications might or might not bear on recognition by T cell receptors. Modifications may increase recognition of the antigenic epitope (e.g. lead to recognition by previously non-recognizing subsets of T cell receptors).
  • [0034]
    The intervening chemical structure, or spacer, has been discussed above. Where the intervening chemical structure comprises one or more epitopes/determinants, the overall length within defined limits is dictated to a large extent by the identity and of the epitope/determinant. In the case in which the intervening chemical structure is antigenically neutral, the teachings of U.S. application Ser. No. 09/396,813, now U.S. Pat. No. 6,432,409, apply. As indicated, the spacer is preferably less than the length of a peptidyl backbone of 9 amino acids linearly arranged. Optimally, spacer length is the length of a peptidyl backbone of between 4 and 6 amino acids, linearly arranged. Preferably, the spacer is unable to hydrogen bond in any spatially distinct manner to other distinct elements of the enhancing hybrid peptide.
  • [0035]
    Again, with respect to antigenically neutral spacer elements, various chemical groups may be incorporated in the spacer segment instead of amino acids. Examples are described in U.S. Pat. No. 5,910,300, the contents of which are incorporated herein by reference. In a preferred embodiment the spacer is comprised of an aliphatic chain optimally interrupted by heteroatoms, for example a C2-C6 alkylene, or ═N—(CH2)2-6—N═. Alternatively, a spacer may be composed of alternating units, for example of hydrophobic, lipophilic, aliphatic and aryl-aliphatic sequences, optionally interrupted by heteroatoms such as O, N, or S. Such components of a spacer are preferably chosen from the following classes of compounds: sterols, alkyl alcohols, polyglycerides with varying alkyl functions, alkyl-phenols, alkyl-amines, amides, hydroxyphobic polyoxyalkylenes, and the like. Other examples are hydrophobic polyanhydrides, polyorthoesters, polyphosphazenes, polyhydroxy acids, polycaprolactones, polylactic, polyglycolic polyhydroxy-butyric acids. A spacer may also contain repeating short aliphatic chains, such as polypropylene, isopropylene, butylene, isobutylene, pentamethlyene, and the like, separated by oxygen atoms.
  • [0036]
    Additional peptidyl sequences which can be used in a spacer are described in U.S. Pat. No. 5,856,456, the contents of which are incorporated herein by reference. In one embodiment, the spacer has a chemical group incorporated within which is subject to cleavage. Without limitation, such a chemical group may be designed for cleavage catalyzed by a protease, by a chemical group, or by a catalytic monoclonal antibody. In the case of a protease-sensitive chemical group, tryptic targets (two amino acids with cationic side chains), chymotryptic targets (with a hydrophobic side chain), and cathepsin sensitivity (B, D or S) are favored. The term ‘tryptic target’ is used herein to describe sequences of amino acids which are recognized by trypsin and trypsin-like enzymes. The term ‘chymotryptic target’ is used herein to describe sequences of amino acids which are recognized by chymotrypsin and chymotrypsin-like enzymes. In addition, chemical targets of catalytic monoclonal antibodies, and other chemically cleaved groups are well known to persons skilled in the art of peptide synthesis, enzymic catalysis, and organic chemistry in general, and can be designed into the hybrid structure and synthesized, using routine experimental methods.
  • [0037]
    Not all embodiments of the present invention include immunogenic neutrality of the intervening chemical structure, or spacer. That is, the present invention includes embodiments in which the intervening chemical structure, or spacer, is selected from the group consisting of: 1) an MHC Class I epitope, or a portion thereof; and 2) an antibody-recognized determinant, or a portion thereof. In particular, this embodiment is important in connection with the anticipated filing of a counterpart International Application for which the continuation-in-part provisions of the U.S. patent law are inapplicable.
  • [0038]
    The hybrids of the present invention vary from totally peptide in character to substantially non-peptide in character. In view of the fact that some homologs are substantially reduced or non-peptide in character, they will be more likely to have favorable properties, for example, penetration through cellular membranes, solubility, resistance to proteolysis, resistance to inactivation by conjugation, oral bioavailability, and longer half life in vivo.
  • [0039]
    Also included within the scope of this invention are pharmaceutically acceptable salts of the hybrid molecule, when an acidic or basic group is present in the structure. The term ‘pharmaceutically acceptable salt’ is intended to include all acceptable salts such as acetate, ammonium salt, benzenesulfonate, benzoate, borate, bromide, calcium edetate, camsylate, carbonate, chloride/dihydrochloride, citrate, clavulanate, edetate, edisylate, estolate, esylate, fumarate, hexylresorcinate, hydrabamine, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N-methylglucamide, oleaste, oxalate, pamoate, palmitate, panoate, pantothenate, phosphate/diphosphate, polygalacturonate, subacetate, sulfate, tartrate, tosylate, triethiodide, valerate, and the like. The pharmaceutically acceptable salt can be used as a dosage form for modifying the solubility or hydrolysis characteristics, or can be used in a sustained release or pro-drug formulation. Depending on the particular functionality for the compound of the present invention, pharmaceutically acceptable salts of the compounds of this invention may be formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc and from bases such as ammonia, arginine, chloroprocaine, choline, diethanolamine, diethylamine, ethylenediamine, lysine, N-methyl-glutamine, ornithine, N,N′-dibenzylethylenediamine, N-benzylphenethylamine, piperazine, procaine, tris(hydroxymethyl)aminomethane, and tetramethylenediamine hydroxide, and the like. These salts may be prepared by standard procedures, for example, by reacting a free acid with suitable organic or inorganic base. When a basic group is present, such as an amino, and acidic salt, i.e., acetate, hydrobromide, hydrochloride, pamoate, and the like, can be used as the dosage form.
  • [0040]
    Also in the case of an acid (—COOH) or alcohol group being present, pharmaceutically acceptable esters can be employed, for example, acetate, maleate, pivaloyloxymethyl, and the like and those esters known in the art for modifying solubility or hydrolysis characteristics for use as sustained release or prodrug formulations.
  • [0041]
    The hybrid molecules of this present invention or components thereof may have chiral centers, and therefor may occur as racemates, racemic mixtures, and as individual enantiomers or diastereomers, with all such isomeric forms being included in the present invention as well as mixtures thereof. Furthermore, some of the crystalline forms of hybrid compounds of the present invention may exist as polymorphous and as such are intended to be included in the present invention. In addition, some of the compounds of the present invention may form solvates with water or common organic solvents. Such solvates are also encompassed within the scope of this invention.
  • [0042]
    The enhancing hybrid of the present invention may be composed of peptide or peptidomimetic or additional chemical groups which may be synthesized and selected by methods which have been developed for the synthesis and selection of antigenic peptides. Those methods and compounds are presented in the following patents: U.S. Pat. No. 4,708,871; U.S. Pat. No. 5,194,392; U.S. Pat. No. 5,270,170; U.S. Pat. No. 5,382,513; U.S. Pat. No. 5,539,084; U.S. Pat. No. 5,556,762; (1997) U.S. Pat. No. 5,595,915; U.S. Pat. No. 5,747,334; and U.S. Pat. No. 5,874,214, the contents of which are incorporated herein by reference.
  • [0043]
    The disclosure presented above relates primarily to antigen presentation enhancing hybrid peptides. In another aspect, the present invention relates to nucleic acid sequences which encode such enhancing peptides. It is noted that the scope of the enhancing hybrid peptide disclosure is somewhat broader than the corresponding nucleic acid sequence disclosure in light of the fact that enhancing hybrid peptides produced using recombinant DNA techniques from an encoding nucleic acid sequence must be produced from one of the 20 naturally occurring amino acids. A much broader range of substitutions is available when an enhancing hybrid peptide is produced by chemical synthetic techniques.
  • [0044]
    A wide variety of delivery systems are available for use in delivering the enhancing hybrid of the present invention to a target cell in vitro and in vivo. Such delivery systems include, for example, viral and non-viral systems. Examples of suitable viral systems include, for example, adenoviral vectors, adeno-associated virus, retroviral vectors, vaccinia, herpes simplex virus, HIV, the minute virus of mice, hepatitis B virus and influenza virus. Non-viral delivery systems may also be used, for example using, uncomplexed DNA, DNA-liposome complexes, DNA-protein complexes and DNA-coated gold particles, bacterial vectors such as salmonella, and other technologies such as those involving VP22 transport protein, Co-X-gene, and replicon vectors.
  • [0045]
    One option for expressing a nucleic acid sequence of interest in an animal cell is the adenovirus system. Adenovirus possesses a double-stranded DNA genome, and replicates independently of host cell division. Adenoviral vectors offer a variety of advantages relative to alternative methods for introducing expressible constructs into cells. For example, adenoviral vectors are capable of transducing a broad spectrum of human tissues and high levels of gene expression can be obtained in dividing and nondividing cells. Adenoviral vectors are characterized by a relatively short duration of transgene expression due to immune system clearance and dilutional loss during target cell division. Several routes of administration can be used including intravenous, intrabiliary, intraperitoneal, intravesicular, intracranial and intrathecal injection, and direct injection of a target organ or tissue. Thus, it is recognized in the art that targeting based on anatomical boundaries is achievable.
  • [0046]
    The adenoviral genome encodes about 15 proteins and infection involves a fiber protein which binds to a cell surface receptor. This receptor interaction results in internalization of the virus. Viral DNA enters the nucleus of the infected cell and transcription is initiated in the absence of cell division. Expression and replication is under control of the E1A and E1B genes (see Horwitz, M. S., In Virology, 2.sup.nd ed., 1990, pp. 1723-1740). Removal of E1 genes renders the virus replication-incompetent.
  • [0047]
    Adenoviral serotypes 2 and 5 have been extensively used for vector construction. Bett et al. (Proc. Nat. Acad. Sci. U.S.A., 1994, 91: 8802-8806) have used an adenoviral type 5 vector system with deletions of the E1 and E3 adenoviral genes. The 293 human embryonic kidney cell line has been engineered to express E1 proteins and can thus transcomplement the E1-deficient viral genome. The virus can be isolated from 293 cell media and purified by limiting dilution plaque assays (Graham, F. L. and Prevek, L. In Methods in Molecular Biology: Gene Transfer and Expression Protocols, Humana Press 1991, pp. 109-128). Recombinant virus can be grown in 293 cell line cultures and isolated by lysing infected cells and purification by cesium chloride density centrifugation. A problem associated with the 293 cells for manufacture of recombinant adenovirus is that due to additional flanking regions of the E1 genes, they may give rise to replication competent adenovirus (RCA) during the viral particle production. Although this material is only wild-type adenovirus, and is not replication competent recombinant virus, it can have significant effects on the eventual yield of the desired adenoviral material and lead to increased manufacturing costs, quality control issues for the production runs and acceptance of batches for clinical use. Alternative cell lines such as the PER.C6 which have more defined E1 gene integration than 293 cells (i.e. contain no flanking viral sequence) have been developed which do not allow the recombination events which produce RCA and thus have the potential to overcome above viral production issues.
  • [0048]
    Adeno-associated virus (AAV) (Kotin, R. M., Hum. Gene Ther., 1994, 5: 793-801) are single-stranded DNA, nonautonomous parvoviruses able to integrate into the genome of nondividing cells of a very broad host range. AAV has not been shown to be associated with human disease and does not elicit an immune response. AAV has two distinct life cycle phases. Wild-type virus will infect a host cell, integrate and remain latent. In the presence of adenovirus, the lytic phase of the virus is induced, which depends on the expression of early adenoviral genes, and leads to active virus replication. The AAV genome is composed of two open reading frames (called rep and cap) flanked by inverted terminal repeat (ITR) sequences. The rep region encodes four proteins which mediate AAV replication, viral DNA transcription, and endonuclease functions used in host genome integration. The rep genes are the only AAV sequences required for viral replication. The cap sequence encodes structural proteins that form the viral capsid. The ITRs contain the viral origins of replication, provide encapsidation signals, and participate in viral DNA integration. Recombinant, replication-defective viruses that have been developed for gene therapy lack rep and cap sequences. Replication-defective AAV can be produced by co-transfecting the separated elements necessary for AAV replication into a permissive 293 cell line. U.S. Pat. No. 4,797,368 contains relevant disclosure and such disclosure is incorporated herein by reference.
  • [0049]
    Retroviral vectors are useful for infecting dividing cells, and are composed of an RNA genome that is packaged in an envelope derived from host cell membrane and viral proteins. Retroviral gene expression involves a reverse transcription step in which its positive-strand RNA genome is employed as a template to direct the synthesis of double-stranded DNA, which is then integrated into the host cell DNA. The integrated provirus is able to use host cell machinery for gene expression.
  • [0050]
    Murine leukemia virus is a commonly employed retrovirus species (Miller et al., Methods Enzymol., 1993, 217: 581-599). Retroviral vectors are typically constructed by deletion of the gag, pol and env genes. The deletion of these sequences provides capacity for insertion of nucleic acid sequences of interest, and eliminates the replicative functions of the virus. Genes encoding antibiotic resistance often are included as a means of selection. Promoter and enhancer functions also may be included, for example, to provide for tissue-specific expression following in vivo administration. Promoter and enhancer functions contained in long terminal repeats may also be used.
  • [0051]
    Such viruses, and modifications of such viruses which carry an exogenous nucleic acid sequence of interest, can only be produced in viral packaging cell lines. The packaging cell line may be constructed by stably inserting the deleted viral genes (gag, pol and env) into the cell such that they reside on different chromosomes to prevent recombination. The packaging cell line is used to construct a producer cell line that will generate replication-defective retrovirus containing the nucleic acid sequence of interest by inserting the recombinant proviral DNA. Plasmid DNA containing the long terminal repeat sequences flanking a small portion of the gag gene that contains the encapsidation sequence and the genes of interest is transfected into the packaging cell line using standard techniques for DNA transfer and uptake (electroporation, calcium precipitation, etc.). Variants of this approach have been employed to decrease the likelihood of production of replication-competent virus (Jolly, D., Cancer Gene Therapy, 1994, 1, 51-64). The host cell range of the virus is determined by the envelope gene (env) and substitution of env genes with different cell specificities can be employed. Incorporation of appropriate ligands into the envelope protein may also be used for targeting.
  • [0052]
    Administration of recombinant retroviral vectors may be accomplished by any suitable technique. Such techniques include, for example, ex vivo transduction of patients' cells, direct injection of virus into tissue, and by the administration of the retroviral producer cells. ex vivo approaches require the isolation and maintenance in tissue culture of the patient's cells. In this context, a high ratio of viral particles to target cells can be achieved and thus improve the transduction efficiency (see, e.g., U.S. Pat. No. 5,399,346, the disclosure of which is incorporated herein by reference). U.S. Pat. No. 4,650,764 contains disclosure relevant to the use of retroviral expression systems and the disclosure of this referenced patent is incorporated herein by reference.
  • [0053]
    In some cases direct introduction of virus in vivo is necessary or preferred. Retroviruses have been used to treat brain tumors wherein the ability of a retrovirus to infect only dividing cells (tumor cells) may be particularly advantageous. The administration of a retrovirus producer cell line directly into a brain tumor in a patient has also been proposed (see e.g., Oldfield et al., Hum. Gene Ther., 1993, 4: 39-69). Such a producer cell would survive within the brain tumor for a period of days, and would secrete retrovirus capable of transducing the surrounding brain tumor.
  • [0054]
    Pox virus-based systems for expression have been described (Moss, B. and Flexner, C., Annu. Rev. Immunol., 1987, 5: 305-324; Moss, B., In Virology, 1990, pp. 2079-2111). Vaccinia, for example, are large, enveloped DNA viruses that replicate in the cytoplasm of infected cells. Nondividing and dividing cells from many different tissues are infected, and gene expression from a nonintegrated genome is observed. Recombinant virus can be produced by inserting the transgene into a vaccinia-derived plasmid and transfecting this DNA into vaccinia-infected cells where homologous recombination leads to the virus production. A significant disadvantage is that it elicits a host immune response to the 150 to 200 virally encoded proteins making repeated administration problematic.
  • [0055]
    The herpes simplex virus is a large, double-stranded DNA virus that replicates in the nucleus of infected cells. This virus is adaptable for use in connection with exogenous nucleic acid sequences (see Kennedy, P. G. E. and Steiner, I., Q. J. Med., 1993, 86: 697-702). Advantages include a broad host cell range, infection of dividing and nondividing cells, and large sequences of foreign DNA can be inserted into the viral genome by homologous recombination. Disadvantages are the difficulty in rendering viral preparations free of replication-competent virus and a potent immune response. Deletion of the viral thymidine kinase gene renders the virus replication-defective in cells with low levels of thymidine kinase. Cells undergoing active cell division (e.g., tumor cells) possess sufficient thymidine kinase activity to allow replication.
  • [0056]
    A variety of other viruses, including HIV, the minute virus of mice, hepatitis B virus, and influenza virus, have been disclosed as vectors for gene transfer (see Jolly, D., Cancer Gene Therapy, 1994, 1: 51-64). Nonviral DNA delivery strategies are also applicable. These DNA delivery strategies relate to uncomplexed plasmid DNA, DNA-lipid complexes, DNA-liposome complexes, DNA-protein complexes, DNA-coated gold particles and DNA-coated polylactide coglycolide particles. Purified nucleic acid can be injected directly into tissues and results in transient gene expression for example in muscle tissue, particularly effective in regenerating muscle (Wolff et al., Science, 1990, 247: 1465-1468). Davis et al. (Hum. Gene Ther., 1993, 4: 733-740) has published on direct injection of DNA into mature muscle (skeletal muscle is generally preferred).
  • [0057]
    Plasmid DNA on gold particles can be “fired” into cells (e.g. epidermis or melanoma) using a gene-gun. DNA is coprecipitated onto the gold particle and then fired using an electric spark or pressurized gas as propellant (Fynan et al., Proc. Natl. Acad. Sci. U.S.A., 1993, 90: 11478-11482). Electroporation has also been used to enable transfer of DNA into solid tumors using electroporation probes employing multi-needle arrays and pulsed, rotating electric fields (Nishi et al., in Cancer Res., 1996, 56:1050-1055). High efficiency gene transfer to subcutaneous tumors has been claimed with significant cell transfection enhancement and better distribution characteristics over intra-tumoral injection procedures.
  • [0058]
    Lipid-mediated transfections are preferred for both in vitro and in vivo transfections (Horton et al., J. Immunology, 162:6378, 1999). Lipid-DNA complexes are formed by mixing DNA and lipid 1 to 5 minutes before injection, using commercially available lipids such as DMRIE-C reagent.
  • [0059]
    Liposomes work by surrounding hydrophilic molecules with hydrophobic molecules to facilitate cell entry. Liposomes are unilamellar or multilamellar spheres made from lipids. Lipid composition and manufacturing processes affect liposome structure. Other molecules can be incorporated into the lipid membranes. Liposomes can be anionic or cationic. Nicolau et al. (Proc. Natl. Acad. Sci. U.S.A., 1983, 80: 1068-1072) has published work relating to insulin expression from anionic liposomes injected into rats. Anionic liposomes mainly target the reticuloendothelial cells of the liver, unless otherwise targeted. Molecules can be incorporated into the surface of liposomes to alter their behavior, for example cell-selective delivery (Wu, G. Y. and Wu, C. H., J. Biol. Chem., 1987, 262: 4429-4432).
  • [0060]
    Felgner et al. (Proc. Nat. Acad. Sci. U.S.A., 1987, 84: 7413-7417) has published work relating to cationic liposomes, demonstrated their binding of nucleic acids by electrostatic interactions and shown cell entry. Intravenous injection of cationic liposomes leads to transgene expression in most organs on injection into the afferent blood supply to the organ. Cationic liposomes can be administered by aerosol to target lung epithelium (Brigham et al., Am. J. Med. Sci., 1989, 298: 278-281). In vivo studies with cationic liposome transgene delivery have been published (see, e.g., Nabel, G., Rev. Hum. Gene Ther., 1994, 5: 79-92; Hyde et al., Nature, 1993, 362: 250-255 and; Conary et al., J. Clin. Invest., 1994, 93: 1834-1840).
  • [0061]
    Microparticles are being studied as systems for delivery of DNA to phagocytic cells such approaches have been reported by Pangaea Pharmaceuticals. Such a DNA microencapsulation delivery system has been used to effect more efficient transduction of phagocytic cells, such as macrophages, which ingest the microspheres. The microspheres encapsulate plasmid DNA encoding potentially immunogenic peptides which, when expressed, lead to peptide display via MHC molecules on the cell surface which can stimulate immune response against such peptides and protein sequences which contain the same epitopes. This approach is presently aimed towards a potential role in anti-tumor and pathogen vaccine development but may have other possible gene therapy applications.
  • [0062]
    Natural viral coat proteins which are capable of homogeneous self-assembly into virus-like particles (VLPs) have also been used to package DNA for delivery. The major structural coat protein (VP1) of human polyoma virus can be expressed as a recombinant protein and is able to package plasmid DNA during self-assembly into a VLP. The resulting particles can be subsequently used to transduce various cell lines.
  • [0063]
    Improvements in DNA vectors have also been made and are likely applicable to many of the non-viral delivery systems. These include the use of supercoiled minicircles (which do not have bacterial origins of replication nor antibiotic resistance genes and thus are potentially safer as they exhibit a high level of biological containment), episomal expression vectors (replicating episomal expression systems where the plasmid amplifies within the nucleus but outside the chromosome and thus avoids genome integration events) and T7 systems (a strictly a cytoplasmic expression vector in which the vector itself expresses phage T7 RNA polymerase and the therapeutic gene is driven from a second T7 promoter, using the polymerase generated by the first promoter). Other, more general improvements to DNA vector technology include use of cis-acting elements to effect high levels of expression, sequences derived from alphoid repeat DNA to supply once-per-cell-cycle replication and nuclear targeting sequences.
  • [0064]
    In other aspects, the present invention relates to methods for enhancing presentation of an MHC Class II-presented antigenic peptide to a T-lymphocyte. As discussed in U.S. Pat. No. 6,432,409, the MHC Class II-restricted antigenic epitope is appropriately incorporated into the C-terminus of an enhancing hybrid of the present invention, described above. The produced enhancing hybrid is then contacted under physiological conditions to an MHC Class II expressing antigen presenting cell which is in contact with or is then contacted to a T cell which is responsive to the presentation of the antigenic epitope by an MHC Class II molecule of the antigen presenting cell. This method is suitable for use with all antigenic epitopes which conform to the above listed description of an antigenic epitope. Examples of methods to assay such enhancement in vitro are detailed in the Exemplification section below, and in U.S. patents listed in the present disclosure.
  • [0065]
    In one aspect, the subject invention relates to a method to improve the potency of peptide vaccines containing MHC Class II-presented epitopes of antigens of interest to activate CD4+ immunoregulatory T cells for therapeutic or diagnostic purposes. A wide range of diseases and conditions in humans will benefit from the application of the compounds and methods of this invention to activate CD4+ immunoregulatory T cells. Such CD4+ immunoregulatory T cells can either augment or suppress the immune response to antigens of clinical interest in cancer, infectious disease, allergy, autoimmunity, graft rejection, and other clinical processes.
  • [0066]
    Antigens of clinical interest in the treatment or modification of various diseases and conditions as presented herein, are recognized by the T cells of the immune system as small peptide fragments, which are presented by Major Histocompatibility Complex (MHC) molecules on the surfaces of antigen presenting cells. MHC Class I molecules present such antigenic peptides to CD8+ cytotoxic or killer T cells. Most cells of the body express cell surface MHC Class I-presented peptides which have been drawn from the repertoire of cellular proteins and bound into the MHC Class I molecules of those cells at the time of their synthesis in the endoplasmic reticulum (the “immunological survey of self”). After viral infection or malignant transformation, the CD8+, cytotoxic T cells recognize the novel or “foreign” endogenously derived peptides in the MHC Class I molecules and kill the presenting cells.
  • [0067]
    MHC Class II molecules present antigenic peptides to CD4+ T immunoregulatory cells, which regulate the immune response by augmenting or suppressing various effector mechanisms of that response. Such effector mechanisms include, for example, cytotoxic T cell killing of target cells, antibody production by B cells and plasma cells, and dendritic cell activation. Because they regulate directly or indirectly almost all mechanisms in the immune response, CD4+ T immunoregulatory cells have been called the conductors of the immune response orchestra. MHC Class II molecules are expressed on only a subset of the cells of the body, such as macrophages, dendritic cells, and B-cells that have specialized mechanisms to internalize and process antigens of the environment. At the time of synthesis in the endoplasmic reticulum, the antigenic peptide-binding site of MHC Class II molecules is filled with the Ii protein. After transport of that complex to a post-Golgi, antigen charging compartment, the Ii protein is removed by proteases with the concerted insertion of antigenic peptides from foreign proteins, which have been internalized and processed by the antigen processing cells (Cresswell P. Cell. 1996 84:505-7; Hudson A W. Exp Cell Res. 2002 272:1-7; Bryant P W. Adv Immunol. 2002 80:71-114). The Ii-Key segment of the Ii protein interacts with an allosteric site on the MHC Class II molecule to induce lability of the antigenic peptide binding site during release of the Ii protein and binding of a selected antigenic peptide. After dissociation/destruction of the Ii-Key segment, the antigenic peptide is tightly bound in the MHC Class II molecule, for extended expression in the antigenic peptide binding site of those molecules. After transport to the cell surface, such MHC Class II-antigen peptide complexes are recognized by specialized receptors on CD4+ T immunoregulatory cells. Activation of those cells regulates the immune response in various ways, which are considered later in terms of individual therapeutic objectives. In brief, subsets of CD4+ cells may be activated along Th1, Th2, or Th2 pathways, which are characterized by differential induction of cytokines and other genes. Those regulatory cells either induce or suppress immune responses in an antigen-specific manner. Furthermore, CD4+ T cells can be induced to be a long-lived population of memory T cells.
  • [0068]
    The allosteric site at which the Ii-Key segment of the Ii protein interacts is accessible to the environment in cell surface-expressed MHC Class II molecules. This fact is of considerable value clinically because Ii-Key/antigenic epitope hybrids peptides can be administered in a simple manner in a fluid phase, for example subcutaneously, intravenously, intrathecally, intraperitoneally, transmucosally and as an aerosol to the respiratory tract, and can contact the target MHC Class II molecules without traversing membranes or undergoing any special intracellular or metabolic processing or modification. Furthermore, the fact that the allosteric site of MHC Class II molecules is expressed on the surfaces of living, or even paraformaldehyde-fixed antigen presenting cells has facilitated in vitro studies of the mechanism of action of Ii-Key peptides and of Ii-Key/antigenic epitope hybrid peptides, as presented both herein and previously in U.S. Pat. No. 5,559,028 (1996) and U.S. Pat. No. 5,919,639 (1999).
  • [0069]
    In addition to the favored property of contacting cell surface-expressed with MHC Class II molecules after a simple fluid phase administration, the Ii-Key/antigenic epitope hybrid peptides can also be taken up in an antigen processing and presenting cell, such as a macrophage or dendritic cell, and contacted to MHC Class II molecules in the course of their transversing a post-Golgi, antigen charging compartment. Selective use of either these two, very different pathways for antigen to contact MHC Class II molecules is useful during the treatment of various diseases and conditions as described herein. For example, intravenous administration at a low concentrations over a long period of time, will favor epitope presentation in a manner yielding immunosuppression, which is favored for example in the case of peptide epitopes from antigens related to multiple sclerosis or rheumatoid arthritis. Or, on the other hand, in the case of augmenting the immune response to a subsequently administered DNA vaccine for an antigen relevant to therapy of either a cancer or an infectious disease, administration of an Ii-Key/antigenic epitope incorporating an epitope coded by the DNA vaccine with an adjuvant cytokine or other stimulant promotes development of a Th1-mediated response.
  • [0070]
    The method of enhancing presentation of an MHC Class II-restricted antigenic epitope to a T lymphocyte finds wide application in the diagnosis and therapy of diseases. T cell responses to diagnostic antigenic epitopes are often measured in the diagnosis of diseases, particularly with respect to etiological infectious agents. The use of enhancing hybrids of the present invention which have such diagnostic antigenic epitopes incorporated will increase substantially the sensitivity of these in vitro diagnostic assays. In the case of infectious diseases and cancer, antigenic epitopes which are identified as pathogen or cancer specific can be incorporated into an enhancing hybrid of the present invention and the hybrid then used to initiate a Th response to a pathogen or cancer specific MHC Class II-presented antigenic epitope. This response leads to activation and expansion of T helper cells which in turn activate or ‘license’ dendritic cells, to prime an effective MHC Class I restricted cytotoxic T lymphocyte response toward the invading organism. In the case of autoimmune diseases, allergy, and graft rejection, specific antigenic epitopes which trigger the pathogenic immune response are identified and then incorporated into an enhancing hybrid of the present invention. The hybrid is then used to stimulate T cells in a manner leading to a Th2 response which will down regulate T cell responses. In this case, stimulation of a suppressor cell response is used to down regulate a pathogenic immune response. Methods for identifying enhancing hybrids which specifically stimulate a predetermined subset of T lymphocytes are described below. Additional methods and utilities of such hybrids in the therapy of disease are considered below.
  • [0071]
    In another aspect, the Ii-Key antigenic epitope hybrids increase the repertoire of MHC Class II alleles, and therefore the reaction of individuals in the vaccinated population who can be immunized with any given MHC Class II-presented epitope. Since the potency of an antigenic epitope presented within an Ii-Key/antigenic epitope hybrid is much larger than that of the same epitope presented as a peptide, mammals with low responder MHC Class II alleles for that given epitope may be stimulated to a level equivalent to mammals with high responder MHC Class II alleles. The development of immunoregulatory T cell clones recognizing that epitope will lead to enhanced subsequent presentation of the same epitope from an antigen of interest, for example of a malignant or virus-infected cell. This expansion of the repertoire of MHC Class II alleles promoting a therapeutic response to any one epitope, leads to a greater portion of the population being protected by immunizing with any given epitope. Thus, a “basket of peptides” vaccine, i.e., one containing peptides with various epitopes, is not needed. That is, without the use of the Ii-Key/antigenic epitope hybrid, a much larger number of individual antigenic epitope peptides must be used in a T helper peptide vaccine.
  • [0072]
    In another aspect, Ii-Key/antigenic epitope hybrids enhance responses to DNA vaccines. Vaccines containing the cDNA sequence for one or more antigens from either a pathogen or a tumor specific or tumor-associated antigen are being tested clinically. However, in many instances, high levels of protective antibodies, or long duration immunological memory, or maximal cytotoxic T cell responses, are not found. This lack of potency has been ascribed to weak helper T cell responses to such immunization. T helper cells can therefore be primed with Ii-Key/antigenic epitope hybrids to MHC Class II-presented epitopes in the cDNA vaccine in a suitable temporal schedule to maximize immunization with the cDNA vaccine.
  • [0073]
    In another aspect, addition of the Ii-Key-linker to each of a member of a library of peptides, overlapping through the sequence of an antigen of interest, increases the sensitivity of picking up MHC Class II epitopes. Given the increased potency of presentation of epitope in such hybrids, weakly antigenic epitopes, and epitopes with other limitations in inducing a particular pathway of biological response, for example those mediated by IgE, might be better recognized. Furthermore, in the case of combinatorial libraries of peptides synthesized with homology to a given experimental antigenic epitope, or a sequence only partially identified, for example by HLPC separation and tandem mass spectrography, the potency of peptides in such libraries can be enhanced by synthesizing the Ii-Key motif and linker at the N-terminus of such peptides. The fact that the synthesis of such peptides proceeds from the C- to the N-termini is favorable because either, sequentially ava, then K, then M, then R, then L (LRMK in reverse) (SEQ ID NO: 8) can be added, or Ac-LRMK-ava (SEQ ID NO: 9) can be added terminally as a unit.
  • [0074]
    In another aspect Ii-Key/antigenic epitope technology can be applied in the discovery, validation and use of cryptic antigenic epitopes. Cryptic antigenic epitopes have been defined empirically to be those epitopes, which are recognized upon immunization of a mammal with a peptide from an antigenic protein, but not upon immunization of a genetically identical mammal with the intact antigenic protein. In extensive experimental studies by Sercarz and colleagues, a procedure was established to discover most cryptic epitopes in a given antigenic protein, with respect to a given strain of mice. A library of peptides, for example each 15 amino acids in length with overlapping terminal segment of 6 amino acids, was created through the primary amino acid sequence of an antigen protein of interest, for example hen egg lysozyme. One mouse of a given strain was immunized with lysozyme and the proliferative response of splenic T cells to each of the peptides in the lysozyme library was tested. The epitopes in peptides stimulating the proliferative response were termed dominant epitopes. When additional mice of that strain were immunized with each of the respective peptides of the library of lysozyme peptides, all of the dominant epitopes were found to be immunogenic in isolated peptides, but additional epitopes were also discovered. These additional experiments were termed cryptic epitopes. Sercarz and colleagues demonstrated a series of mechanisms by which cryptic epitopes are not immunogenic when presented within the intact proteins. The clinical value of cryptic epitopes lies in part in the fact that a given individual is very unlikely to have previously recognized such epitope immunologically and therefore has not been tolerized to that epitope. Upon presentation of such cryptic epitopes within Ii-Key hybrids, therefore, a robust immune response can be developed, if the dose, route, schedule and adjuvants are designed toward that end. In the case of cancer, and even in the case of some infectious agents, tolerance can be developed to one or more epitopes, with the end result being that an effective immune response of the host is blocked. Cryptic epitope offer a novel repertoire of antigenic epitopes for such therapeutic purposes. Likewise such epitopes from allergens offer targets to develop therapeutic Th1 response while an IgE-promoting Th2 response had been developed toward dominant epitopes of the allergen. In such cases, Ii-Key/antigenic epitope hybrids containing dominant epitopes might exacerbate the pathological allergic responses.
  • [0075]
    In another aspect, the Ii-Key/antigenic epitope hybrids are favored in clinical diagnostic or therapeutic immunizations of patient for responses to epitopes in antigen of interest. That is, immunizing with Ii-Key/antigenic epitope hybrids as opposed to the epitope peptide, is favored because the dose required to obtain a clinically significant result is greatly reduced. Concomitantly the likelihood of a fatal anaphylactic response to the antigen, either in the case of an allergen, or otherwise, is reduced.
  • [0076]
    Additional assay systems can be used to measure the effect of incorporating an antigenic epitope other than a single MHC Class II epitope into an enhancing hybrid of the present invention. Assays with alternative readouts include, without limitation, measuring efficacy of immunoglobulin production from B cells, measuring efficacy of cytotoxic T cell generation, and the use of native T cells from animals which are outbred, inbred, congenic, transgenic for a T cell receptor or another biologically relevant molecule.
  • [0077]
    Methods for modulating the immune response of an individual finds application in the therapeutic treatment of an individual with a disease or condition. An antigenic epitope to which an enhanced immune response is considered to be beneficial in treatment of the patient is first selected. In one embodiment, the molecule from which the antigenic epitope is derived plays a role in pathogenesis. Alternatively, the antigenic epitope may be an epitope found on a harmful agent such as a pathogen, or on a pathogen infected cell. The term ‘therapeutic treatment’ as used herein is intended to include ameliorating the signs or symptoms of disease, or arresting the progression of disease in an individual identified or considered to be suffering from a disease. The term ‘prevention’ as used herein is intended to include ameliorating the underlying cause to, or associated factor predisposing to, a disease, in an individual who might not have begun to experience recognizable signs or symptoms of a disease.
  • [0078]
    The disease may be an infectious disease caused or associated with infection by a bacterium, a virus, a parasite, a fungus, a rickettsia, or other infectious agent, or combination of such agents. The therapy may be directed against the toxin of a disease or against a receptor for a toxin of a disease. Preferred toxins for epitope derivation include, without limitation, staphylococcal enterotoxins, toxic shock syndrome toxin, retroviral antigens (e.g. antigens derived from human immunodeficiency virus), streptococcal antigens, mycoplasma, mycobacterium, and herpes viruses. Highly preferred toxins are anthrax toxin (lethal factor, edema factor and protective antigen), SEA, SEB, SE1-3, SED and SEE.
  • [0079]
    The disease or condition may be considered to be an autoimmune process, for example rheumatoid arthritis, multiple sclerosis, lupus erythematosus, diabetes mellitus, myasthenia gravis, autoimmune thyroiditis, scleroderma, dermatomyositis, pemphigus, and other similar processes. Examples of such model systems for autoimmune diseases which can be used to evaluate the effects of the compounds and methods of the present invention are systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, insulin dependent diabetes mellitus, and experimental allergic encephalomyelitis. The procedures for conducting these experiments are presented in Clark et al., (1994) U.S. Pat. No. 5,284,935, the contents of which are incorporated herein by reference.
  • [0080]
    The disease or condition may be considered to be an allergic process, for example asthma, hayfever, allergic rhinitis, topical dermatitis, colitis, and other such processes initiated or associated with particular allergens or no defined allergen. Examples of such allergens are plant, animal, bacterial, parasitic allergens and metal-based allergens that cause contact sensitivity. Preferred allergens for use in the present invention are weed, grass, peanut, mite, flea and cat antigens.
  • [0081]
    Alternatively, the disease or condition may be a proliferative or malignant process, for example cancer, benign prostatic hypertrophy, psoriasis, adenomas or other cellular proliferations of intrinsic origin, or in response to a viral or other infectious, irritative, or environmental process.
  • [0082]
    The term ‘mammal’ as used herein is meant to encompass the human species as well as all other mammalian species. The compounds and methods of this invention may be applied in the treatment of diseases and conditions occurring in individuals of all mammalian species. The term ‘individual’ as used herein refers to one of any mammalian species, including the human species. The diseases and conditions occurring in individuals of the human species, and mentioned herein by way of example, shall include comparable diseases or conditions occurring in another species, whether caused by the same organism or pathogenic process, or by a related organism or pathogenic process, or by unknown or other known, organism and/or pathogenic process. The term ‘physician’ as used herein also encompasses veterinarians, or any individual participating in the diagnosis and/or treatment of an individual of a mammalian species.
  • [0083]
    The present invention also provides for the administration of a compound, as a drug, a prodrug of the compound, or a drug-metabolite of the compound, in a suitable pharmaceutical formulation. The terms ‘administration of’ or ‘administering a’ compound is understood to mean providing a compound of the invention, as a drug, a prodrug of the compound, or a drug-metabolite of the compound, to an individual in need of treatment or prevention of a disease. Such a drug which contains one or more of the hybrid polypeptides of the present invention, as the principal or member active ingredient, for use in the treatment or prevention of one or more of the above-noted diseases and conditions, can be administered in a wide variety of therapeutic dosage forms in the conventional vehicles for topical, oral, systemic, and parenteral administration. The route and regimen of administration will vary depending upon the disease or condition to be treated, and is to be determined by the skilled practitioner. For example, the compounds can be administered in such oral dosage forms for example as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection. Likewise, they may also be administered in intravenous (either by bolus or infusion methods), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form. All of these is forms are well known to those of ordinary skill in the pharmaceutical arts.
  • [0084]
    The daily dose of the products may be varied over a range from 0.001 to 1,000 mg per adult per day. For oral administration, the compositions are preferably provided in the form of tables containing from 0.001 to 1,000 mg, preferably 0.001, 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 10.0, 20.0, 50.0, 100.0 milligrams of active ingredient for the symptomatic adjustment of dosage according to signs and symptoms of the patient in the course of treatment. An effective amount of drug is ordinarily supplied at a dosage level of from about 0.0001 mg/kg to about 50 mg/kg of body weight per day. The range is more particular from about 0.0001 mg/kg to 7 mg/kg of body weight per day.
  • [0085]
    Advantageously, suitable formulations of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses for example of two, three, or four times daily. The enhancing hybrid polypeptide of the present invention may be used to prepare a medicament or agent useful for the treatment of the diseases or conditions listed above. Furthermore, compounds of the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in the art. To be administered in the form of a transdermal delivery system, the dosage administration will, or course, be continuous rather than intermittent throughout the dosage regimen.
  • [0086]
    For treatment and prevention of disease, the hybrid polypeptide of the present invention may be administered in a pharmaceutical composition comprising the active compound in combination with a pharmaceutically acceptable carried adopted for topical administration. Topical pharmaceutical compositions may be, for example, in the form of a solution, cream, ointment, gel, lotion, shampoo, or aerosol formulation adapted for application to the skin. These topical pharmaceutical composition containing the compounds of the present invention ordinarily include about 0.005% to 5% by weight of the active compound in admixture with a pharmaceutically acceptable vehicle.
  • [0087]
    For the treatment and prevention of disease and conditions, for example listed above, the hybrid polypeptide of the present invention may be used together with other agents known to be useful in treating such diseases and conditions. For combination treatment with more than one active agent, where the active agents can be administered concurrently, the active agents can be administered concurrently, or they can be administered separately at staggered times.
  • [0088]
    The dosage regimen utilizing the compositions of the present invention is selected in accordance with a variety of factors, including for example type, species, age, weight, sex and medical condition of the patient, the severity of the condition to be treated, and the particular compound thereof employed. A physician of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter, or arrest the progress of the disease or condition. Optimal precision in achieving concentration of drug with the range that yields efficacy either without toxicity or with acceptable toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This process involves a consideration of the distribution, equilibrium, and elimination of the drug, an is within the ability of the skilled practitioner.
  • [0089]
    In the methods of the present invention, the compounds herein described in detail can form the active ingredient and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carders (collectively referred to herein as ‘carder materials’) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups, and the like, and consistent with conventional pharmaceutical practices. For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, aga, bentonite, xanthan gum and the like.
  • [0090]
    The liquid forms may be suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl cellulose and the like. Other dispersing agents which may be employed are glycerin and the like. For parental administration, sterile suspensions an solutions are desired. Isotonic predations which generally contain suitable preservatives are employed when intravenous administration is desired.
  • [0091]
    Topical preparations containing the active drug component can be admixed with a variety of carrier materials well known in the art, such as, for example, alcohols, aloe vera gel, allatoin, glycerine, vitamins A or E oils, mineral oil, PPG2 myristyl propionate, and the like, to form, for example, alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations.
  • [0092]
    The hybrid polypeptide of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilameller vesicles and multilamellar vesicles. Liposomes can be formed from a variety of compounds, including for example cholesterol, stearylamine, and various phosphatidylcholines.
  • [0093]
    The hybrid polypeptide or formulation thereof of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihyrdo-pyrans, polycyanoacrylates, and cross-linked or amphipathic block copolymers of hydrogels.
  • [0094]
    The hybrid polypeptides of the present invention and formulations thereof can be prepared using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants which are themselves known to those of ordinary skill in this art, but are not mentioned in greater detail herein.
  • [0095]
    As an alternative to administering the enhancing hybrid of the present invention directly to an individual to enhance the MHC Class II presentation of an antigenic epitope to T lymphocytes of the individual, a population of antigen presenting cells may be obtained from the individual and treated ex vivo with the enhancing hybrid of the present invention. These cells are treated with the enhancing hybrid under conditions appropriate for binding of the hybrid to an MHC Class II molecule of the antigen presenting cells. Once treated, the antigen presenting cells are administered to the individual under conditions which promote physical contact of the treated cells with T lymphocytes of the individual. As described above, the effect on the immune response, enhancement or suppression, will depend upon which subset of T cells are preferentially stimulated by the enhancing hybrid. Enhancement of the immune response may have a favorable effect upon the cytotoxic response against, for example, either a cancer cell or an infectious organism. Alternately, enhancement of the T suppressor cell response may have the effect of suppressing the immune response to a specific molecule. Such suppression may have a therapeutic effect when utilizing antigenic epitopes from etiological antigens of autoimmune diseases, for example, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, or lupus erythematosus. The methods and procedures for the ex vivo treatment of cells from a patient with the compounds and methods of the present invention may be adapted from the following patents, the contents of which are incorporated herein by reference: Rosenberg (1998) U.S. Pat. No. 5,126,132; Chada et al., (1997) U.S. Pat. No. 5,693,522; Kriegler et al., (1998) U.S. Pat. No. 5,849,586; Gruber et al., (1999) U.S. Pat. No. 5,856,185; and Kriegler et al., (1999) U.S. Pat. No. 5,874,077.
  • [0096]
    In another respect, the compounds and methods of the present invention can be used under ex vivo conditions to promote the generation of cytotoxic T lymphocytes, using the compounds and methods described in Celis et al., (1998) U.S. Pat. No. 5,846,827, the contents of which are incorporated herein by reference.
  • [0097]
    As discussed above, a non-comprehensive discussion of specific examples of epitopes/determinants useful as elements in the enhancing hybrids of the present invention is provided in the Exemplification section. Also found in the corresponding Exemplification section is a discussion of methods for using an enhancing hybrid containing such an element. One skilled in the art, through the application of no more than routine experimentation, can incorporate experimentally-determined or predicted epitopes/determinants into an enhancing hybrid for application to a wide range of disease or conditions.
  • [0098]
    In another aspect this invention relates to a method to identify and exploit naturally occurring Ii-Key/MHC Class II antigenic epitopes which have in the sequence a primary sequence motif which functions during the processing and binding of such peptides to MHC Class II molecules in the classical exogenous pathway, as does the synthetic Ii-Key/antigenic epitope hybrids.
  • [0099]
    Given the identification of the presence or absence of such Ii-Key motifs comprising, one can modify the amino acid sequence of the protein in a manner to introduce such a motif when one was not present, or to delete such a motif when one was present. Such modifications are obtained for example trough manipulation of the genes coding of the antigenic protein in a manner to substitute a functionally accepted amino acid in the Ii-Key motif. In some instances a deletion or insertion of amino acids can obtain the same end, for example when the antigenic epitope occurs at or near the N-terminus of the protein. Such modifications to change the immunogenecity of the protein have favorable clinical properties. For example, vaccine promoters can behave increased potency. Certain therapeutic proteins can have decrease immunogenecity.
  • [0100]
    In another aspect, the present invention relates to methods for selecting biologically active MHC Class II-presented epitopes and altering the immune response to such epitopes in antigenic proteins or polypeptides. Specifically, this disclosure provides method to identify in the amino acid sequence of a protein the presence or absence of a Ii-Key immunoregulatory motif of 5 amino acids preceding an experimentally determined or algorithm-predicted, MHC Class II-presented, antigenic epitope. This immunoregulatory Ii-Key motif enhances charging of the antigenic epitope, which follows it into the antigenic peptide binding site of MHC Class II molecules. Given predictions of antigenic epitopes within a protein, identifying the subset of those epitopes preceded by an Ii-Key motif improves greatly the efficiency of vaccine peptide selection. Also, by modifying the sequence of a protein or polypeptide, for example, either to introduce or to eliminate an Ii-Key motif before selected MHC Class II-presented epitopes, the immunological response to that protein can be altered.
  • [0101]
    Adverse immunological responses to a therapeutic protein can limit the use of such a protein. Such adverse immunological responses can be lessened either by decreasing immunogenecity of some of the MHC Class II-presented epitopes or by inducing immunosuppression. For either case, insertion or alteration(s) at the location of an Ii-Key motif appropriately spaced before an MHC Class II epitope can achieve that endpoint without alteration of the MHC Class II epitope itself. It may not be possible to alter residues within the MHC Class II-epitope without loss of the biological function of the therapeutic protein. The following procedure is followed in designing sequence modifications in a therapeutic protein of interest to alter its immunogenecity and/or the immune response to that protein.
  • [0102]
    The sequence of a protein, or a fragment thereof, is established by one of several methods. The protein or fragment thereof can be experimentally sequenced, or the sequence can be deduced from either the sequence of either the gene coding for the protein or a cDNA created from the RNA coding for the protein. Given that primary amino acid sequence, the experimentally determined or algorithm-predicted MHC Class II epitopes are specified. The experimentally determined epitopes are known from prior investigations. The algorithm-predicted epitopes are found by several methods, such as the ProPred MHC Class-II Binding Peptide Prediction Server (Raghava G P. Nat Biotechnol. 1999 17:555-61); Singh, H. Bioinformatics 2001 17:1236-7 (access via: http://www.imtech.res.in/raghava/propred/index.html)). An alternative program is the SYFPEITHI program (Rammensee H-G. Immunogenetics 1999 50: 213-219 (access via: http://134.2.96.221/scripts/MHCServer.dll/Ep.html)). These epitopes are also characterized with respect to the MHC Class II alleles, which are either known or predicted to present them to the immune system of humans or an experimental animal such as the mouse. Thus, differing sets of predicted epitopes are obtained, according to the relevant presenting MHC Class II allele. Some epitopes are presented by multiple MHC Class II alleles and are, therefore, preferred.
  • [0103]
    This disclosure presents a method for the identification of an Ii-Key immunoregulatory motif. Specifically, in the sequence of a protein, the immunoregulatory, Ii-Key motif is a segment of 5 contiguous amino acids containing at least two amino acids of the group comprising Leu, Ile, Val, Phe, and Met, and at least one of the group comprising His, Lys, and Arg, where that contiguous 5 amino acid segment is separated by 5 to 11 amino acids from the N-terminal residue of the MHC Class II-presented epitope.
  • [0104]
    The subset of such antigenic epitopes with the presence of an appropriately spaced Ii-Key motif lead to vaccine peptides to enhance the potency of the CD4+ T cell immune response. Such epitopes are considered to be more likely to be dominant or biologically active. Peptides with such epitopes are favored as vaccine protect against infectious diseases and cancer, and to immunosuppressive vaccines to allergy. The compositions and methods of the present invention relate to non-naturally occurring proteins or polypeptides which contain: 1) a C-terminal element comprising an MHC Class II-presented epitope; 2) an N-terminal element comprising an Ii-key motif; and 3) an intervening element comprising a sequence from about 4 to about 11 amino acid residues. The use of the term non-naturally occurring is intended to require that the protein or polypeptide is modified. Generally, the modification is by recombinant DNA techniques, and the modification or modifications take place within elements 2) or 3) as defined above. The designations “N- and C-terminal” are meant to refer only to the relationship of these elements in the 3-part segment specifically recited. One of skill in the art will recognize that if such a 3-part segment is located within a protein, it is likely that additional residues will extend in the C-terminal direction from the C-terminal element, and in the N-terminal direction from the N-terminal element. In addition to proteins or polypeptides as described above, the present application is also directed toward expressible nucleic acid sequences which encode such proteins or polypeptides.
  • [0105]
    In preferred embodiments, the non-naturally occurring protein or polypeptide is a modified form of a naturally occurring protein or polypeptide. Therapeutic proteins represent a particularly important class. Such modified proteins or polypeptides stimulate an immune response which differs from that induced by their non-modified, naturally-occurring counterparts. Such products include therapeutic proteins, such as hormones, cytokines, or other molecules interacting with cell surface receptors. Modifications of an Ii-Key motif can be made to eliminate its function, or a site N-terminal to a putative antigenic epitope can be modified to introduce an Ii-Key motif. Such modifications suppress a deleterious immune response to the therapeutic protein. Such products include the therapeutic protein, and fragments thereof, and genetic constructs leading to their expression.
  • [0106]
    Modifications most likely not to disturb the biological function of a therapeutic protein to be engineered to alter immunogenecity include the following. Presence is scored of sequences, and even individual amino acid residues, which are known from the crystallographic structure of the protein to be superficially exposed on the protein, and thus more likely to accept a mutation without loss of function. In the case of therapeutic proteins for which the three dimensional structure has not been determined, various methods are applied to predicting acceptance of mutations to engineer an Ii-Key box appropriately spaced from an antigenic epitope. Distances from the N-terminus and from the C-terminus of the protein are determined. Upon modestly denaturing conditions, N-terminal and C-terminal antigenic epitopes can be presented by MHC Class II molecules. Epitopes at the N-terminus of the protein are favored over epitopes at the C-terminus, in part because the to-be-designed Ii-Key box is more distal. Also sequence alterations are more likely to be accepted in a sequence, which is predicted to be on the surface of the protein, preferably in relatively loose configuration. Such segments can be identified in homologous proteins with a relatively higher frequency of naturally occurring mutations. Segments are identified containing residues, which in site-directed mutational studies, have been shown to accept amino acid substitutions. By the preceding and additional methods, one skilled in the art will predict segments of a protein that are more likely to accept without loss of function, amino acid substitutions at residue positions resulting in the creation of Ii-Key box motifs at appropriate N-terminal displacements from the N-terminus of an antigenic epitope. The following peptide sequences are targeted for Ii-Key box manipulations, in rank order: epitopes known to be MHC Class II-presented, epitopes predicted to be MHC Class II-presented by MHC Class II alleles present either in the highest frequency among humans or in the animal strain of experimental interest. Some of these methods are presented in U.S. Pat. No. 5,679,527 (1997), the content of which is incorporated herein by reference.
  • [0107]
    In addition to the above site-specific engineered replacements, one skilled in the art will use additional combinatorial molecular biological methods to generate mutations within sets of residue positions to create an Ii-Key box motif spaced 4 to 8 amino acids N-terminal to a selected, either known or putative antigenic epitope. Such methods may encompass the preparations of multiple products, which are screened for altered immunogenecity with or without retention of biological activity.
  • [0108]
    Each experimentally determined or algorithm-predicted epitope in the protein of interest is examined for the presence in its primary sequence of a segment of 5 contiguous amino acids containing at least two amino acids of the group comprising Leu, Ile, Val, Phe, and Met, and at least one of the group comprising His, Lys, and Arg, where that contiguous 5 amino acid segment is separated by 4 to 12 amino acids from the N-terminal residue of a MHC Class II-presented epitope. The subset of all experimentally or algorithm-predicted epitopes meeting these criteria are preferred for developmental work, including for example synthesis of peptide vaccines, chemical modifications of those vaccines for a favorable therapeutic effect, experimental study in animals, and clinical studies. In this disclosure the standard single letter nomenclature of the International Union of Pure and Applied Chemists is used to identify amino acids within the sequence of proteins or peptides.
  • [0109]
    More preferable is the set of experimentally or algorithm-predicted epitopes with a 4 to 8 amino acids separation from the N-terminal residue of the MHC Class II-presented epitope, of the segment of 5 contiguous amino acids containing at least two amino acids of the group comprising Leu, Ile, Val, Phe, and Met, and at least one of the group comprising His, Lys, and Arg. This set constitutes a subset of the set of experimentally or algorithm-predicted epitopes with a 0 to 12 amino acids separation from the N-terminal residue of the MHC Class II-presented epitope, of the segment of 5 contiguous amino acids containing at least two amino acids of the group comprising Leu, Ile, Val, Phe, and Met, and at least one of the group comprising His, Lys, and Arg. An important utility in this method is the reduction in the number of candidate epitopes subject to study toward therapeutic or diagnostic development objectives. In each of the Exemplification Tables of this disclosure presenting either experimentally determined or algorithm-predicted epitopes, the presence of an Ii-Key box separated by 4-10 amino acids from the N-terminus of the MHC Class II-presented epitope is indicated, with the number of intervening residue positions. Among these, preference is ranked according to the length of the separating interval, with shorter being better, among spacers of 4 or more amino acids.
  • [0110]
    Peptides which are chosen for synthesis have within the natural primary sequence of the protein, an MHC Class II-presented epitope and a segment of 5 contiguous amino acids containing at least two amino acids of the group comprising Leu, Ile, Val, Phe, and Met, and at least one of the group comprising His, Lys, and Arg, the intervening segment of 0 to 12 amino acids, and the antigenic epitope, comprising a total length of 12 to 34 amino acids. More preferred is a synthetic peptide synthesized according to the primary sequence of the protein, including the segment of 5 contiguous amino acids containing at least two amino acids of the group comprising Leu, Ile, Val, Phe, and Met, and at least one of the group comprising His, Lys, and Arg, the intervening segment of 4 to 11 amino acids, and the antigenic epitope, comprising a total length of 15 to 25 amino acids. The sequence of the synthetic peptide will usually be the natural sequence of a mammalian protein, but also the protein may be a non-natural sequence, such as that generated by methods using a combinatorial library with selections for a useful function. Furthermore, the sequence of the protein may include modifications of the sequence of natural protein, including for example substitution, insertion, or deletion of one or more amino acids, including the use of non-natural amino acids.
  • [0111]
    The selected peptides, including the segment of 5 contiguous amino acids containing at least two amino acids of the group comprising Leu, Ile, Val, Phe, and Met, and at least one of the group comprising His, Lys, and Arg, the intervening segment of 4 to 8 amino acids, and the antigenic epitope, comprising a total length of 12 to 34 amino acids will be modified to obtain favorable biological and pharmacokinetic properties. These medications are selected from the group consisting of: a) acetylation of the N-terminus, b) amidation of the C-terminus; c) replacement of an amino acid with another natural or synthetic amino acid, d) replacement of an L-amino acid with a D-amino acid, e) inversion of the amino acid sequence and use of D-amino acids in each residue positions, f) modifications to limit proteolysis or clearance (inactivation), and g) modifications to improve solubility, transport and half-life. Methods of chemical modification of therapeutic peptides for favorable therapeutic properties are presented, for example, in U.S. Pat. No. 5,679,527, the disclosure of which is incorporated herein by reference.
  • [0112]
    The method to design such modifications start with a list of identified epitopes, ranked according to each of several characteristics in order to identify segments of the therapeutic protein, which are more likely to accept without loss of function, amino acid substitutions which create an Ii-Key box motif appropriately spaced from the N-terminus of an antigenic epitope. The characteristics by which the epitopes are ranked include, without limitation, the following. Presence is scored of sequences, and even individual amino acid residues, which are known from the crystallographic structure of the protein to be superficially exposed on the protein, and thus more likely to accept a mutation without loss of function. In the case of therapeutic proteins for which the three dimensional structure has not been determined, various methods are applied to predicting acceptance of mutations to engineer an Ii-Key box appropriately spaced from an antigenic epitope. Distances from the N-terminus and from the C-terminus of the protein are determined. Upon modestly denaturing conditions, N-terminal and C-terminal antigenic epitopes can be presented by MHC Class II molecules. Epitopes at the N-terminus of the protein are favored over epitopes at the C-terminus, in part because the to-be-designed Ii-Key box is more distal. Presence in a sequence motif, which is predicted to be on the surface of the protein, preferably in relatively loose configuration. Segments are identified which in homologous proteins have a relatively higher frequency of naturally occurring mutations. Segments are identified containing residues, which in site-directed mutational studies have been shown to accept amino acid substitutions. By the preceding and additional methods, one skilled in the art will predict segments of a protein which are more likely to accept without loss of function, amino acid substitutions at residue positions which create Ii-Key box motifs at appropriate N-terminal displacements from the N-terminus of an antigenic epitope which is highly ranked according to the following ranking scheme: epitopes known to be MHC Class II-presented, epitopes predicted to be MHC Class II-presented by MHC Class II alleles present either in the highest frequency among humans or in the animal strain of experimental interest. Some of these methods are presented in U.S. Pat. No. 5,679,527 (1997) the disclosures of which are incorporated herein by reference.
  • [0113]
    The Ii-Key box/spacer identifying algorithm is applied within the amino acid sequence of the protein to examine regions N-terminal to each of the above experimentally determined or predicted MHC Class II-presented epitopes, in a manner to identify three categories: a) presence of an Ii-Key box motif spaced by 4 to 8 amino acids, N-terminal to the antigenic epitope, b) presence of an Ii-Key box motif spaced by 4 to 8 amino acids, N-terminal to the antigenic epitope if one or more amino acids were exchanged for a member of the group Leu, Ile, Val, Phe, Met and/or one or more amino acids were exchanged for a member of the group His, Lys and Arg in the primary sequence.
  • [0114]
    In addition to the above site-specific engineered replacements, one skilled in the art will use additional combinatorial molecular biological methods to generate mutations within sets of residue positions to create an Ii-Key box motif spaced 4 to 8 amino acids N-terminal to a selected, either known or putative antigenic epitope. Such methods may encompass the preparations of multiple products, which are screened for altered immunogenecity with or without retention of biological activity.
  • [0115]
    Many uses of Ii-Key antigenic epitope hybrids can be described with respect to individual antigenic proteins. Such uses are presented in the Examples, in varying degrees of detail. The concepts, which are presented in the context of one Example, apply nevertheless in the cases of all Examples when appropriate, even when they are not repeated in the context of each individual Example. While such specific examples well present methods to design and synthesize Ii-Key antigenic epitope hybrids of specific proteins by which such Ii-Key antigenic epitope hybrids can be created and used with respect to other proteins of interest, as the need might arise from to time.
  • [0116]
    In another aspect, this invention relates to the use of Ii-Key/antigenic epitope hybrids to enhance protective immune responses to a subsequently administered DNA vaccine or against an attenuated infectious pathogen vaccine. Such adjuvant vaccine preparations can be referred to as PreVaccines™. One example is the use of Ii-Key antigenic epitope hybrids in vaccination protocols to protect against variola. Uses in protecting against smallpox virus are considered in relatively greater detail in a corresponding section of the Exemplification section which follows. Considerations detailed herein also serve to model applications directed toward other pathogens. In the case of smallpox vaccination, Ii-Key antigenic epitope hybrids are used to elicit a Th1 response to one or more MHC Class II-presented epitopes of the gp42 extracellular envelope protein coded by the B5R viral gene of vaccinia. Individuals so vaccinated will have an anamnestic response which is more rapid and of higher potency in terms of antibody titers and isotype an affinity maturation, CTL and memory responses to challenge by cDNA vaccines for the B5R gene, by vaccinia, or by variola. In a related application, such PreVaccines™ can be used before vaccination with recombinant vaccinia virus containing either Ii-RGC genes or CIITA plus Ii-RGC genes. The recombinant vaccinia virus containing an Ii-RGC gene, upon infection within a professional antigen presenting cell such as a dendritic cell, will lead to MHC Class II-restricted T helper cell responses in those cells as described. In the case of recombinant vaccinia virus containing both an Ii-RGC gene and a CIITA gene, such a virus upon infecting cells which do not normally express MHC Class II molecules, such as dendritic cells, will express MHC Class II molecules without Ii protein. A wide repertoire of MHC Class II-presented epitopes are thus represented and the response to those epitopes is further enhanced by prior expansion of responses to the MHC Class II epitope in the PreVaccine™. Such a use can be further augmented by prior immunization of mammals with Ii-Key antigenic epitope hybrids in an appropriate dose, vehicle, route and schedule. Ii-Key/antigenic epitope hybrids can thus be used either as a stand-alone protective vaccine or as a PreVaccine™ used in conjunction with vaccines for other viruses and infectious pathogens, for example, without limitation, HIV, Bacillus anthracis, EBOLA virus and Marburg virus.
  • EXEMPLIFICATION Example 1 Ii-Key/Ara h 1 Antigenic Epitope Hybrids
  • [0117]
    In one aspect this invention relates to therapeutic modulation of pathological allergic responses of some humans to peanuts and other edible nuts. Such responses include potentially fatal asthmatic or anaphylactic reactions. Good progress has been made in identifying and sequencing the principal protein allergens in peanuts and other nuts mediating these pathological responses. Crossed-radioimmunoelectrophoresis has identified 16 allergenic fractions in raw peanut and sodium dodecylsulfate polyacrylamide gel electrophoresis has revealed 32 protein bands (Barnett D. J Allergy Clin Immunol. 1983 72:61-68). Three major allergens have been identified. Ara h 1 of 64.5 kDa is a member of the vicilin family of seed storage proteins (Burks A W. J Allergy and Clin Immunol. 1991 88:172-9). Ara h 2 of 17.5 kDa is a member of the conglutin family of seed storage proteins (Burks A W. J Allergy and Clin Immunol. 1992 90:962-9). Ara h 3 of 60 kDa, a preproglobulin, is a member of the glycinin-like seed storage proteins (Rabjohn P. J Clin Invest. 1999 103:535-42). For Ara h 1, 23 IgE-recognized epitopes have been mapped, with 4 being dominant. For Ara h 2, 10 IgE-recognized epitopes have been mapped, with 3 being dominant. For Ara h 3, 4 IgE-recognized epitopes have been mapped, with 1 being dominant. For each of these three allergens, the respective cDNAs have been isolated and expressed. The deduced protein sequences are presented below (Tables 1.1, 2.1 and 3.1).
  • [0118]
    Development of allergy-inducing IgE antibodies is regulated by a subset of CD4+ T cells, the receptors of which recognize antigenic peptides presented by MHC Class II-molecules. The recognition of such epitopes by CD4+ T cells can lead either to a Th1 response, in which the responding T cells are characterized by synthesis of predominantly certain cytokines such as IFN-γ, or to a Th2 response, in which the responding T cells are characterized by synthesis of predominantly other cytokines such as IL-4 and IL-10. In patients with allergen-induced asthma, a Th2 pattern of response enhances synthesis of IgE molecules recognizing many different surface epitopes of the offending allergen(s). Binding of IgE to such allergens activates a cascade of biological mediators resulting in the asthmatic symptoms. The compounds and methods of the invention can be applied to the modification of responses in a Th1 or Th2 pathway-specific manner to obtain clinically desired effects. Such modifications can be illustrated for the control of asthma.
  • [0119]
    In animal studies of asthmatic allergic responses to protein antigens, it was discovered that substitution of one or more amino acids within the MHC Class II antigenic epitope leads to potential therapeutic agents inducing an altered T cell immune response. Specifically, such altered antigenic peptides modified a predominantly Th2 response, which promotes asthmatic responses, to a predominantly Th1 response (Janssen E. J Immunol. 2000 164:1580-8; Janssen E M. J Immunol. 2000 165:7207-14). Such immunodeviation from a Th2 to a Th1 pattern functionally suppresses the asthmatic response. However replacement of individual amino acids in a MHC Class II-presented epitope of an offending allergen is expected to alter potency of binding of the antigenic peptides in the antigenic peptide binding site as well as the repertoire of T cell receptors responding to the antigenic peptide. Affinity of the antigenic epitope peptide for a patient's MHC Class II alleles can be decreased by such structural manipulations. One significant advantage of the method of this invention is the ability to immunodeviate the pattern of Th subset activation from the Th2 pathway to the Th1 pathway, without changing the sequence of the antigenic epitope. Since MHC Class II molecules demonstrate allele-specific preferences for some antigenic peptides and not for other antigenic peptides (which might nevertheless be well presented by other MHC Class II alleles), there is no issue of potentially decreased potency of Ii-Key/antigenic epitope hybrids. In fact, given the increase in potency of presentation of epitopes within Ii-Key/antigenic epitope hybrids, one can expect presentation by a wider range of MHC Class II alleles. Another clinically preferred characteristic of the Ii-Key/antigenic epitope hybrids over sequence-modified antigenic epitope peptides is that the dose required to achieve immunodeviation is much less (by a factor of 10 to 100) and therefore potentially fatal anaphylaxis is much less likely to occur.
  • [0120]
    In another aspect, this invention relates to the design of Ii-Key/Ara h 1 antigenic epitope hybrids. Such Ii-Key/Ara h 1 antigenic epitope hybrids comprise the Ii-Key motif LRMK (SEQ ID NO: 3) and acceptable modifications, linked through a simple, flexible linker to a MHC Class II-presented epitopes of the Arachis hypogaea 1 (Ara h 1) major allergen protein found in peanuts and some additional edible nuts. The amino acid sequence of this allergen (626 amino acids) is presented in Table 1.1. The sequence of Ara h 1 was taken from GenBank entry gi/11683gi/ allergen Ara h 1. MHC Class II-presented epitopes within this protein sequence were identified with the Singh ProPred MHC Class-II Binding Peptide Prediction Server (Raghava G P. Nat Biotechnol. 1999 17:555-61; Singh, H. Bioinformatics 2001 17:1236-7 (access via: http://www.imtech.res.in/raghava/propred/index.html)). The ProPred program evaluates sequences for presentation by many common MHC Class II alleles. An alternative program is the SYFPEITHI program (Rammensee H-G. Immunogenetics 1999 50: 213-219 (access via: http://www.uni-tuebingen.de/uni/kxi/)). Epitopes with highest scores were identified for their presentation by 51 HLA-DR alleles, that cover more than 90% of the MHC Class II alleles. The highest scoring epitopes predicted with the ProPred program are likely to be experimentally antigenic. The peptides listed in Table 1.2 have the highest scoring epitopes, in the ProPred program analysis for Ara h 1. Ii-Key/Ara h 1 hybrids containing some of the predicted MHC Class II-presented Ara h 1 epitopes of Table 1.2 are listed in Table 1.3. Experimentally defined IgE-binding Ara h 1 epitopes which overlap with predicted MHC Class II-presented Ara h 1 epitopes are listed in Table 1.4. Ii-Key/Ara h 1 hybrids containing predicted MHC Class II Ara h 1 epitopes and experimentally determined IgE-binding Ara h 1 epitopes are listed in Table 1.5.
  • [0000]
    TABLE 1.1
    Deduced amino acid sequence of Ara h 1.
      1 mrgrvsplml llgilvlasv sathaksspy qkktenpcaq rclqscqqep (SEQ ID NO: 10)
     51 ddlkqkaces rctkleydpr cvydprghtg ttnqrsppge rtrgrqpgdy
    101 dddrrqprre eggrwgpagp rerereedwr qpredwrrps hqqprkirpe
    151 gregeqewgt pgshvreets rnnpfyfpsr rfstrygnqn grirvlqrfd
    201 qrsrqfqnlq nhrivqieak pntlvlpkha dadnilviqq gqatvtvang
    251 nnrksfnlde ghalripsgf isyilnrhdn qnlrvakism pvntpgqfed
    301 ffpassrdqs sylqgfsrnt leaafnaefn eirrvlleen aggeqeergq
    351 rrwstrssen negvivkvsk ehveeltkha ksvskkgsee egditnpinl
    401 regepdlsnn fgklfevkpd kknpqlqdld mmltcveike galmlphfns
    451 kamvivvvnk gtgnlelvav rkeqqqrgrr eeeededeee egsnrevrry
    501 tarlkegdvf impaahpvai nasselhllg fginaennhr iflagdkdnv
    551 idqiekqakd lafpgsgeqv ekliknqkes hfvsarpqsq sqspsspeke
    601 spekedqeee nqggkgplls ilkafn
  • [0000]
    TABLE 1.2
    Predicted MHC Class II-presented epitopes of
    Ara h 1.
    PEPTIDE SEQ ID
    NO: Pos. Sequence Score Ii-Key NO:
    1.2.1 417 V K P D K K N P Q 6.00 11
    1.2.2 193 I R V L Q R F D Q 6.00 12
    1.2.3 313 L Q G F S R N T L 6.00 13
    1.2.4 453 M V I V V V N K G 6.00 3 14
    1.2.5 457 V V N K G T G N L 5.20 15
    1.2.6 498 V R R Y T A R L K 5.30 16
    1.2.7 209 L Q N H R I V Q I 5.30 8 17
    1.2.8 206 F Q N L Q N H R I 4.40 5 18
    1.2.9   9 M L L L G I L V L 5.30 3 19
    1.2.10  11 L L G I L V L A S 5.50 4 20
    1.2.11   1 M R G R V S P L M 4.25 21
    1.2.12  15 L V L A S V S A T 4.20 22
    1.2.13 429 L D M M L T C V E 5.10 9 23
    1.2.14 264 L R I P S G F I S 5.00 5 24
    1.2.15 270 F I S Y I L N R H 4.48 -/11 25
    1.2.16 275 L N R H D N Q N L 4.10 6 26
    1.2.17 325 F N A E F N E I R 4.30 27
    1.2.18 329 F N E I R R V L L 4.60 28
    1.2.19 335 V L L E E N A G G 4.20 29

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0121]
    In Table 1.2, PEPTIDES: 1.2.1, 1.2.3, 1.2.6, 1.2.5, and 1.2.18 overlap to some degree with experimentally defined IgE-binding epitopes of Table 1.4. PEPTIDES 1.2.9, 1.2.10, 1.2.11, 1.2.12 are peptides with altered amino acid sequences in a recombinant, mutated Ara h 1 (Burks A W. Eur J. Immunol. 1997 245:334-9). IgE epitopes were defined further in the work of Shin et al. (J Biol. Chem. 1998 273:13753-9).
  • [0000]
    TABLE 1.3
    Ii-Key/Ara h 1 hybrids containing some of the
    predicted MHC Class II-presented Ara h 1
    epitopes of Table 1.2.
    SEQ
    ID
    PEPTIDE Pos. Sequence NO:
    1.3.1 192 Ac-LRMK-ava-IRVLQRFDQ-NH2 30
    1.3.2   1 Ac-LRMK-ava-MRGRVSPLM-NH2 31
    1.3.3   1/8/10/14 Ac-LRMK-ava-MRGRVSPLML 32
    LLGILVLASV SAT-NH2
    1.3.4 205 Ac-LRMK-ava-FQNLQNHRI-NH2 33
    1.3.5 205/208 Ac-LRMK-ava-FQNLQNHRIVQI- 34
    NH2
    1.3.6 428 Ac-LRMK-ava-LDMMLTCVE-NH2 35
    1.3.7 263 Ac-LRMK-ava-LRIPSGFIS-NH2 36
    1.3.8 263/269/274 Ac-LRMK-ava- 37
    LRIPSGFISYILNRHDNQNL-NH2

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 1.2. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0122]
    The activity of additional Ii-Key/Ara h antigenic epitope hybrids are tested with one residue of ä-aminovaleric acid as a spacer because, in previous studies of a series of hybrids with systematic variation of spacer structures, the hybrid with one ava residue was no less active than any hybrid with a more complex spacer sequence. In the Ara h hybrids, the Ii-Key-spacer (LRMK-ava) (SEQ ID NO: 9) sequence was linked to the first amino acid of the ProPred-identified peptide, which amino acid is thought to fit into pocket 1 of the antigenic peptide-binding site of the MHC Class II molecules.
  • [0123]
    The peptides of Table 1.3 are characterized as follows. PEPTIDE 1.3.1 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 1.2.2. PEPTIDE 1.3.2 is a composite of the first two MHC Class II-presented epitopes (PEPTIDE 1.2.9; PEPTIDE 1.2.11), overlapping by two amino acids. PEPTIDE 1.3.3 is a composite of the first four MHC Class II-presented epitopes (PEPTIDE 1.2.11, PEPTIDE 1.2.9, PEPTIDE 1.2.10, PEPTIDE 1.2.12). PEPTIDES 1.3.2 and 1.3.3 are peptides with altered amino acid sequences in the recombinant, mutated Ara h 1 (Burks A W. Eur J Immunol. 1997 245:334-9). PEPTIDE 1.3.4 contains the ProPred-predicted MHC Class II-presented epitopes PEPTIDE 1.2.8. PEPTIDE 1.3.5 is the composite of two MHC Class II-predicted epitopes (PEPTIDE 1.2.7 and PEPTIDE 1.2.8), overlapping by six amino acids. PEPTIDE 1.3.6 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 1.2.13. PEPTIDE 1.3.7 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 1.2.14. PEPTIDE 1.3.8 is the composite of three MHC Class II-predicted epitopes (PEPTIDE 1.2.14, PEPTIDE 1.2.15 and PEPTIDE 1.2.16), overlapping by three and four amino acids, respectively.
  • [0000]
    TABLE 1.4
    Experimentally defined IgE-binding Ara h 1
    epitopes which overlap with predicted MHC Class
    II-presented Ara h 1 epitopes.
    PEPTIDE Pos. Sequence SEQ ID NO:
    1.4.1 409 NNFGKLFEVK 38
    1.4.2 311 SYLQEFSRNT 39
    1.4.3 498 RRYTARLKEG 40
    1.4.4 325 FNAEFNEIRR 41
    1.4.5 461 GTGNLELVAV 42

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0000]
    TABLE 1.5
    Ii-Key/Ara h 1 hybrids containing predicted MHC
    Class II Ara h 1 epitopes and experimentally
    determined IgE-binding Ara h 1 epitopes.
    SEQ ID
    PEPTIDE Pos. Sequence NO:
    1.5.1 416 Ac-LRMK-ava-NNFGKLFEVKPDKKNPQ- 43
    NH2
    1.5.2 312 Ac-LRMK-ava-LQGFSRNTL-NH2 44
    1.5.3 496 Ac-LRMK-ava-VRRYTARLK-NH2 45
    1.5.4 452 Ac-LRMK-ava-MVIVVVNKG-NH2 46
    1.5.5 456 Ac-LRMK-ava-VVNKGTGNL-NH2 47
    1.5.6 452 Ac-LRMK-ava- 48
    MVIVVVNKGTGNLELVAV-NH2
    1.5.7 324 Ac-LRMK-ava-FNAEFNEIR-NH2 49
    1.5.8 328 Ac-LRMK-ava-FNEIRRVLL-NH2 50
    1.5.9 334 Ac-LRMK-ava-VLLEENAGG-NH2 51
    1.5.10 324/ Ac-LRMK-ava- 52
    328/ FNAEFNEIRRVLLEENAGG-NH2
    334

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the proposed hybrid containing a predicted MHC Class II-presented epitope of Table 1.2 and an IgE binding epitope of Table 1.4. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0124]
    The PEPTIDES of Table 1.5 are characterized as follows. PEPTIDES 1.5.1, 1.5.6, and 1.5.10 include residues of an experimentally defined, IgE-binding epitope. PEPTIDES 1.5.1, 1.5.2, 1.5.4, 1.5.6, 1.5.9, and 1.5.10 have residues of a ProPred-predicted MHC Class II-presented epitopes. PEPTIDES 1.5.2, 1.5.3, 1.5.4, 1.5.5, 1.5.6, 1.5.7, 1.5.8 and 1.5.10 share amino acids between overlapping IgE binding and MHC Class II-presented epitopes. PEPTIDES 1.5.4, 1.5.5, 1.5.6, 1.5.8, 1.5.9, and 1.5.10 share amino acids between overlapping MHC Class II-presented epitopes.
  • [0125]
    The peptides of Table 1.5 are characterized as follows. PEPTIDE 1.5.1 is the composite of MHC Class II-presented epitope with the highest ProPred predictive binding score (PEPTIDE 1.2.1) and IgE binding epitope (PEPTIDE 1.4.1), overlapping by 2 amino acids. PEPTIDE 1.5.2 is the composite of MHC Class II-presented epitope SEQ ID NO 44 and IgE binding epitope PEPTIDE 1.4.2, overlapping by 8 amino acids. PEPTIDE 1.5.3 is the composite of MHC Class II-presented epitope PEPTIDE 1.2.6 and IgE binding epitope PEPTIDE 1.4.3, overlapping by 8 amino acids. PEPTIDE 1.5.4 contains MHC Class II-predicted epitope PEPTIDE 1.2.4 and an IgE binding epitope PEPTIDE 1.4.5, overlapping by 1 amino acid. PEPTIDE 1.5.5 contains MHC Class II-predicted epitope PEPTIDE 1.5 and an IgE binding epitope PEPTIDE 1.4.5, overlapping by 5 amino acids. PEPTIDE 1.5.6 is the composite of the two MHC Class II-predicted epitopes, PEPTIDE 1.2.4 and PEPTIDE 1.2.5, overlapping by 5 amino acids. Additionally, there is a 5 amino acids overlap with IgE binding epitope (PEPTIDE 1.4.5). PEPTIDE 1.5.7 contains MHC Class II-predicted epitope PEPTIDE 1.2.17 and an IgE binding epitope PEPTIDE 1.4.4, overlapping by 9 amino acids. PEPTIDE 1.5.8 contains MHC Class II-predicted epitope PEPTIDE 1.18 and an IgE binding epitope PEPTIDE 1.4.4, overlapping by 6 amino acids. PEPTIDE 1.5.9 contains MHC Class II-predicted epitope PEPTIDE 1.2.19. PEPTIDE 1.5.10 is the composite of the three MHC Class II-predicted epitopes PEPTIDES 1.2.17, 1.2.18, and 1.2.19 and IgE binding epitope PEPTIDE 3.1.5. PEPTIDE 1.5.5 is the composite of three MHC Class II-predicted epitopes (PEPTIDES 1.2.17, 1.2.18 and 1.2.19), overlapping by 5 and 3 amino acids, respectively. Additionally, there is a 9 amino acid overlap with IgE binding epitope (PEPTIDE 1.4.4).
  • Example 2 Ii-Key/Ara h 2 Peanut Antigenic Epitope Hybrids
  • [0126]
    In another aspect, this invention relates to the design of Ii-Key/Ara h 2 antigenic epitope hybrids. Sampson, WO 0052154, a series of Ara h 2 MHC Class II-presented epitopes, which had been experimentally identified by Burks A W. (J Allergy Clin Immunol. 1992 90:962-7). Ara h 2-specific T cell lines were established from the peripheral blood of 12 atopic and 4 nonatopic individuals. All of the T cell lines were predominantly CD 4+ T cells. Reactivity of each of these T cell lines was tested against individual peptides from a library of overlapping Ara h 2 peptides. Four immunodominant T cell epitopes were identified for Ara h 2: epitope 1 (amino acids 18-28), epitope 2 (amino acids 45-55), epitope 3 (amino acids 95-108), and epitope 4 (amino acids 134-144). Epitopes 1, 2, and 4 have overlapping sequences with IgE antibody-recognized epitopes while epitope 3 does not overlap IgE binding epitopes. Bannon and colleagues suggested that such sequences provide for the possibility for the development of a non-anaphylactic, T cell-directed immunotherapeutic (Bannon G A. Int Arch Allergy Immunol. 2001 124:70-72). The sequence of Ara h 2 in Table 2.1 was taken from GenBank gi/15418705/allergen II [Arachis hypogaea]. Experimentally defined MHC Class II-presented Ara h 2 epitopes are listed in Table 2.2. Ii-Key/Ara h 2 hybrids containing some of the experimentally defined MHC Class II-presented Ara h 2 epitopes of Table 2.2 are listed in Table 2.3. Predicted MHC Class II epitopes of Ara h 2 are listed in Table 2.4. Ii-Key/Ara h 2 hybrids containing some of the predicted MHC Class II-presented Ara h 2 epitopes of Table 2.4 are listed in Table 2.5. Experimentally defined IgE-binding Ara h 2 epitopes, which overlap with predicted MHC Class II-presented Ara h 2 epitopes from Table 2.4 are listed in Table 2.6. Hybrids containing predicted MHC Class II Ara h 2 epitopes and overlapping experimentally determined IgE-binding Ara h 2 epitopes are listed in Table 2.7.
  • [0000]
    TABLE 2.1
    Deduced amino acid sequence of Ara h 2.
      1 makltilval alfllaahas arqqwelqgd rrcqsqlera nlrpceqhlm (SEQ ID NO: 53)
     51 qkiqrdedsy erdpyspsqd pyspspydrr gagssqhqer ccnelnefen
    101 nqrcmcealq qimenqsdrl qgrqqeqqfk relrnlpqqc glrapqrcdl
    151 dvesgg
  • [0000]
    TABLE 2.2
    Experimentally defined MHC Class II-presented
    Ara h 2 epitopes.
    SEQ ID
    PEPTIDE Pos. Sequence Ii-Key NO:
    2.2.1  22 RQQWE LQGDRRCQSQ 3 54
    2.2.2  42 LRPCEQHLMQKIQRDEDSYE 55
    2.2.3   7 HQERCCNELN 56
    2.2.4 102 QRCMCEALQQ 57
    2.2.5 137 PQQCGLRAPQ 58

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of an experimentally determined MHC Class II-presented epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0127]
    The PEPTIDES of Table 2.2 are characterized as follows. PEPTIDES 2.2.1, 2.2.2, 2.2.4, and 2.2.5 are ProPred-predicted MHC Class II-presented sequences. PEPTIDE 2.2.1 contains an IgE binding epitopes. PEPTIDES 2.2.1 and 2.2.2 have overlapping amino acids of the IgE binding epitope and MHC Class II-presented epitope. Pos. is the residue number in the primary amino acid sequence of the first amino acid of the epitope. Many of the experimentally predicted epitopes are also predicted with the ProPred algorithm, either entirely or partially.
  • [0000]
    TABLE 2.3
    Ii-Key/Ara h 2 hybrids containing some of the
    experimentally defined MHC Class II-presented
    Ara h 2 epitopes of Table 2.2.
    SEQ
    ID
    PEPTIDE Pos. Sequence NO:
    2.3.1  19 Ac-LRMK-ava-RQQWE LQGDRRCQSQ-NH2 59
    2.3.2  39 Ac-LRMK-ava- 60
    LRPCEQHLMQKIQRDEDSYE-NH2
    2.3.3  84 Ac-LRMK-ava-HQERCCNELN-NH2 61
    2.3.4  99 Ac-LRMK-ava-QRCMCEALQQ-NH2 62
    2.3.5 135 Ac-LRMK-ava-PQQCGLRAPQ-NH2 63

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 2.2. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0128]
    The PEPTIDES of Table 2.3 are characterized as follows. PEPTIDE 2.3.1 contains an experimentally defined, IgE-binding epitope. PEPTIDES 2.3.1 and 2.3.2 share amino acids between overlapping IgE binding and MHC Class II-presented epitopes. PEPTIDES 2.3.2 and 2.3.3 are peptides with altered amino acid sequences in the modified Ara h 1 of Burks and colleagues (Burks A W. Eur J Immunol. 1997 245:334-9). Pos. is the residue number in the primary amino acid sequence of the first amino acid of the epitope.
  • [0000]
    TABLE 2.4
    Predicted MHC Class II epitopes of Ara h 2.
    SEQ ID
    PEPTIDE Pos. Sequence Score Ii-key NO:
    2.4.1   5 I L V A L A L F L 6.10 64
    2.4.2  26 L Q G D R R C Q S 5.80 8 65
    2.4.3   3 L T I L V A L A L 5.30 66
    2.4.4  49 L M Q K I Q R D E 4.10 67
    2.4.5  12 L F L L A A H A S 3.30 4 68
    2.4.6   7 L V A L A L F L L 4.70 69
    2.4.7  42 L R P C E Q H L M 3.60 70
    2.4.8  10 L A L F L L A A H 3.30 2 71
    2.4.9 133 L R N L P Q Q C G 2.70 72
    2.4.10  37 L E R A N L R P C 2.20 73
    2.4.11  13 F L L A A H A S A 1.90 5 74
    2.4.12  77 Y D R R G A G S S 1.90 75
    2.4.13  98 F E N N Q R C M C 1.70 76

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0129]
    The PEPTIDES of Table 2.4 are characterized as follows. PEPTIDES 2.4.1, 2.4.5, 2.4.6, 2.4.8, and 2.4.11 are peptides not preserved in an Ara h 2 modified to decrease allergic IgE binding. In PEPTIDE 2.4.4 R54 is replaced by A. In PEPTIDE 2.4.7 P43 and Q46 are each replaced by A. PEPTIDES 2.4.2, 2.4.4, 2.4.9, 2.4.10, and 2.4.13 are experimentally defined T cell epitopes. PEPTIDES 2.4.2, 2.4.4, 2.4.7, 2.4.10, and 2.4.11 have amino acids of an IgE binding epitope.
  • [0000]
    TABLE 2.5
    Ii-Key/Ara h 2 hybrids containing some of the
    predicted MHC Class II-presented Ara h 2
    epitopes of Table 2.4.
    SEQ
    ID
    PEPTIDE Pos. Sequence NO:
    2.5.1   5 Ac-LRMK-ava-ILVALALFL-NH2 77
    2.5.2   3 Ac-LRMK-ava-LTILVALAL-NH2 78
    2.5.3   6 Ac-LRMK-ava-LVALALFLL-NH2 79
    2.5.4   3/5/6 Ac-LRMK-ava-LTILVALALFLL-NH2 80
    2.5.5 132 Ac-LRMK-ava-LRNLPQQCG-NH2 81
    2.5.6  76 Ac-LRMK-ava-YDRRGAGSS-NH2 82
    2.5.7  97 Ac-LRMK-ava-FENNQRCMC-NH2 83

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 2.4. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0130]
    The PEPTIDES of Table 2.5 are characterized as follows. PEPTIDES 2.5.1, 2.5.2, 2.5.3, 2.5.4 are peptides not preserved in the modified Ara h 2. PEPTIDES 2.5.5 and 2.5.7 are experimentally defined CD4+ T cell epitopes.
  • [0131]
    In another aspect, this invention provides for the immunodeviation of an allergic patient's antibody response from an IgE pattern to an IgG or IgG subtype pattern. The decrease synthesis of IgE antibodies to the allergen and/or the synthesis of IgG antibodies, which block the binding of IgE antibodies, has a desired therapeutic effect. To this end MHC Class II epitopes of the allergen are joined with an IgE binding peptide sequence in an Ii-Key/MHC Class II epitope/IgE epitope hybrid peptide. The sequences so combined may be taken from different segments of the primary amino acid sequence of the allergen. For example, a MHC Class II epitope with a high ProPred score can be coupled to a peptide from an IgE-recognized site on the allergen. Preferably, however, those two respective MHC Class II-presented and IgE-recognized sites overlap in the primary sequence of the allergen. Such hybrids combining MHC Class II-presented Ara h2 2 epitopes from Table 2.4 and experimentally determined IgE binding epitopes of Table 2.6 are presented in Table 2.7.
  • [0000]
    TABLE 2.6
    Experimentally defined IgE-binding Ara h 2
    epitopes, which overlap with predicted MHC
    Class II-presented Ara h 2 epitopes from
    Table 2.4.
    PEPTIDE Pos. SEQUENCE Ii-Key SEQ ID NO:
    2.6.1  18 HASARQQWEL 10 84
    2.6.2  24 QWELQGDRRC  5 85
    2.6.3  30 DRRCQSQLER 11 86
    2.6.4  42 LRPCEQHLMQ 87
    Θ
    2.6.5  52 KIQRDEDSYE 88
    2.6.6 130 KRELRNLPQQ 89

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of an experimentally determined IgE binding epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0000]
    TABLE 2.7
    Hybrids containing predicted MHC Class II Ara h
    2 epitopes and overlapping experimentally
    determined IgE-binding Ara h 2 epitopes.
    SEQ
    ID
    PEPTIDE Pos. Sequence NO:
    2.7.1 26 Ac-LRMK-ava-LQGDRRCQS-NH2 90
    2.7.2 48 Ac-LRMK-ava-LMQKIQRDE-NH2 91
    2.7.3 41 Ac-LRMK-ava-LRPCEQHLM-NH2 92
    2.7.4 41/48 Ac-LRMK-ava-LRPCEQHLMOKIQRDE-NH2 93
    2.7.5 36 Ac-LRMK-ava-LERANLRPCEQHLMQΘ-NH2 94
    2.7.6 12 Ac-LRMK-ava-FLLAAHASARQQWEL-NH2 95

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 2.4 and an IgE binding epitope of Table 2.6. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0132]
    The PEPTIDES of Table 2.7 are characterized as follows. PEPTIDE 2.7.1 contains predicted and experimentally defined MHC Class II epitope PEPTIDE 7.2, which coincides with IgE binding epitope PEPTIDES 2.6.2 and 2.6.3. PEPTIDE 2.7.2 contains predicted and experimentally defined MHC Class II epitope PEPTIDE 7.4, which coincides with IgE binding epitope PEPTIDES 2.6.4 and 2.6.5. PEPTIDE 2.7.3 contains predicted MHC Class II epitope PEPTIDE 7.7, which coincides with IgE binding epitope PEPTIDE 2.6.4. PEPTIDE 2.7.4 contains MHC Class II epitopes PEPTIDE 7.4 and 7.7, and IgE binding epitopes PEPTIDE 2.6.4 and 2.6.5. PEPTIDE 2.7.5 contains MHC Class II epitope PEPTIDE 7.10 overlapping with IgE binding epitope PEPTIDE 2.6.4. PEPTIDE 2.7.6 contains MHC Class II epitope PEPTIDE 7.11, overlapping with IgE binding epitope PEPTIDE 2.6.1.
  • Example 3 Ii-Key/Ara h 3 Peanut Antigenic Epitope Hybrids
  • [0133]
    In another aspect, this invention relates to the design of Ii-Key/Ara h 3 antigenic epitope hybrids. Rabjohn et al. reported the molecular cloning and T cell epitope analysis of the peanut allergen Ara h3 (J Clin Invest. 1999 103:535-42). The sequence of Ara h 3 in Table 3.1 was taken from GenBank gi/3703107/glycin. Predicted MHC Class II epitopes of Ara h 3 are listed in Table 3.2. Ii-Key/Ara h 3 hybrids containing some of the MHC Class II-presented epitopes of Table 3.2 are listed in Table 3.3. Experimentally defined IgE-binding Ara h 3 epitope, overlapping with a predicted MHC Class II-presented Ara h 3 epitope are listed in Table 3.4. Hybrids containing predicted MHC Class II Ara h 3 epitopes overlapping experimentally defined IgE-binding epitopes Table 3.5.
  • [0000]
    TABLE 3.1
    Deduced amino acid sequence of Ara h 3.
      1 rqqpeenacq fqrlnaqrpd nrieseggyi etwnpnnqef ecagvalsrl (SEQ ID NO: 96)
     51 vlrrnalrrp fysnapqeif iqqgrgyfgl ifpgcprhye ephtqgrrsq
    101 sqrpprrlqg edqsqqqrds hqkvhrfdeg dliavptgva fwlyndhdtd
    151 vvavsltdtn nndnqldqfp rrfnlagnte qeflryqqqs rqsrrrslpy
    201 spyspqsqpr qeerefsprg qhsrreragq eeeneggnif sgftpefleq
    251 afqvddrqiv qnlrgetese eegaivtvrg glrilspdrk rradeeeeyd
    301 edeyeydeed rrrgrgsrgr gngieetict asakknigrn rspdiynpqa
    351 gslktandln llilrwlgps aeygnlyrna lfvahyntna hsiiyrlrgr
    401 ahvqvvdsng nrvydeelqe ghvlvvpqnf avagksqsen feyvafktds
    451 rpsianlage nsvidnlpee vvansyglqr eqarqlknnn pfkffvppsq
    501 qsprava
  • [0000]
    TABLE 3.2
    Predicted MHC Class II epitopes of Ara h 3.
    SEQ ID
    PEPTIDE Pos. Sequence Score Ii-Key NO:
    3.2.1 395 Y R L R G R A H V 6.10 -  97
    3.2.2 393 I I Y R L R G R A 4.70  6  98
    3.2.3 446 F K T D S R P S I 5.70  99
    3.2.4 278 V R G G L R I L S 5.40 100
    3.2.5 274 I V T V R G G L R 5.00 10 101
    3.2.6 282 L R I L S P D R K 4.70 102
    3.2.7 252 F Q V D D R Q I V 5.20 103
    3.2.8 364 L R W L G P S A E 5.00 104
    3.2.9 362 L I L R W L G P S 4.80 105
    3.2.10 173 F N L A G N T E Q 4.80 106
    3.2.11 424 L V V P Q N F A V 4.70 107
    3.2.12 403 V Q V V D S N G N 4.50  4 108
    3.2.13 405 V V D S N G N R V 4.10  6 109
    3.2.14 382 F V A H Y N T N A 4.40 110

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0000]
    TABLE 3.3
    Ii-Key/Ara h 3 hybrids containing some of the
    MHC Class II-presented epitopes of Table 3.2.
    SEQ ID
    PEPTIDE Pos. Sequence NO:
    3.3.1 393 Ac-LRMK-ava-IIYRLRGRA-NH2 111
    3.3.2 395 Ac-LRMK-ava-YRLRGRAHV-NH2 112
    3.3.3 393/395 Ac-LRMK-ava-IIYRLRGRAHV-NH2 113
    3.3.4 446 Ac-LRMK-ava-FKTDSRPSI-NH2 114
    3.3.5 362 Ac-LRMK-ava-LILRWLGPS-NH2 115
    3.3.6 364 Ac-LRMK-ava-LRWLGPSAE-NH2 116
    3.3.7 362/364 Ac-LRMK-ava-LILRWLGPSAE-NH2 117
    3.3.8 252 Ac-LRMK-ava-FQVDDRQIV-NH2 118
    3.3.9 173 Ac-LRMK-ava-FNLAGNTEQ-NH2 119
    3.3.10 424 Ac-LRMK-ava-LVVPQNFAV-NH2 120
    3.3.11 403 Ac-LRMK-ava-VQVVDSNGN-NH2 121
    3.3.12 405 Ac-LRMK-ava-VVDSNGNRV-NH2 122
    3.3.13 403/405 Ac-LRMK-ava-VQVVDSNGNRV-NH2 123
    3.3.14 382 Ac-LRMK-ava-FVAHYNTNA-NH2 124

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 3.2.
  • [0134]
    PEPTIDE 3.3.1 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 3.2.2. PEPTIDE 3.3.2 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 3.2.1. PEPTIDE 3.3.3 is a composite of two ProPred-predicted MHC Class II-presented epitopes (PEPTIDES 3.2.1 and 3.2.2), overlapping by 7 amino acids. PEPTIDE 3.3.4 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 3.2.3. PEPTIDE 3.3.5 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 3.3.6. PEPTIDE 3.2.6 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 3.2.8. PEPTIDE 3.3.7 is a composite of two ProPred-predicted MHC Class II-presented epitopes (PEPTIDES 3.2.9 and 3.2.8), overlapping by 7 amino acids. PEPTIDE 3.3.8 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 3.2.7. PEPTIDE 3.3.9 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 3.2.10. PEPTIDE 3.3.10 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 3.2.11. PEPTIDE 3.3.11 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 3.2.11. PEPTIDE 3.3.12 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 3.2.13. PEPTIDE 3.3.13 is a composite of two ProPred-predicted MHC Class II-presented epitopes (PEPTIDES 3.2.12 and 3.2.13), overlapping by 7 amino acids. PEPTIDE 3.3.14 contains the ProPred-predicted MHC Class II-presented epitope PEPTIDE 3.2.14.
  • [0000]
    TABLE 3.4
    Experimentally defined IgE-binding Ara h 3
    epitope, overlapping with a predicted MHC Class
    II-presented Ara h 3 epitope.
    PEPTIDE Pos. Sequence Ii-Key SEQ ID NO:
    3.4.1 276 VTVRGGLRILSPDRK 11 125

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0000]
    TABLE 3.5
    Hybrids containing predicted MHC Class II Ara h
    3 epitopes overlapping experimentally defined
    IgE-binding epitopes.
    SEQ
    ID
    PEPTIDE POS. Sequence NO:
    3.5.1 274 Ac-LRMK-ava-IVTVRGGLR-NH2 126
    3.5.2 277 Ac-LRMK-ava-VRGGLRILS-NH2 127
    3.5.3 281 Ac-LRMK-ava-IVTVRGGLRILSPDRK-NH2 128
    3.5.4 274/ Ac-LRMK-ava-IVTVRGGLRILSPDRK-NH2 129
    277/
    281

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 3.2 and an IgE binding epitope of Table 3.4.
  • [0135]
    The PEPTIDES of Table 3.5 are characterized as follows. PEPTIDES 3.5.1-3.5.4 share amino acids between IgE binding and MHC Class II-presented epitopes. PEPTIDE 3.5.1 contains the predicted MHC Class II epitope PEPTIDE 3.2.5, which coincides with experimentally defined IgE binding epitope PEPTIDE 13.1. PEPTIDE 3.5.2 contains the predicted MHC Class II epitope PEPTIDE 3.5.4, which coincides with experimentally defined IgE binding epitope PEPTIDE 3.4.1. PEPTIDE 3.4.3 contains the predicted MHC Class II epitope PEPTIDE 3.2.6, which coincides with experimentally defined IgE binding epitope PEPTIDE 3.4.1. PEPTIDE 3.4.4 is a composite of three ProPred-predicted MHC Class II-presented epitopes (PEPTIDE 3.2.4, 3.2.5 and 3.2.6), which coincide with IgE binding epitope PEPTIDE 3.4.1.
  • Example 4 Ii-Key/Fel d 1 Cat Dander Antigenic Epitope Hybrids
  • [0136]
    In another aspect, this invention relates to the design of Ii-Key/Fel d 1 antigenic epitope hybrids. Such Ii-Key/Fel d 1 antigenic epitope hybrids comprise the Ii-Key motif LRMK (SEQ ID NO: 3) and modifications, joined through a functionally acceptable linker to a MHC Class II-presented epitopes of the Fl d 1 major allergen protein of cat dander. The amino acid sequences of Fel d 1 chains 1 and 2 in Table 4.1 were taken from GenBank gi/1082945/chain 1 and gi/1082946/chain 2 (Morgenstern J P. Proc Natl Acad Sci USA. 1991 88:9640-4). The MHC Class II-presented epitopes of Fel d 1 (chain 1) listed in Table 4.2 include those with the highest scores in the ProPred program analysis. Table 4.3 presents Ii-Key/Fel d 1 (chain 1) hybrids containing some of the predicted MHC Class II-presented epitopes of Table 2. Table 4.4 presents Fel d 1 (chain 1) MHC Class II-presented peptides which elicit allergic responses in cat dander-atopic humans (Haselden B M. J Exp Med. 1999 189: 1885-94). Table 4.5 presents Ii-Key/Fel d 1 (chain 1) hybrids containing some of the experimentally defined MHC Class II-presented epitopes of Table 4.4. Table 4.6 presents predicted MHC Class II-presented epitopes of Fel d 1 (chain 2). Table 4.7 presents designed Ii-Key/Fel d 1 (chain 2) hybrids containing some of the MHC Class II-presented epitopes of Table 4.6. Table 4.8 presents Fel d 1 (chain 2) MHC Class II-presented peptides which elicit allergic responses in cat dander-atopic humans (Haselden B M. J Exp Med. 1999 189: 1885-94). Table 4.9. presents designed Ii-Key/Fel d 1 (chain 2) hybrids containing some of the MHC Class II-presented epitopes of Table 4.8. Since some of the epitope peptides alone can induce hyporesponsiveness to cat dander allergen challenge in the clinic (Oldfield W L. J Immunol. 2001 167:1734-9; Mazzarella G. Allergy 2000 61:6-9), the corresponding hybrids will be more potent and less susceptible to induce anaphylaxis during clinical testing or therapy. Methods for such analyses and therapy are presented in Larche M. WO99/34826 and U.S. Pat. No. 6,120,769 (2000), which are incorporated herein by reference.
  • [0137]
    Asthma is a complex inflammatory disease of the lung characterized by variable airflow obstruction, bronchial hyper-responsiveness, and airway inflammation. Inflammation in asthma consists of airway infiltration by mast cells, lymphocytes, and eosinophils. There is accumulating evidence that CD4+ cells with a Th2-cytokine pattern play a pivotal role in the pathogenesis of asthma. These cells orchestrate the recruitment and activation of the primary effector cells of the allergic response (mast cells and eosinophils), through the release of cytokines such as IL-4, IL-5, and IL-13. Allergic inflammation is also implicated in airway epithelium changes, although the mechanisms by which inflammatory cells and, in particular, T cells interact with the epithelium are not completely clarified.
  • [0138]
    Treatment of mice in an ovalbumin-induced asthmatic response with superagonistic Th1-skewing peptide 336E-A (ISQAVHAAHAEINAAGR) (SEQ ID NO: 130) resulted in a Th1-like cytokine profile and a significant decrease in airway eosinophilia and OVA-specific IL-4 and IL-5 production (Janssen E. J Immunol. 2000 164:1580-8; Janssen E M. J Immunol. 2000 165:7207-14). In these studies the wild type sequence (ISQAVHAAHAEINEAGR) (SEQ ID NO: 131) was modified in homologs each with a single alanine substitutions at all non-alanine residue positions.
  • [0139]
    In extension of the principles of this study Pene et al. examined the effects of immunotherapy with Fel d 1 peptides on the response to bronchial provocation tests with a standardized Fel d 1 cat extract on Fel d 1-specific serum IgE and IgG levels and in vitro IL-4 and IFN-ã production (Pene J. J Allergy Clin Immunol. 1998; 102:571-8). Patients allergic to cats received 6 weekly injections of low dose, medium dose, or high dose of Fel d 1 peptides or a placebo. Six weeks after ending immunotherapy, posttreatment PD20Forced Expiratory Volume/1sec was not significantly different between the treated and placebo groups. However, in the medium- and high-dose groups there was a significant improvement between baseline and posttreatment days. IL-4 release was significantly reduced in the high dose-treated group whereas it was unchanged in the low or medium dose- and in the placebo-treated groups. In all groups, IFN-γ, IgE, and IgG levels remained unchanged. The investigators concluded there was no correlation between the improvement of PD20FEV1 and the decrease in IL-4 production. They suggested that peptide immunotherapy might act by shifting the Fel d 1-induced response of peripheral blood mononuclear cells in vitro from the Th2-like to the Th0-like phenotype. The Ii-Key/antigenic epitope hybrids presented below include some of the experimentally tested MHC Class II epitopes of these investigators and can be predicted to be of greater potency and have a wider margin of safety in diagnostic and therapeutic applications, for reasons presented previously.
  • [0140]
    The preceding studies followed the preparation of ALLERVAX CAT, a peptide vaccine containing two peptides of 27 amino acids containing regions of multiple MHC Class II-presented epitopes from the Felis domesticus cat allergen Fel d1 chain I were produced FC1P1, LFLTGTPDEYVEQVAQY (SEQ ID NO: 132); FC1P2, EQVAQYKALPVVLENA (SEQ ID NO: 133); and FC1P3, KALP-VVLENARILKNCV (SEQ ID NO: 134).
  • [0141]
    The deduced amino acid sequence of Fel d 1 Chain 1 and Chain 2 are presented in Table 4.1 (>gi|1082945|pir∥B56413 major allergen Fel dI chain 1 short form—cat; >gi|1082946|pir∥C56413 major allergen Fel dI chain 2 precursor—cat). Predicted MHC Class-II-presented epitopes of Fel d 1 (chain 1) are listed in Table 4.3. Designed Ii-Key/Fel d 1 (chain 1) hybrids containing some of the predicted MHC Class II-presented epitopes of Table 4.2 are listed in Table 4.3. Experimentally defined Fel d 1 chain 1 MHC Class II-presented epitopes are listed in Table 4.4. Designed Ii-Key/Fel d 1 (chain 1) hybrids containing some of the MHC Class II-presented epitopes of Table 4.4 are listed I Table 4.5. Predicted MHC Class II-presented epitopes of Fel d 1 (chain 2) are listed in Table 4.6. Designed Ii-Key/Fel d (chain 2) hybrids containing some of the MHC Class II-presented epitopes of Table 4.6 are listed in Table 4.7. Experimentally determined/Fel d 1 (chain 2) MHC Class II-presented epitopes are listed in Table 4.8. Designed Ii-Key/Fel d 1 (chain 2) hybrids containing some of the MHC Class II-presented epitopes of Table 4.8 are listed in Table 4.9.
  • [0000]
    TABLE 4.1
    Deduced amino acid sequence of Fel d 1.
    Chain 1.
    >gi|1082945|pir||B56413 major allergen Fel dI chain 1 short
    form - cat (SEQ ID NO: 135)
      1 mldaalppcp tvaatadcei cpavkrdvdl fltgtpdeyv eqvaqykalp
     51 vvlenarilk ncvdakmtee dkenalslld kiytsplc
    Chain 2.
    >gi|1082946|pir||C56413 major allergen Fel dI chain 2
    precursor - cat (SEQ ID NO: 136)
      1 mrgallvlal lvtqalgvkm aetcpifydv ffavangnel lldlsltkvn
     51 atepertamk kiqdcyveng lisrvldglv mttissskdc mgeavqntve
    101 dlklntlgr
  • [0000]
    TABLE 4.2
    Predicted MHC ClassII-presented epitopes of Fel
    d 1 (chain 1).
    SEQ ID
    PEPTIDE Pos. Sequence Score Ii-Key NO:
    4.2.1 46 Y K A L P V V L E 4.20 137
    4.2.2 51 V V L E N A R I L 4.80 138
    4.2.3 43 V A Q Y K A L P V 4.70 139
    4.2.4 39 Y V E Q V A Q Y K 3.60  8 140
    4.2.5 24 V K R D V D L F L 3.4 141
    4.2.6 20 I C P A V K R D V 3.1 142
    4.2.7 76 L S L L D K I Y T 2.70  8 143
    4.2.8 79 L D K I Y T S P L 2.52 11 144
    4.2.9 30 L F L T G T P D E 2.40 145

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0000]
    TABLE 4.3
    Designed Ii-Key/Fel d 1 (chain 1) hybrids
    containing some of the predicted MHC Class II-
    presented epitopes of Table 4.2.
    SEQ
    ID
    PEPTIDE Pos. Sequence NO:
    4.3.1 43 Ac-LRMK-ava-VAQYKALPV-NH2 146
    4.3.2 46 Ac-LRMK-ava-YKALPVVLE-NH2 147
    4.3.3 51 Ac-LRMK-ava-VVLENARIL-NH2 148
    4.3.4 39 Ac-LRMK-ava-YVEQVAQYK-NH2 149
    4.3.5 39/43/46/51 Ac-LRMK-ava- 150
    YVEQVAQYKALPVVLENARIL-NH2
    4.3.6 20 Ac-LRMK-ava-ICPAVKRDV-NH2 151
    4.3.7 24 Ac-LRMK-ava-VKRDVDLFL-NH2 152
    4.3.8 30 Ac-LRMK-ava-LFLTGTPDE-NH2 153
    4.3.9 20/24/30 Ac-LRMK-ava- 154
    ICPAVKRDVDLFLTGTPDE-NH2
    4.3.10 76 Ac-LRMK-ava-LSLLDKIYT-NH2 155
    4.3.11 79 Ac-LRMK-ava-LDKIYTSPL-NH2 156
    4.3.12 76/79 Ac-LRMK-ava-LSLLDKIYTSPL- 157
    NH2

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0000]
    TABLE 4.4
    Experimentally defined Fel d 1 chain 1 MHC
    Class II-presented epitopes.
    PEPTIDE Pos. Sequence Ii-Key SEQ ID NO:
    4.4.1 20 EICPAVKRDVDLFLTGT 158
    4.4.2 30 LFLTGTPDEYVEQVAQY 159
    4.4.3 41 EQVAQYKALPVVLENA 10 160
    4.4.4 47 KALPVVLENARILKNCV 161
    4.4.5 57 RILKNCVDAKMTEEDKE  5 162
    4.4.6 66 KMTEEDKENALSLLDK  2 163
    4.4.7 72 KENALSVLDKIYTSPL 164

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of certain peptides found to elicit responses in patients with allergy to cat dander (Haselden B M. J Exp Med. 1999 189: 1885-94). Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope. PEPTIDE 4.4.2 is from FC1P1; PEPTIDE 4.4.3 is from FC1P2; PEPTIDE 4.4.4 is from FC1P3 (Haselden B M. J Exp Med. 1999 189:1885-94).
  • [0000]
    TABLE 4.5
    Designed Ii-Key/Fel d 1 (chain 1) hybrids
    containing some of the MHC Class II-presented
    epitopes of Table 4.4.
    SEQ
    ID
    PEPTIDE Pos. Sequence NO:
    4.5.1 30 Ac-LRMK-ava-LFLTGTPDEYVEQVAQY- 165
    NH2
    4.5.2 41 Ac-LRMK-ava-EQVAQYKALPVVLENA- 166
    NH2
    4.5.3 47 Ac-LRMK-ava-KALPVVLENARILKNCV- 167
    NH2
    4.5.4 57 Ac-LRMK-ava-RILKNCVDAKMTEEDKE- 168
    NH2
    4.5.5 41/47/57 Ac-LRMK-ava- 169
    QVAQYKALPVVLENARILKNCVDAKMTEED
    KE-NH2

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0000]
    TABLE 4.6
    Predicted MHC Class II-presented epitopes of
    Fel d 1 (chain 2)
    PEPTIDE Pos. Sequence Score Ii-Key SEQ ID NO:
    4.6.1 10 LLVTQALGV 6.10  3 170
    4.6.2 40 LLLDLSLTK 4.60 171
    4.6.3 79 LVMTTISSS 4.37 172
    4.6.4 18 VKMAETCPI 4.80 11 173
    4.6.5  5 LVLALLVTQ 4.50 174
    4.6.6  4 LLVLALLVT 3.90 175

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0000]
    TABLE 4.7
    Designed Ii-Key/Fel d (chain 2) hybrids
    containing some of the MHC Class II-presented
    epitopes of Table 4.6.
    SEQ
    ID
    PEPTIDE Pos. Sequence NO:
    4.7.1  9 Ac-LRMK-ava-LLVTQALGV-NH2 176
    4.7.2  5 Ac-LRMK-ava-LVLALLVTQ-NH2 177
    4.7.3  4 Ac-LRMK-ava-LLVLALLVT-NH2 178
    4.7.4  4/5/9 Ac-LRMK-ava-LLVLALLVTQALGV-NH2 179
    4.7.5 17 Ac-LRMK-ava-VKMAETCPI-NH2 180
    _4.7.6 39 Ac-LRMK-ava-LLLDLSLTK-NH2 181
    4.7.7 78 Ac-LRMK-ava-LVMTTISSS-NH2 182

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope.
  • [0000]
    TABLE 4.8
    Experimentally determined Fel d 1 (chain 2) MHC
    Class II-presented epitopes.
    PEPTIDE Pos. Sequence Ii-Key SEQ ID NO:
    4.8.1 46 LTKVNATEPERTAMKK 183
    4.8.2 57 TAMKKIQDCYVENGLI 6 184
    4.8.3 65 CYVENGLISRVLDGLV 185
    4.8.4 84 ISSSKDCMGEAVQNTV 5 186
    4.8.5 94 AVQNTVEDLKLNTLGR 187

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of an experimentally determined MHC Class II-presented epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0000]
    TABLE 4.9
    Designed Ii-Key/Fel d 1 (chain 2) hybrids
    containing some of the MHC Class II-presented
    epitopes of Table 4.8.
    SEQ
    ID
    PEPTIDE Pos. Sequence NO:
    4.9.1 46 Ac-LRMK-ava-LTKVNATEPERTAMKK-NH2 188
    4.9.2 57 Ac-LRMK-ava-TAMKKIQDCYVENGLI-NH2 189
    4.9.3 65 Ac-LRMK-ava-CYVENGLISRVLDGLV-NH2 190
    4.9.4 84 Ac-LRMK-ava-ISSSKDCMGEAVQNTV-NH2 191
    4.9.5 57/65 Ac-LRMK-ava- 192
    TAMKKIQDCYVENGLISRVLDGLV-NH2

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope.
  • Example 5 Ii-Key/Phl p 1 Pollen Antigenic Epitope Hybrids
  • [0142]
    In another aspect this invention relates to the design and use of Ii-Key/Phl p 1 pollen antigenic epitope hybrids. Laffer and colleagues obtained the cDNA for the major allergen Phl p I from timothy grass (Phleum pratense) and found that the recombinant protein Phl p I inhibits IgE binding to group I allergens prepared form eight different grass species (Laffer S. J Allergy Clin Immunol. 1994 94:689-98). In a study of the T-cell epitopes of Phl p 1, major pollen allergen of timothy grass (Phleum pratense) Schenk S, and colleagues found evidence for crossreacting and non-crossreacting T-cell epitopes within grass group I allergens (Schenk S. J Allergy Clin Immunol. 1995 96:986-96). Immunological characterization of various purified recombinant timothy grass pollen (Phleum pratense) allergens (Phl p 1, Phl p2, Phl p 5) were characterized with respect to such cross reactions (Vrtala S. J Allergy Clin Immunol. 1996 97:781-7). Various nonanaphylactic synthetic peptides were obtained from antibody-recognized epitopes of the major grass pollen allergen, Phl p 1, for allergy vaccination (Focke M. FASEB J. 2001 15:2042-4). Some of these epitopes are incorporated in the Ii-Key/MHC Class II epitope/IgG epitope hybrids of Tables 5.7. In related work, Blaher et al. identified MHC Class II-presented epitopes of Lol p 9, a major allergen of ryegrass (Lolium perenne) pollen (Blaher B. J Allergy Clin Immunol. 1996 98:124-32).
  • [0143]
    The sequence of Phl p I allergen [Phleum pratense] in Table 5.1 was taken from GenBank 473360, Phl p I allergen. Predicted MHC class-II-presented epitopes of Phl p 1 are listed in Table 5.2. Ii-Key/Phl p 1 hybrids containing some of the MHC Class II-presented Phl p 1 epitopes of Table 5.2 are listed in Table 5.3. Experimentally defined MHC Class II-presented epitopes of Phl p 1 are listed in Table 5.4. Ii-Key/Phl p 1 hybrids containing some of the experimentally defined MHC Class II-presented epitopes of Table 5.4 are listed in Table 5.5. Experimentally defined IgE-binding epitopes of Phl p 1 overlapping with MHC Class II-presented Phl p 1 epitopes are listed in Table 5.6. A hybrid peptide including an experimentally defined MHC Class II and IgE binding Php 1 epitope is listed in Table 5.7.
  • [0000]
    TABLE 5.1
    Deduced amino acid sequence of Phl p 1 pollen
    protein. (SEQ ID NO: 193)
      1 massssvllv vvlfavflgs aygipkvppg pnitatygdk wldakstwyg
     51 kptgagpkdn ggacgykdvd kppfsgmtgc gntpifksgr gcgscfeikc
    101 tkpeacsgep vvvhitddne epiapyhfdl sghafgamak kgdeqklrsa
    151 gelelqfrrv kckypegtkv tfhvekgsnp nylallvkyv ngdgdvvavd
    201 ikekgkdkwi elkeswgaiw ridtpdkltg pftvrytteg gtkteaedvi
    251 pegwkadtsy esk
  • [0000]
    TABLE 5.2
    Predicted MHC class II-presented epitopes of
    Phl P 1.
    PEPTIDE Pos. Sequence Score Ii-Key SEQ ID NO:
    5.2.1   0 MASSSSVLL 6.30 194
    5.2.2 220 WRIDTPDKL 5.86 5 195
    5.2.3   9 VVVLFAVFL 5.80 196
    5.2.4  10 VVLFAVLG 6.00 197
    5.2.5   6 VLLVVVLFA 5.10 198
    5.2.6  96 FEIKCTKPE 5.80 6 199
    5.2.7  15 VFLGSAYGI 4.80 200
    5.2.8 186 LVKYVNGDG 4.50 9 201
    5.2.9 185 LLVKYVNGD 4.50 8 202

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0000]
    TABLE 5.3
    Ii-Key/Phl p 1 hybrids containing some of the
    MHC Class II-presented Phl p 1 epitopes of
    Table 5.2.
    SEQ
    ID
    PEPTIDE Pos. Sequence NO:
    5.3.1   0 Ac-LRMK-ava-MASSSSVLL-NH2 203
    5.3.2   6 Ac-LRMK-ava-VLLVVVLFA-NH2 204
    5.3.3   9 Ac-LRMK-ava-VVVLFAVFL-NH2 205
    5.3.4  10 Ac-LRMK-ava-VVLFAVFLG-NH2 206
    5.3.5   0/6/9/10 Ac-LRMK-ava- 207
    MASSSSVLLVVVLFAVFLG-NH2
    5.3.6 219 Ac-LRMK-ava-WRIDTPDKL-NH2 208
    5.3.7  95 Ac-LRMK-ava-FEIKCTKPE-NH2 209
    5.3.8  15 Ac-LRMK-ava-VFLGSAYGI-NH2 210
    5.3.9 184 Ac-LRMK-ava-LLVKYVNGD-NH2 211
    5.3.10 185 Ac-LRMK-ava-LVKYVNGDG-NH2 212
    5.3.11 184/185 Ac-LRMK-ava-LLVKYVNGDG-NH2 213

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope.
  • [0000]
    TABLE 5.4
    Experimentally defined MHC Class II-presented
    epitopes of Phl p 1.
    Ii- SEQ ID
    PEPTIDE Pos. Sequence Key NO:
    5.4.1  96 F E I K C T K P E A C S 6 214
    5.4.2 123 I A P Y H F D L S G H A 5 215

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0144]
    The experimentally defined MHC Class II epitopes of Phl p 1, cross react within grass group I allergens—Lol p 1 (ryegrass, Lolium perenne), Sec c 1 (rye, secale cereale) (Schenk S. J Allergy Clin Immunol. 1995 96:986-96). Specifically the epitope of PEPTIDE 5.4.1 cross reacts with Lol p 1 (A97 is replaced by S97) and cross reacts with Sec c 1 (189 is replaced by L89). The epitope of PEPTIDE 5.4.2 cross reacts with Lol p 1 and sec c 1 (A124 is replaced by D124).
  • [0000]
    TABLE 5.5
    Ii-Key/Phl p 1 hybrids containing some of the
    experimentally defined MHC Class II-presented
    epitopes of Table 3.
    SEQ ID
    PEPTIDE Pos. Sequence NO:
    5.4.1  96 Ac-LRMK-ava-FEIKCTKPEACS-NH2 216
    5.4.2 123 Ac-LRMK-ava-IAPYHFDLSGHA-NH2 217

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0000]
    TABLE 5.6
    Experimentally defined IgE-binding epitopes of
    Phl p 1 overlapping with MHC Class II-presented
    Phl p 1 epitopes.
    SEQ
    ID
    PEPTIDE Pos. Sequence NO:
    5.6.1  24 IPKVPPG PNITATYGDK WLDAKSTWYG KPT 218
    5.6.2  65 GYKDVD KPPFSGMTGC GNTPIFKSGR G 219
    5.6.3 109 EP VVVHITDDNE EPIAPYHFDL SGHAFGAMA 220
    5.6.4 173 HVEKGSNP NYLALLVKYV NGDGDVVAV 221
    5.6.5 235 RYTTEG GTKTEAEDVI PEGWKADTSY ESK 222

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope. The peptides containing IgE-binding epitopes were defined by Focke and colleagues (Focke M. FASEB J. 2001 15:2042-4). PEPTIDE 5.6.3 includes experimentally defined MHC Class II epitopes of Phl p 1 (Schenk S. J Allergy Clin Immunol. 1995 96:986-96) overlapping with IgE epitope containing peptides (Focke M. FASEB J. 2001 15:2042-4).
  • [0000]
    TABLE 5.7
    A hybrid peptide including an experimentally
    defined MHC Class II and IgE binding Php 1
    epitope.
    SEQ ID
    PEPTIDE Pos. Sequence NO:
    5.7.1 109 Ac-LRMK-ava- 223
    FEIKCTKPEACSGEPVVVHITDDNE
    EPIAPYHFDLSGHAFGAMA-NH2

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope. PEPTIDE 5.7.1 includes Phl p 1 experimentally defined IgE epitopes (Focke M. FASEB J. 2001 15:2042-4) and experimentally defined MHC Class II epitopes of Phl p 1 (Schenk S. J Allergy Clin Immunol. 1995 96:986-96).
  • Example 6 Ii-Key/Phl p 5a Birch Pollen Antigenic Epitope Hybrids
  • [0145]
    In another aspect this invention relates to the design and use of Ii-Key/Phl p 5a birch pollen antigenic epitope hybrids. Multiple T cell epitopes on Bet v I, the major birch pollen allergen, have been determined using specific T cell clones and overlapping peptides (Ebner C. J Immunol. 1993 150:1047-54). Vrtala and colleagues found that the major birch pollen allergen, Bet v 1, can be divided into two fragments each of which contained nonanaphylactic T cell epitopes and are candidates for suppressive immunotherapy (Vrtala S. Int Arch Allergy Immunol. 1997 113:246-8; Vrtala S. J Clin Invest. 1997 99:1673-81). Friedl-Hajek R. and colleagues characterized a highly promiscuous, HLA allele-specific T-cell epitope in the birch major allergen Bet v 1Five Bet v 1-specific T cell clones derived from two birch pollen-allergic individuals and specific for Bet v 1 (Friedl-Hajek R. Clin Exp Allergy. 1999 29:478-87). One of these T cell clones reacted with a Bet v 1 peptide containing amino acid residues 21-33 (BP21), the other two T cell clones reacted with a minimal peptide containing residues 37-45 (BP37). While BP37-specific T cell clones were restricted by a HLA-DQA1*0301/DQB1*0603 heterodimer, BP21 was recognized in a highly promiscuous manner. T cell clones recognizing this sequence were restricted by HLA-DPB1*0201, a HLA-DQA1*0201/DQB1*0201 heterodimer, or HLA-DRB3*0101.
  • [0146]
    The sequence of Phl p 5a birch pollen protein in Table 6.1 was taken from GenBank 2851456 (Bufe A. J Allergy Clin Immuno. 1994 94:173-81). Predicted MHC Class II-presented epitopes of Phl p 5a are listed in Table 6.2. Experimentally defined, cross reacting MHC Class II isoepitopes of Phl p 5a and Phl p 5b are listed in Table 6.3 (Muller W. Clin Exp Allergy. 1998 28:1538-48). Designed Ii-Key/antigenic epitope hybrids containing Phl p 5 MHC Class II-presented epitopes are listed in Table 6.4.
  • [0000]
    TABLE 6.1
    Deduced amino acid sequence of Phl p 5a birch
    pollen. (SEQ ID NO: 224)
      1 adlgygpatp aapaagytpa tpaapagada agkatteeqk liekinagfk
     51 aalagagvqp adkyrtfvat fgpasnkafa eglsgepkga aessskaalt
    101 skldaaykla yktaegatpe akydayvatl sealriiagt levhavkpaa
    151 eevkvipage lqviekvdaa fkvaataana apandkftvf eaafndeika
    201 stggayesyk fipaleaavk qayaatvata pevkytvfet alkkaitams
    251 eaqkaakpaa aatatataav gaatgaataa tggykv
  • [0000]
    TABLE 6.2
    Predicted MHC Class II-presented epitopes of
    Phl p 5a.
    Ii- SEQ ID
    PEPTIDE Pos. Sequence Score Key NO:
    6.2.1 126 Y V A T L S E A L 8.40 225
    6.2.2 153 V K V I P A G E L 5.10 5 226
    6.2.3 134 L R I I A G T L E 5.00 227
    6.2.4 209 Y K F I P A L E A 4.80 228
    6.2.5 206 Y E S Y K F I P A 4.00 229
    6.2.6 171 F K V A A T A A N 4.10 2 230
    6.2.7  64 Y R T F V A T F G 4.00 231

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0000]
    TABLE 6.3
    Experimentally defined, cross reacting MHC
    Class II isoepitopes of Phl p 5a and Phl p 5b.
    Ii- SEQ ID
    PEPTIDE Pos. Sequence Key NO:
    6.3.1 209 Y K F I P A L E A A V K 232
    6.3.2 161 L Q V I E K V D A A F K 2 233

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of an experimentally determined MHC Class II-presented epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope. PEPTIDE 6.3.1 corresponds to peptide Phl p 5b(184-195; YKCIPSLEAAVK) (SEQ ID NO: 234) and PEPTIDE 6.3.2 corresponds to peptide Phl p 5b(136-147; LQIIDKIDAAFK (SEQ ID NO: 235) (Muller W. Clin Exp Allergy. 1998 28:1538-48).
  • [0000]
    TABLE 6.4
    Hybrids containing Phl p 5 MHC Class II-
    presented epitopes.
    SEQ
    ID
    PEPTIDE Pos. Sequence NO:
    A. Non-overlapping epitopes.
    6.4.1 126 Ac-LRMK-ava-YVATLSEAL-NH2 236
    6.4.2 153 Ac-LRMK-ava-VKVIPAGEL-NH2 237
    6.4.3 134 Ac-LRMK-ava-LRIIAGTLE-NH2 238
    6.4.4  64 Ac-LRMK-ava-YRTFVATFG-NH2 239
    B. Overlapping epitopes
    6.4.5 209 Ac-LRMK-ava-YKFIPALEA-NH2 240
    6.4.6 206 Ac-LRMK-ava-YESYKFIPA-NH2 241
    6.4.7 206/209 Ac-LRMK-ava-YESYKFIPALEA-NH2 242
    6.4.8 161 Ac-LRMK-ava-LQVIEKVDAAFK-NH2 243
    6.4.9 171 Ac-LRMK-ava-FKVAATAAN-NH2 244
    6.4.10 161/171 Ac-LRMK-ava- 245
    LQVIEKVDAAFKVAATAAN-NH2

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • Example 7 Ii-Key/Phospholipase A-2 Bee Venom Antigenic Epitope Hybrids
  • [0147]
    In another aspect this invention relates to the design and use of Ii-Key/Phospholipase A-2 bee venom antigenic epitope hybrids. Muller and colleagues successful induced specific T-cell anergy in patients allergic to bee venom with immunotherapy with T-cell recognized peptides of bee venom phospholipase A2 (Muller U. J Allergy Clin Immunol. 1998 101:747-54). Five patients with IgE-mediated systemic allergic reactions to bee stings were treated with a mixture of three T-cell epitope peptides of PLA. Ten patients allergic to BV receiving whole BV immunotherapy served as control subjects. Increasing doses of the peptide mixture, up to a maintenance dose of 100 micrograms, were administered subcutaneously within 2 months. The patients were then challenged with PLA and 1 week later with a bee sting. The cellular and humoral immune response was measured in vitro. No allergic side effects were caused by the peptide immunotherapy, and all patients tolerated the challenge with PLA without systemic allergic symptoms. Two patients developed mild systemic allergic reactions after the bee sting challenge. After peptide immunotherapy, specific proliferative responses to PLA and the peptides in peripheral blood mononuclear cells were decreased in successfully treated patients. The production of TH2 and TH1 cytokines was inhibited, and B cells were not affected in their capacity to produce specific IgE and IgG4 antibodies. Their levels increased after allergen challenge in favor of IgG4. The investigators concluded that immunotherapy of BV allergy with short T-cell peptides of PLA induces epitope-specific anergy in peripheral T cells and changes the specific isotype ratio in a fashion similar to that of conventional immunotherapy in successfully treated patients. Additional MHC Class II-presented candidate epitopes have been identified (Texier C. J Immunol 2000 164:3177-84).
  • [0148]
    The sequence of bee venom phospholipase A-2 in Table 7.1 was taken from GenBank 129501 allergen Api m1 (Kuchler K. Eur J Biochem. 1989 184:249-54). Predicted MHC Class II-presented epitopes of the major bee venom allergen phospholipase A-2 are listed in Table 7.2. Table 7.3. Experimentally defined MHC Class II-presented epitopes of the major bee venom allergen phospholipase A-2 are listed in Table 7.3. Ii-Key/PHL A2 hybrids containing some of the MHC Class II-presented PHL A2 epitopes of Table 1 and 2 (nonoverlapping and overlapping epitopes) are listed in Table 7.4.
  • [0000]
    TABLE 7.1
    Deduced amino acid sequence of Phospholipase A-2
    bee venom. (SEQ ID NO: 246)
      1 gslfllllst shgwqirdri gdneleerii ypgtlwcghg nkssgpnelg
     51 rfkhtdaccr thdmcpdvms ageskhgltn tashtrlscd cddkfydclk
    101 nsadtissyf vgkmyfnlid tkcyklehpv tgcgertegr clhytvdksk
    151 pkvyqwfdlr ky
  • [0000]
    TABLE 7.2
    Predicted MHC Class II-presented epitopes of
    the major bee venom allergen Phospholipase A-2.
    SEQ ID
    PEPTIDE Pos. Sequence Score Ii-Key NO:
    7.2.1  14 W Q I R D R I G D 7.80 247
    7.2.1   4 F L L L L S T S H 5.70 248
    7.2.1 110 F V G K M Y F N L 5.50 249
    7.2.1 118 L I D T K C Y K L 5.30 250
    7.2.1   6 L L L S T S H G W 4.80 251
    7.2.1 116 F N L I D T K C Y 4.50 252
    7.2.1   5 L L L L S T S H G 4.40 253
    7.2.1  52 F K H T D A C C R 4.10 254
    7.2.1 124 Y K L E H P V T G 4.08 255

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0000]
    TABLE 7.3
    Experimentally defined MHC Class II-presented
    epitopes of the major bee venom allergen
    Phospholipase A-2.
    Ii- SEQ ID
    PEPTIDE Pos. Sequence Key NO:
    7.3.1 113 K M Y F N L I D T K C Y K 256
    7.3.2 122 K C Y K L E H P V T G C G  4 257
    7.3.3 109 Y F V G K M Y F N L I D T 258
    7.3.4 141 C L H Y T V D K S K P K 10 259
    7.3.5  73 E S K H G L T N T A S H T 260
    RLSCD

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of an experimentally determined MHC Class II-presented epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope. The above epitopes were defined by Texier and colleagues and Carballido and colleagues (Texier C. J Immunol. 2000 164:3177-84; Carballido J. J Immunol. 1993 150:3582-91).
  • [0000]
    TABLE 7.4
    Ii-Key/PHL A2 hybrids containing some of the
    MHC Class II-presented PHL A2 epitopes of Table
    1 and 2.
    SEQ
    ID
    PEPTIDE Pos. Sequence NO:
    Nonoverlapping epitopes
    7.4.1  14 Ac-LRMK-ava-WQIRDRIGD-NH2 261
    7.4.2  52 Ac-LRMK-ava-FKHTDACCR-NH2 262
    Overlapping epitopes
    7.4.3   4 Ac-LRMK-ava-FLLLLSTSH-NH2 263
    7.4.4   5 Ac-LRMK-ava-LLLLSTSHG-NH2 264
    7.4.5   6 Ac-LRMK-ava-LLLSTSHGW-NH2 265
    7.4.6   4/5/6 Ac-LRMK-ava-FLLLLSTSHGW-NH2 266
    7.4.7 110 Ac-LRMK-ava-FVGKMYFNL-NH2 267
    7.4.8 116 Ac-LRMK-ava-FNLIDTKCY-NH2 268
    7.4.9 118 Ac-LRMK-ava-LIDTKCYKL-NH2 269
    7.4.10 110/116/ Ac-LRMK-ava-FVGKMYFNLIDTKCYKL- 270
    118 NH2

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • Example 8 Bla g 5 glutathione-S-transferase (GST) cockroach antigenic epitope hybrids
  • [0149]
    In another aspect this invention relates to the design and use of Ii-Key/Bla g 5 glutathione-S-transferase (GST) cockroach antigenic epitope hybrids. Sensitization to cockroach allergens is associated with the development of asthma. This antigenic protein was found to induce positive skin test reactions in 70% of patients who are allergic to cockroaches. A 23-kDa allergen, glutathione S-transferase (EC 2.5.1.18; GST) was purified from German cockroach (Blattella germanica) by glutathione affinity chromatography (Arruda L K. J Biol Chem. 1997 272:20907-12). The purified protein and recombinant protein had a high level of IgE antibody binding activity. This GST caused positive immediate skin tests in cockroach-allergic patients using as little as 3 pg of recombinant protein. Bla g 1 and has a molecular structure composed of multiple tandem amino-acid repeats. Two consecutive repeats are not identical but form a duplex that constitutes a basic molecular unit of Bla g 1 (Pomes A. Am J Respir Crit Care Med, 2002 165:391-7). The presence of two repeats was not a requirement for IgE antibody binding (Pomes A. Eur J Biochem. 2002 269:3086-3092). That fact supports the view that single linear chains of one repeat in the antigen can be found to express IgE binding epitopes. Bla g 2 is one of the most potent cockroach allergens (prevalence of IgE responses of 60 to 80%) and shows homology to the aspartic proteinase family of enzymes. Mutational destruction of the enzymatic activity did not alter allergenicity (Pomes A. Am J Respir Crit Care Med, 2002 165:391-7). Recombinant proteins of cockroach allergens Bla g 2, Bla g 4 and Bla g 5 and mite group 5 allergens were produced in bacterial expression vectors and demonstrated strong immediate skin and serum IgE antibody responses in cockroach-allergic patients (Chapman M D. Int Arch Allergy Immunol. 1997 113:102-4). Each of the recombinant allergens retained biologic activity and the investigators suggested that cocktails of two to four recombinant allergens could be used for diagnostic or therapeutic purposes. No dominant linear IgE-binding epitopes have been reported, but when they become determined, Ii-Key/Bla g MHC Class II epitope/BLA g IgE-recognized hybrid peptides can be designed and tested.
  • [0150]
    The sequence of cockroach allergen BLA g 5 in Table 8.1 was taken from GenBank 6225491 (Arruda L K. J Biol Chem. 1997 272:20907-12). Predicted MHC Class II epitopes of cockroach allergen Bla g 5 are presented in Table 8.2. Experimentally defined MHC Class II epitopes of Bla g 5 (Papouchado B G. Tissue Antigens. 2000 55:303-11) are presented in Table 8.3.
  • [0000]
    TABLE 8.1
    Deduced amino acid sequence of cockroach allergen
    Bla g 5. (SEQ ID NO: 271)
      1 mapsykltyc pvkalgepir fllsygekdf edyrfqegdw pnlkpsmpfg
     51 ktpvleidgk qthqsvaisr ylgkqfglsg kddwenleid mivdtisdfr
    151 aaianyhyda denskqkkwd plkketipyy tkkfdevvka nggylaagkl
    201 twadfyfvai ldylnhmake dlvanqpnlk alrekvlglp aikawvakrp
    251 ptdl
  • [0000]
    TABLE 8.2
    Predicted MHC Class II-presented epitopes of
    cockroach allergen Bla g 5.
    PEPTIDE Sequence Ii- SEQ ID
    NO: Pos. 1 2 3 4 5 6 7 8 9 Score Key NO:
    8.2.1  49 F G K T P V L E I 7.60 272
    8.2.2 245 W V A K R P P T D 5.85 273
    8.2.3  91 M I V D T I S D F 5.57 274
    8.2.4 205 F Y F V A I L D Y 5.40 275
    8.2.5  19 I R F L L S Y G E 5.10 2 276
    8.2.6  55 L E I D G K Q T H 4.60 7 277
    8.2.7  99 F R A A I A N Y H 4.20 278

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0000]
    TABLE 8.3
    Experimentally defined MHC Class II epitopes of
    Bla g 5.
    PEPTIDE SEQ ID
    NO: Pos. Sequence Ii-Key NO:
    8.3.1  92 IVDTISDFRAAIANYHYDAD 279
    8.3.1 212 DYLNHMAKEDLVANQPNLKA 280

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of an experimentally determined MHC Class II-presented epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • Example 9 Ii-Key/CEA Antigenic Epitope Hybrids
  • [0151]
    Carcinoembryonic antigen (CEA) is a tumor associated antigen (TAA) that is expressed in tumors including colon, breast and pancreas. The protein and the cDNA have been used for therapeutic tumor vaccines. A recombinant vaccinia-CEA vaccine has been used to generation cytotoxic T cells specific for human carcinoembryonic antigen epitopes (Tsang K Y. J Natl Cancer Inst. 1995 87:982-90). Ii-Key hybrids can be used to develop T helper cell responses to this tumor-associated antigen prior to DNA vaccines of any form. Thus, the clinical value of such a recombinant vaccinia-CEA construct can be enhanced substantially with the products and methods of this invention, as described in this disclosure. Additional CEA vaccination procedures, in which Ii-Key/CEA antigenic epitope hybrids can be applied, are presented below.
  • [0152]
    Reisfeld and colleagues demonstrated that an oral DNA vaccine against human CEA prevented growth and dissemination of Lewis lung carcinoma in CEA transgenic mice (Niethammer A G. Vaccine 2001 20:421-9). A DNA vaccine encoding human CEA broke peripheral T-cell tolerance toward this antigen expressed by Lewis lung carcinoma stably transduced with CEA in C57BL/6J mice transgenic for CEA. The vaccine was delivered by oral gavage with an attenuated strain of Salmonella typhimurium (SL7207), and boosted with an antibody-IL2 fusion protein. Both CTL and antigen-presenting dendritic cells were activated as indicated by a decisive increase in their respective activation markers CD2, CD25, CD28 as well as CD48 and CD80.
  • [0153]
    Stevenson and colleagues demonstrated that DNA fusion vaccine including MHC Class II epitopes of tetanus toxoid along with a tumor antigen of interest (here CEA) induced cytotoxic T cell responses against defined peptides. Fusion of the fragment C of tetanus toxin to a CEA sequence promoted antibody and CD4+ T cell responses against tested B cell tumors. Using only the first domain of tetanus toxoid, which contains a “universal” helper epitope, followed by two known CTL-recognized epitopes of CEA, they found strong CTL responses to each CTL-recognized peptide to be induced by the engineered construct.
  • [0154]
    Diagnostic assays with Ii-Key/antigenic epitope hybrids can be used to monitor therapy and predict outcomes in patients with CEA-positive tumors, as indicated from the following study. PBMC from two CEA-based vaccine clinical trials were analyzed for T cell responses to the same CEA peptide and to an influenza (Flu) control peptide (Arlen P. Cancer Immunol Immunother. 2000 49:517-29). The first trial consisted of three monthly vaccinations of CEA peptide (designated PPP) in adjuvant. The second trial consisted of cohorts receiving three monthly vaccinations of avipox-CEA recombinant (designated AAA) or cohorts receiving a primary vaccination with recombinant vaccinia-CEA followed by two monthly vaccinations with avipox-CEA (designated VAA). Few, if any, CEA-specific T cell responses were seen in patients receiving PPP vaccinations, while the majority of patients receiving the poxvirus CEA recombinants demonstrated increases in CEA-specific T cell responses and no increases in Flu-specific responses. CEA-specific IgG responses developed in patients following recombinant CEA poxvirus vaccinations. T cell responses to the CEA peptide were significantly increased after immunization with the recombinant poxvirus vaccine, as compared with the peptide vaccine (p=0.028). Clearly poxvirus recombinant-based vaccines are more potent in initiating tumor-antigen-specific T cell responses than are peptide vaccines. Their activity can be further enhanced, by prior vaccination with Ii-Key/CEA antigenic epitope hybrids.
  • [0155]
    In the case of tumor antigens such as CEA, Ii-Key/MHC Class II-presented antigenic epitope hybrids create T helper cell responses that augment the development of immune responses to CEA MHC Class I epitopes, for example through dendritic cell licensing. Such CTL activation by MHC Class I epitopes can be generated also by incorporating such MHC Class I epitopes in an Ii-Key MHC Class II-presented hybrids. Several MHC Class I epitope of CEA have been experimentally determined (Kawashima I. Hum Immunol. 1998 59:1-14; Nukaya I. Int J Cancer. 1999 80:92-7; Table 5). Such peptides have been discovered by various techniques. Nested deletions of the cDNA for the antigen of interest lead to protein products, which can be assayed for stimulation of CD8+ cells lines, which recognize the antigen. Given various cell lines recognizing individual epitopes, the localization of T cell epitopes can be approximated within the primary sequence, by analyzing the reactions of each T cell clone to the nested deletion cDNA constructs. Then a library of overlapping peptides through biologically active target regions can be assayed to define exactly the individual determinants. The binding of such peptides to immunopurified MHC Class I molecules can also be assayed, for example by inhibition of binding of a radiolabeled standard peptide to MHC molecules (Kawashima I. Hum Immunol. 1998 59:1-14). The MHC Class I molecules can be immunopurified and bound into microtiter plates, in which the various components of the assay are added sequentially, with appropriate washings. Alternatively the MHC Class I molecules can be detergent-solubilized without purification, for example from a microsomal membrane preparation of a cultured lymphoblastoid cell line, and the complexes separated in a gel filtration column, with the bound radioactive peptide being separated in the protein complexes from the unbound, free peptide. In the work of Kawashima, initial studies were performed with HLA-A2.1 molecules. The highly reactive peptide 9.5.2, which induced vigorous anti-tumor CTL responses, also bound tightly to other common HLA alleles of the A2 supertype (A2.2, A2.3, A2.6 and A6802), thus demonstrating a potential in providing broad and not ethnically biased population coverage. CTL lines were used to identify peptides 9.5.4 and 9.5.6, which elicited CTL lines that lysed tumor cells expressing HLA-A24 and CEA. The cytotoxicity to tumor cells by the CTL lines was antigen-specific since it was inhibited by peptide-pulsed cold target cells as well as by monoclonal antibodies to MHC Class I and CD3 molecules. Similar methods can be used to characterize the biological responses induced by Ii-Key/MHC Class II epitope/MHC Class I epitope hybrids of this disclosure.
  • [0156]
    Alternatively, such peptides are identified with algorithms for the prediction of MHC Class I and Class II T cell-recognized epitopes (Lu J. Cancer Res. 2000 60:5223-7). These computer-based predictive algorithms, which are available on the Internet (Parker K C. J Immunol. 1992 149:3580-7; Rammensee H G. Immunogenetics. 1995 41:178-228), were used to identify HL4-B7-restricted CTL epitopes for carcinoembryonic antigen (CEA). Of three candidate peptides, CEA9(632) (IPQQHTQVL) (SEQ ID NO: 281) induced primary CTL responses in lymphocytes from HLA-B7+ normal blood donors when dendritic cells were used as antigen-presenting cells. These CTLs were efficient in killing tumor cells that expressed HLA-B7 and produced CEA.
  • [0157]
    Cell lines reflecting the natural T-cell response against MHC Class I epitopes of epithelial cell adhesion molecule, Her-2/neu, and carcinoembryonic antigen in patients with colorectal cancer have been used to identify the respective antigenic epitopes (Nagorsen D. Cancer Res. 2000 60:4850-4). Antigens of epithelial cell adhesion molecule (Ep-CAM), her-2/neu, and CEA were potential targets in antigen-specific vaccination-based cancer therapy. The investigators tested whether a natural specific T-cell response against these antigens already exists in patients with colorectal carcinoma. The IFN-gamma ELISPOT assay was used to detect circulating TAA-reactive T cells directly ex vivo in unstimulated peripheral blood mononuclear cells. They determined that seven of 22 patients, but none of the 8 healthy subjects, had T cells specifically secreting IFN-gamma in response to antigen peptides (n=4, Ep-CAM; n=5, her-2/neu; n=6, CEA). T-cell responses occurred only in patients with metastatic disease (Dukes' stages C and D). The results of this study indicate that natural T-cell responses against tumor antigens occur in approximately one-half of colorectal carcinoma patients with involvement of lymph nodes or distant metastases, but not in colorectal carcinoma patients with disease confined to the bowel tract. Ii-Key/antigenic epitope hybrids containing MHC Class II epitopes can be used to vaccinate patients with localized and metastatic disease against their tumors.
  • [0158]
    The amino acid sequence of CEA was obtained from GenBank as 11386171 carcinoembryonic antigen-related cell adhesion molecule 5; carcinoembryonic antigen [Homo sapiens] (Table 1). The primary sequence of human carcinoembryonic antigen (CEA) was deduced from cDNA sequence (Oikawa S. Biochem Biophys Res Commun. 1987 142:511-8). The carcinoembryonic antigen (CEA) contains multiple immunoglobulin-like domains (Oikawa S. Biochem Biophys Res Commun. 1987 144:634-42). Predicted MHC Class II-presented epitopes of CEA are listed in Table 9.2. Designed Ii-Key/CEA hybrids containing some of the MHC Class II-presented epitopes of CEA in Table 9.2 are listed in Table 9.3. Predicted MHC Class I-presented CEA epitopes are listed in Table 9.4. Experimentally defined MHC Class I epitopes of CEA are listed in Table 9.5. Ii-Key/MHC Class II/MHC Class I CEA hybrids are listed in Table 9.6.
  • [0000]
    TABLE 9.1
    Deduced amino acid sequence of CEA.
      1 mespsapphr wcipwqrlll taslltfwnp pttaklties tpfnvaegke (SEQ ID NO: 282)
     51 vlllvhnlpq hlfgyswykg ervdgnrqii gyvigtqqat pgpaysgrei
    101 iypnaslliq niiqndtgfy tlhviksdlv neeatgqfrv ypelpkpsis
    151 snnskpvedk davaftcepe tqdatylwwv nnqslpvspr lqlsngnrtl
    201 tlfnvtrndt asykcetqnp vsarrsdsvi lnvlygpdap tisplntsyr
    251 sgenlnlsch aasnppaqys wfvngtfqqs tqelfipnit vnnsgsytcq
    301 ahnsdtglnr ttvttitvya eppkpfitsn nsnpvededa valtcepeiq
    351 nttylwwvnn qslpvsprlq lsndnrtltl lsvtrndvgp yecgiqnels
    401 vdhsdpviln vlygpddpti spsytyyrpg vnlslschaa snppaqyswl
    451 idgniqqhtq elfisnitek nsglytcqan nsasghsrtt vktitvsael
    501 pkpsissnns kpvedkdava ftcepeaqnt tylwwvngqs lpvsprlqls
    551 ngnrtltlfn vtrndarayv cgiqnsvsan rsdpvtldvl ygpdtpiisp
    601 pdssylsgan lnlschsasn pspqyswrin gipqqhtqvl fiakitpnnn
    651 gtyacfvsnl atgrnnsivk sitvsasgts pglsagatvg imigvlvgva
    701 li
  • [0000]
    TABLE 9.2
    Predicted MHC Class II-presented epitopes of
    CEA.
    PEPTIDE Pos. Sequence Score Ii-Key SEQ ID NO:
    9.2.1 427 YRPGVNLSL 6.40 283
    9.2.2 535 WVNGQSLPV 6.30 284
    9.2.3 179 WVNNQSLPV 5.50 285
    357 6.30
    9.2.4 627 WRINGIPQQ 5.80 286
    9.2.5 249 YRSGENLNL 5.50 287
    9.2.6  52 LLLVHNLPQ 5.40 288
    9.2.7 449 WLIDGNIQQ 5.10 12 289
    5.78
    9.2.8 591 YGPDTPIIS 5.10 290
    9.2.9 119 FYTLHVIK S 5.10 291
    9.2.10  79 IIGYVIGTQ 5.00 292

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0000]
    TABLE 9.3
    Designed Ii-Key/CEA hybrids containing same of
    the MHC Class II-presented epitopes of CEA in
    Table 9.2.
    A. Non-overlapping epitopes
    PEPTIDE Pos. Sequence SEQ ID NO:
    9.3.1 179 Ac-LRMK-ava-WVNNQSLPV-NH2 293
    357
    535
    9.3.2 427 Ac-LRMK-ava-YRPGVNLSL-NH2 294
    9.3.3 627 Ac-LRMK-ava-WRINGIPQQ-NH2 295
    9.3.4 249 Ac-LRMK-ava-YRSGENLNL-NH2 296

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 9.2. CEA contains seven extracellular domains, which are strikingly homologous to each other. This fact explains the repeated identical epitopes that starts at positions 178, 356, and 534 in this Table (Oikawa S. Biochem Biophys Res Commun. 187 144:634-42).
  • [0000]
    TABLE 9.4
    Predicted MHC Class I-presented CEA epitopes.
    PEPTIDE Pos. Sequence Score SEQ ID NO:
    9.4.1  61 HLFGYSWYK 1350 297
    9.4.2 425 TYYRPGVNL  200 298
    9.4.3 652 TYACFVSNL  200 299
    9.4.4 691 IMIGVLVGV  196 300
    9.4.5 605 YLSGANLNL   98 301

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class I-presented epitope. Score is the T1/2 of disassociation of a peptide containing this subsequence (Tsang K Y. J Natl Cancer Inst. 1995 87:982-90). Peptide 9.3.1 are presented by HLA-A3. Peptides 9.3.2 and 9.3.2 are presented by HLA-A24. Peptides 9.3.4 and 9.3.5 are presented by HLA-A2.1. The MHC Class I-presented epitopes of this Table were predicted with the use of the online program at (http://bimas.dcrt.nih.gov/molbio/hla_bind/).
  • [0000]
    TABLE 9.5
    Experimentally defined MHC Class I epitopes of
    CEA.
    PEPTIDE Pos. Sequence SEQ ID NO:
    9.5.1 691 IMIGVLVGV 302
    9.5.2  24 LMTFWNPPV 303
    9.5.3 605 YLSGANLNL 304
    9.5.4 268 QYSWFVNGTF 305
    9.5.5 652 TYACFVSNL 306
    9.5.6  61 HLFYSWYK 307

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the experimentally defined MHC Class I-presented epitope. Peptides 9.5.1, 9.5.2, 9.5.3 are presented by HLA-A2.1 and 9.5.6 is presented by HLA-A3 (Kawashima I. Hum Immunol. 1998 59:1-14). Peptides 9.5.4 and 9.5.5 are presented by HLA-A24 (Nukaya I. Int J Cancer. 1999 80:92-7). Peptide 9.5.2 is presented by HLA-A2.1 and it and LLTFWNPPV (SEQ ID NO: 308) are engineered CEA epitopes with respect to the wild type sequence LLTFWNPPT (SEQ ID NO: 309).
  • [0000]
    TABLE 9.6
    Ii-Key/MHC Class II/MHC Class I CEA hybrids.
    SEQ
    ID
    PEPTIDE Pos. Sequence NO:
    9.6.1 II: 179, 357, Ac-LRMK-ava-WVNNQSLPV- 310
    535, IMIGVLVGV-NH2
    I: 691
    9.6.2 II: 427, Ac-LRMK-ava-TYYRPGVNLSL- 311
    I: 425 NH2
    9.6.3 II: 249, Ac-LRMK-ava-YRSGENLNL- 312
    I: 268 QYSWFVNGTF-NH2
    9.6.4 II: 52, Ac-LRMK-ava-LLLVHNLPQ- 313
    I: 61 HLFYSWYK-NH2

    Ii-Key/MHC Class II/MHC Class I CEA hybrids. The sequence position of the MHC Class II epitope is indicated: II:residue position of first epitope amino acid, and of the MHC Class I epitope is indicated: I:residue position of first epitope amino acid.
  • Example 10 Ii-Key/Ca-125 Cancer Antigenic Epitope Hybrids
  • [0159]
    The ovarian cancer antigen CA-125 is used in immunotherapeutic vaccinations. In one case, vaccination with a mixed vaccine of autogenous and allogeneic breast cancer cells and tumor associated antigens including the breast cancer antigen CA15.3, the carcinoembryonic antigen (CEA) and the ovarian cancer antigen CA125, resulted in immune and clinical responses in breast cancer patients (Jiang X P. Cancer Biother Radiopharm. 2000 15:495-505). The vaccine induced a significant increase in post-vaccination lymphocyte proliferative responses to AUTOC, CA15.3, CEA and CA125 but not ALLOC, compared to pre-vaccination (p<0.05, p<0.01, p<0.05, p<0.01 and p>0.05, respectively, a paired t Test).
  • [0160]
    The amino acid sequence of CA125 ovarian cancer antigen mucin 16 [Homo sa . . . [gi:14971110] as listed in Genebank is presented in Table 10.1. Predicted MHC Class II-presented epitopes of CA125, ovarian cancer antigen are listed in Table 10.2. Ii-Key/CA 125 hybrids containing some of the MHC Class II-presented epitopes of Table 10.2 are listed in Table 10.3. Predicted MHC Class I-presented epitopes of CA 125 are listed in Table 10.4. Ii-Key/MHC II epitope/MHC I epitope hybrids are listed in Table 10.5.
  • [0000]
    TABLE 10.1
    Deduced amino acid sequence of CA125 ovarian
    cancer antigen (SEQ ID NO: 314)
       1 rvdpigpgld rerlywelsq ltnsitelgp ytldrdslyv ngfnpwssvp
      51 ttstpgtstv hlatsgtpss lpghtapvpl lipftlnfti tnlhyeenmq
     101 hpgsrkfntt ervlqgllkp lfkstsvgpl ysgcrltllr pekhgaatgv
     151 daictlrldp tgpgldrerl ywelsqltns vtelgpytld rdslyvngft
     201 hrssvpttsi pgtsavhlet sgtpaslpgh tapgpllvpf tlnftitnlq
     251 yeedmrhpgs rkfnttervl qgllkplfks tsvgplysgc rltllrpekr
     301 gaatgvdtic thrldplnpg ldreqlywel skltrgiiel gpylldrgsl
     351 yvngfthrnf vpitstpgts tvhlgtsetp sslprpivpg pllvpftlnf
     401 titnlqyeea mrhpgsrkfn ttervlqgll rplfkntsig plysscrltl
     451 lrpekdkaat rvdaicthhp dpqspglnre qlywelsqlt hgitelgpyt
     501 ldrdslyvdg fthwspiptt stpgtsivnl gtsgippslp ettatgpllv
     551 pftlnftitn lqyeenmghp gsrkfnites vlqgllkplf kstsvgplys
     601 gcrltllrpe kdgvatrvda icthrpdpki pgldrqqlyw elsqlthsit
     651 elgpytldrd slyvngftqr ssvpttstpg tftvqpetse tpsslpgpta
     701 tgpvllpftl nftiinlqye edmhrpgsrk fnttervlqg llmplfknts
     751 vsslysgcrl tllrpekdga atrvdavcth rpdpkspgld rerlywklsq
     801 lthgitelgp ytldrhslyv ngfthqssmt ttrtpdtstm hlatsrtpas
     851 lsgpttaspl lvlftinfti tnlryeenmh hpgsrkfntt ervlqgllrp
     901 vfkntsvgpl ysgcrltllr pkkdgaatkv daictyrpdp kspgldreql
     951 ywelsqlths itelgpytld rdslyvngft qrssvpttsi pgtptvdlgt
    1001 sgtpvskpgp saaspllvlf tlnftitnlr yeenmqhpgs rkfnttervl
    1051 qgllrslfks tsvgplysgc rltllrpekd gtatgvdaic thhpdpkspr
    1101 ldreqlywel sqlthnitel gpyaldndsl fvngfthrss vsttstpgtp
    1151 tvylgasktp asifgpsaas hllilftlnf titnlryeen mwpgsrkfnt
    1201 tervlqgllr plfkntsvgp lysgcrltll rpekdgeatg vdaicthrpd
    1251 ptgpgldreq lylelsqlth sitelgpytl drdslyvngf thrssvptts
    1301 tgvvseepft lnftinnlry madmgqpgsl kfnitdnvmq hllsplfqrs
    1351 slgarytgcr vialrsvkng aetrvdllct ylqplsgpgl pikqvfhels
    1401 qqthgitrlg pysldkdsly lngynepgpd eppttpkpat tflpplseat
    1451 tamgyhlktl tlnftisnlq yspdmgkgsa tfnstegvlq hllrplfqks
    1501 smgpfylgcq lislrpekdg aatgvdttct yhpdpvgpgl diqqlywels
    1551 qlthgvtqlg fyvldrdslf ingyapqnls irgeyqinfh ivnwnlsnpd
    1601 ptsseyitll rdiqdkvttl ykgsqlhdtf rfclvtnltm dsvlvtvkal
    1651 fssnldpslv eqvfldktln asfhwlgsty qlvdihvtem essvyqptss
    1701 sstqhfypnf titnlpysqd kaqpgttnyq rnkrniedal nqlfrnssik
    1751 syfsdcqvst frsvpnrhht gvdslcnfsp larrvdrvai yeeflrmtrn
    1801 gtqlqnftld rssvlvdgys pnrnepltgn sdlpfwavil iglagllgli
    1851 tclicgvlvt trrrkkegey nvqqqcpgyy qshldledlq
  • [0000]
    TABLE 10.2
    Predicted MHC Class II-presented epitopes of
    CA125, ovarian cancer antigen.
    PEPTIDE Pos. Sequence Score Ii-Key SEQ ID NO:
    10.2.1 1630 FRFCLVTNL 7.40  9 315
    10.2.2 1018 VLFTLNFTI 7.10 316
    10.2.3 1174 ILFTLNFTI 7.10 317
    10.2.4  156 LRLDPTGPG 6.80 318
    10.2.5 1017 LVLFTLNFT 6.20 319
    10.2.6  861 LVLFTINFT 6.05 320
    10.2.7  527 IVNLGTSGI 5.70 321
    10.2.8 1318 LRYMADMGQ 5.70 322
    10.2.9 1029 LRYEENMQH 5.68 323
    10.2.10  873 LRYEENMHH 5.68 324
    10.2.11 1663 VFLDKTLNA 5.60 10 325
    10.2.12 1172 LLILFTLNF 5.60 326
    10.2.13  936 YRPDPKSPG 5.57  2 327
    10.2.14  393 LVPFTLNFT 5.50  3 328
    10.2.15  430 LRPLFKNTS 5.40 329
    10.2.16 1185 LRYEENMWP 5.30  9 330
    10.2.17 1634 LVTNLTMDS 5.30 331
    10.2.18  360 FVPITSTPG 5.20 11 332
    10.2.19 1209 LRPLFKNTS 5.10 333
    10.2.20  898 LRPVFKNTS 5.10 334
    10.2.21 1531 YHPDPVGPG 5.10 335

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope. Because CA 125/MUC 16 is characterized by nine partially conserved tandem repeats (156 amino acids each) in an N-terminal region, similar predicted epitopes have different starting positions (e.g., start position 1017, 1173, 860, or 897, 1208, or 1184, 872, 1028).
  • [0000]
    TABLE 10.3
    Ii-Key/CA125 hybrids containing some of the MHC
    Class II-presented epitopes of Table 10.2.
    SEQ ID
    PEPTIDE Pos. Sequence NO:
    A. Conserved tandem-repeats epitopes
    10.3.1 1017 Ac-LRMK-ava-VLFTLNFTI-NH2 336
    10.3.2 1173 Ac-LRMK-ava-ILFTLNFTI-NH2 337
    10.3.3  860 Ac-LRMK-ava-LVLFTINFT-NH2 338
    10.3.4 1028 Ac-LRMK-ava-LRYEENMQH-NH2 339
    10.3.5  872 Ac-LRMK-ava-LRYEENMHH-NH2 340
    10.3.6 1184 Ac-LRMK-ava-LRYEENMWP-NH2 341
    B. Overlapping MHC II epitopes
    10.3.7 1629 Ac-LRMK-ava-FRFCLVTNL-NH2 342
    10.3.8 1633 Ac-LRMK-ava-LVTNLTMDS-NH2 343
    10.3.9 1629/ Ac-LRMK-ava- 344
    1633 FRFCLVTNLTMDS-NH2

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 10.2.
  • [0000]
    TABLE 10.4
    Predicted MHC Class I-presented epitopes of CA
    125.
    PEPTIDE Pos. Sequence Score SEQ ID NO:
    10.4.1 1675 WLGSTYQLV 478 345
    10.4.2 1018 VLFTLNFTI 381 346
    10.4.3 1174 ILFTLNFTI 381 347
    10.4.4  862 VLFTINFTI 381 348
    10.4.5  344 LLDRGSLYV 260 349
    10.4.6 1506 YLGCQLISL 226 350
    10.4.7 1668 TLNASFHWL 223 351
    10.4.8 1555 GVTQLGFYV 194 352
    10.4.9 1845 GLLGLITCL 182 353
    10.4.10   32 KLTRGIIEL 172 354

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted HLA-A2.1-resented epitope. Score is the T1/2 of disassociation of a peptide containing this subsequence (Tsang K Y. J Natl Cancer Inst. 1995 87:982-90). The MHC Class I-presented epitopes of this Table were predicted with the use of the online program at (http://bimas.dcrt.nih.gov/molbio/hla_bind/).
  • [0000]
    TABLE 10.5
    Ii-Key/MHC II epitope/MHC I epitope hybrids.
    Ii-Key/MHC Class
    SEQ
    ID
    PEPTIDE Pos. Sequence NO:
    10.5.1 1630 Ac-LRMK-ava-FRFCLVTNL- 355
    NH2
    10.5.2 II: 392 Ac-LRMK-ava-LVPFTLNFTI- 356
    I: 394, 238, NH2
      82, 550

    Ii-Key/MHC Class II/MHC Class I CEA hybrids. The sequence position of the MHC Class II epitope is indicated: II:residue position of first epitope amino acid, and of the MHC Class I epitope is indicated: I:residue position of first epitope amino acid. In peptide 10.5.1 the MHC Class II-predicted and the MHC Class I-predicted epitopes overlap precisely. In peptide 10.5.2 an MHC Class-predicted epitope starting at residue position 392 overlaps with the sequence of a MHC Class I-predicted epitope which starts (and is repeated at) residue positions 394, 238, 82, 550.
  • Example 11 Ii-Key/PSA Antigenic Epitope Hybrids
  • [0161]
    The identification of T cell specific epitopes within the coding sequence of PSA has led to the development of various vaccine strategies that target PSA in an attempt to treat established prostate cancer (Kaufman H L. Expert Opin Biol Ther. 2002 2:395-408). These strategies have included HLA-restricted PSA peptides, dendritic cells pulsed with PSA, recombinant viruses expressing PSA and combinations with different cytokines and cell interaction molecules. Many of these methods are enhanced by use of the products and methods of this disclosure.
  • [0162]
    PSA-recombinant pox vaccine constructs are immunogenic and induce antibody responses to a multitude of surface antigens on prostate tumor cell lines by epitope or determinant spreading after stimulation of the immune system by PSA immunization (Cavacini L A. Clin Cancer Res. 2002 8:368-73). Determinant spreading in the antibody responses to prostate cell surface antigens was observed in patients immunized with prostate-specific antigen encoded by recombinant pox vectors. The serum IgG response to cell surface antigens expressed on LNCAP (PSA-positive) and PC-3 (PSA-negative) prostate cancer cell lines were analyzed in individuals with advanced disease receiving vaccinia- or fowlpox-expressed PSA (v-PSA or f-PSA, respectively). Sera from all seven patients in a Phase I study of v-PSA, collected prior to the third immunization, reacted with both prostate tumor cell lines. The majority of individuals (n=12) in a Phase II trial of v-PSA and f-PSA developed sustainable antibody responses to cell surface antigens on the prostate tumor cell lines. The magnitude and kinetics of these responses depended on the immunization schedule.
  • [0163]
    Whiteside and colleagues demonstrated recovery of zeta-chain expression and changes in spontaneous IL-10 production after PSA-based vaccines in patients with prostate cancer (Meidenbauer N. Br J Cancer. 2002 86:168-78). In order to determine a mechanism by which circulating T lymphocytes of patients with prostate cancer have been reported to have functional deficits, including low or absent zeta-chain expression, 10 patients treated with recombinant human prostate specific antigen plus GM-CSF and eight others receiving PSA plus oil emulsion were evaluated. Prior to therapy, the patients had significantly lower zeta-chain expression in circulating CD3+ cells and a higher percentage of zeta-chain negative CD3+ and CD4+ cells than normal donors. The patients' peripheral blood mononuclear cells spontaneously produced more IL-10 ex vivo than those of normal controls. After vaccination, recovery of zeta-chain expression was observed in 50% of patients in both clinical trials. Also, spontaneous IL-10 secretion by peripheral blood mononuclear cells decreased following immunotherapy in patients treated with PSA and GM-CSF. Such therapies will be greatly augmented by products and methods of this disclosure.
  • [0164]
    Mann and colleagues demonstrated enhanced CD4+ and CD8+ T cell responses after exposure to PSA alone, PSA targeted to the mannose receptor (mannosylated PSA (PSA-m)), or PSA targeted to Fc receptors by combining PSA with an anti-PSA antibody (AR47.47) (Berlyn K A. Clin Immunol. 2001 101:276-83). PSA and PSA-m are processed primarily through pathways that favor MHC Class II presentation, while the PSA/anti-PSA immune complexes are processed through both Class I and Class II pathways in monocyte-derived dendritic cells.
  • [0165]
    Gilboa and colleagues demonstrated that autologous dendritic cells transfected with PSA RNA stimulate CTL responses against metastatic prostate tumors (Heiser A. J Clin Invest. 2002 109:409-17). Autologous dendritic cells transfected with mRNA encoding prostate-specific antigen (PSA) stimulate potent, T cell-mediated antitumor immune responses in vitro. A phase I trial evaluated this strategy for safety, feasibility, and efficacy to induce T cell responses against the PSA in patients with metastatic prostate cancer. In 13 subjects, escalating doses of PSA mRNA-transfected dendritic cells were administered with no evidence of dose-limiting toxicity or adverse effects, including autoimmunity. Induction of PSA-specific T cell responses was consistently detected in all patients, suggesting in vivo bioactivity of the vaccine. Vaccination was further associated with a significant decrease in the log slope of serum PSA levels in six of seven subjects.
  • [0166]
    Schlom and colleagues characterized an agonist epitope designated PSA-3A (“A” for agonist) of the PSA-3 CTL epitope which demonstrated enhanced binding to the HLA-A2 allele and enhanced stability of the peptide-MHC complex (Terasawa H. Clin Cancer Res. 2002 8:41-53). T-cell lines generated with either the PSA-3 or the PSA-3A peptide showed higher levels of lysis of targets pulsed with the PSA-3A peptide than those targets pulsed with the PSA-3 peptide. T cells stimulated with dendritic cells (dendritic cells) pulsed with PSA-3A peptide produced higher levels of IFN-gamma than did dendritic cells pulsed with PSA-3 peptide. Dendritic cells infected with a recombinant vaccinia virus containing the agonist amino acid change within the entire PSA gene (designated rV-PSA-3A) were more effective than dendritic cells infected with the rV-PSA vector in enhancing IFN-gamma production by T cells. Finally, the PSA-3A agonist was shown to induce higher levels of T-cell activation, compared with the PSA-3 peptide, in an in vivo model using HLA-A2.1/K(b) transgenic mice. These studies thus demonstrated the potential use of the PSA-3A agonist epitope in both peptide- and vector-mediated immunotherapy protocols for prostate cancer. Such results can be bettered with the products and methods of this disclosure.
  • [0167]
    Recombinant PSA proteins incorporating 6×His (SEQ ID NO: 357) residues were synthesized for magnetic bead attachment allowing antigen isolation and delivery to APC for processing and presentation (Turner M J. J Immunol Meth. 1998 256:107-19). PSA deletion constructs were generated by amplifying 3′ deletions of PSA using a constant 5′ primer and five individual 3′ primers starting at 736 bp, 610 bp, 505 bp, and 394 bp. The recombinant PSA proteins encoded 261, 231, 189, 154 and 117 amino acids. PSA-specific Class I- and II-restricted T cell hybridomas were generated by fusing Thy-1+tumor infiltrating lymphocytes (TIL) isolated from BALB/c mice challenged with Line 1/PSA/IL-2 tumors to the T cell fusion partner BWZ.36. MHC Class I (PSA 188-197) and Class II (PSA 238-253) T cell epitopes were identified.
  • [0168]
    The amino acid sequence of prostate specific antigen (PSA) as obtained from GenBank 45021731 kallikrein 3 is presented in Table 11.1. A cDNA vaccine for this antigen is available (Kim J J. Oncogene. 1998 17:3125-35). Predicted MHC Class II-presented epitopes of PSA are listed in Table 11.2. Experimentally defined MHC Class II-presented epitopes of PSA are listed in Table 11.3. Ii-Key/PSA hybrids containing some of the MHC Class II-presented epitopes of Tables 11.2 and 11.3 are listed in Table 11.4. Predicted MHC Class I-presented epitopes of PSA are listed in Table 11.5. Experimentally defined MHC Class I-presented epitopes of PSA are listed in Table 11.6. Ii-Key/PSA MHC II-presented epitope/PSA MHC I-presented epitope hybrids are listed in Table 11.7.
  • [0000]
    TABLE 11.1
    Deduced amino acid sequence of PSA.
      1 mwvpvvfltl svtwigaapl ilsrivggwe cekhsqpwqv lvasrgravc (SEQ ID NO: 358)
     51 ggvlvhpqwv ltaahcirnk svillgrhsl fhpedtgqvf qvshsfphpl
    101 ydmsllknrf lrpgddsshd lmllrlsepa eltdavkvmd lptqepalgt
    151 tcyasgwgsi epeefltpkk lqcvdlhvis ndvcaqvhpq kvtkfmlcag
    201 rwtggkstcs gdsggplvcn gvlqgitswg sepcalperp slytkvvhyr
    251 kwikdtivan p
  • [0000]
    TABLE 11.2
    Predicted MHC Class II-presented epitopes of
    PSA.
    PEPTIDE Pos. Sequence Score Ii-Key SEQ ID NO:
    11.2.1  59 WVLTAAHCI 8.80 359
    11.2.2   2 WVPVVFLTL 8.10 360
    11.2.3 124 LRLSEPAEL 6.60 361
    11.2.4  67 IRNKSVILL 5.40 8 362
    11.2.5  74 LLGRHSLFH 5.20 363
    11.2.6  72 VILLGRHSL 4.70 364
    11.2.7 223 LQGITSWGS 4.58 365

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0000]
    TABLE 11.3
    Experimentally defined MHC Class II-presented
    epitopes of PSA.
    SEQ ID
    PEPTIDE Pos. Sequence Ii-Key NO:
    11.3.1 238 ERPSLYTKVVHYRKWI 366

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the experimentally defined MHC Class II-presented epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope. Peptide 11.3.1 was experimentally defined (Turner M J. J Immunol Meth. 2001 256:107-19).
  • [0000]
    TABLE 11.4
    Ii-Key/PSA hybrids containing some of the MHC
    Class II-presented epitopes of Tables 11.2 and
    11.3.
    PEPTIDE Pos. Sequence SEQ ID NO:
    A. Non-overlapping epitopes
    11.4.1   2 Ac-LRMK-ava-WVPVVFLTL-NH2 367
    11.4.2 124 Ac-LRMK-ava-LRLSEPAEL-NH2 368
    11.4.3 223 Ac-LRMK-ava-LQGITSWGS-NH2 369
    B. Overlapping epitopes
    11.4.4  59 Ac-LRMK-ava- 370
    WVLTAAHCI-NH2
    11.4.5  67 Ac-LRMK-ava- 371
    IRNKSVILL-NH2
    11.4.6  72 Ac-LRMK-ava- 372
    VILLGRHSL-NH2
    11.4.7  74 Ac-LRMK-ava- 373
    LLGRHSLFH-NH2
    11.4.8  59, Ac-LRMK-ava- 374
     67, WVLTAAHCIRNKSVILLGR
     72, HSLFH-NH2
     74

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 11.2 and 11.3. Peptide 11.4.8 contains several MHC Class II-presented epitopes each beginning at residue positions 59, 67, 72 and 74.
  • [0000]
    TABLE 11.5
    Predicted MHC Class I-presented epitopes of
    PSA.
    PEPTIDE Pos. Sequence Score SEQ ID NO:
    11.5.1  46 GRAVCGVL 2000 375
    11.5.2  67 IRNKSVILL 2000 376
    11.5.3 124 LRLSEPAEL 2000 377
    11.5.4  18 APLILSRIV  660 378
    11.5.5   7 FLTLSVTWI  607 379
    11.5.6 249 YRKWIKDTI  600 380

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class I-presented epitope. The MHC Class I-presented epitopes were predicted with the use of the online program at (http://bimas.dcrt.nih.gov/molbio/hla_bind/). Score is the T1/2 of disassociation of a peptide containing this subsequence (Tsang K Y. J Natl Cancer Inst. 1995 87:982-90). Peptides 11.5.1, 11.5.2, 11.5.3 and 11.5.6 are presented optimally by HLA-B*2705. Peptide 11.5.4 is presented best by HLA-B*5102 and Peptide 11.5.5 is presented best by HLA-A*0201.
  • [0000]
    TABLE 11.6
    Experimentally defined MHC Class I-presented
    epitopes of PSA.
    PEPTIDE Pos. Sequence SEQ ID NO:
    11.6.1 188 HPQKVTKFML 381

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the experimentally defined MHC Class I-presented epitope.
  • [0000]
    TABLE 11.7
    Ii-Key/PSA MHC II-presented epitope/PSA MHC I-
    presented epitope hybrids.
    A Overlapping epitopes
    PEPTIDE Pos. Sequence SEQ ID NO:
    11.7.1   2 Ac-LRMK-ava- 382
    WVPVVFLTLSVTWI-
    NH2
    11.7.2  67 Ac-LRMK-ava- 383
    IRNKSVILL-NH2
    11.7.3 124 Ac-LRMK-ava- 384
    LRLSEPAEL-NH2

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 11.2 and a MHC Class I epitope of Table 11.5. In each of the example the sequences of the predicted MHC Class II and MHC Class I peptides overlap precisely. The residue position of the first amino acid in the predicted MHC Class II epitopes are reported.
  • Example 12 Ii-Key/Melanocyte Protein Pmel 17 Antigenic Epitope Hybrids
  • [0169]
    Melanoma is a leading target in the development of therapeutic peptide and DNA vaccines because several specific tumor-associated antigens have been identified, efficiency of vaccinating mice with peptide or DNA vaccines in treating melanoma is proved, and use of comparable vaccines in the clinic has had occasionally promising results. The use of Ii-Key/melanoma antigenic epitope hybrids in melanoma vaccination is considered in respective Examples concerning melanocyte protein Pme117, gp100, tyrosinase, and tyrosinase-s related protein.
  • [0170]
    Storkus and colleagues identified several MHC Class II-presented epitopes of gp 100/pme117 and tyrosinase melanocyte-associated antigens and tested the response of tumor-reactive human CD4+ T cells from various melanoma patients against these peptides (Kierstead L S. Br J. Cancer. 2001 85:1738-45). Two known and three novel CD4+ T cell epitopes were found using an IFN-gamma ELISPOT assay. Often freshly-isolated PBMC from HLA-DR4+ melanoma patients that are currently disease-free reveal elevated Th1-type CD4+ T-cells that recognize these peptides. Ii-Key/antigenic epitope hybrids incorporating these epitopes are presented in this Disclosure.
  • [0171]
    One problem in tumor immunotherapy is the fact that hosts can be tolerized to self proteins of the tumor. Intracutaneous immunization of C57BL/6 mice with a human Pme117/gp100 DNA vaccine, but not the murine DNA, induces T cell-mediated B16 melanoma protection in vivo (Wagner S N. J Invest Dermatol. 2000 115:1082-7). This state of unresponsiveness to the autoantigen Pme117/gp100 was broken by immunization with a plasmid DNA construct encoding the autologous form of the molecule. Mice receiving of Pme117/gp100 DNA mounted an antigen-specific cytotoxic T lymphocyte response to M3 melanoma. Furthermore M3 tumors growing in immunized mice lost expression of this melanoma-associated antigen whereas M3 melanomas appearing in control-vector-treated animals were still Pme117/gp100-positive. Ii-Key/antigenic epitope hybrids with appropriate immunization schemes and adjuvants can preferentially induce a Th1 or Th2 pattern of response thereby breaking tolerance.
  • [0172]
    The amino acid sequence of melanocyte protein Pmel 17 was obtained at NCBI, >gi|1125063|gb|AAB00386.11 melanocyte protein Pmel 17 [Homo sapiens]=>gi|639590|gb|AAC60634.1 gp100[Homo sapiens].
  • [0000]
    TABLE 12.1
    Deduced amino acid sequence of gp 100/pmel.
      1 mdlvlkrcll hlavigalla vgatkvprnq dwlgvsrqlr tkawnrqlyp (SEQ ID NO: 385)
     51 ewteaqrldc wrggqvslkv sndgptliga nasfsialnf pgsqkvlpdg
    101 viwvnntii ngsqvwggqp vypqetddac ifpdggpcps gswsqkrsfv
    151 yvwktwgqyw qvlggpvsgl sigtgramlg thtmevtvyh rrgsrsyvpl
    201 ahsssaftit dqvpfsvsvs qlraldggnk hflrnqpltf alqlhdpsgy
    251 laeadlsytw dfgdssgtli sralvvthty lepgpvtaqv vlqaaiplts
    301 cgsspvpgtt dghrptaeap nttagqvptt evvgttpgqa ptaepsgtts
    351 vqvpttevis tapvqmptae stgmtpekvp vsevmgttla emstpeatgm
    401 tpaevsivvl sgttaaqvtt tewvettare lpipepegpd assimstesi
    451 tgslgplldg tatlrlvkrq vpldcvlyry gsfsvtldiv qgiesaeilq
    501 avpsgegdaf eltvscqggl pkeacmeiss pgcqppaqrl cqpvlpspac
    551 qlvlhqilkg gsgtyclnvs ladtnslavv stqlimpgqe aglgqvpliv
    601 gillvlmavv lasliyrrrl mkqdfsvpql phssshwlrl prifcscpig
    651 enspllsgqq v
  • [0000]
    TABLE 12.2
    Predicted MHC Class II-presented epitopes of gp
    100.
    PEPTIDE Pos. Sequence Score Ii-Key SEQ ID NO:
    12.2.1 150 VYVWKTWGQ 6.80 386
    12.2.2 423 WVETTREL 6.40 387
    12.2.3 477 LYRYGSFSV 6.40  3 388
    12.2.4 290 VVLQAAIPL 6.40 10 389
    12.2.5 552 LVLHQILKG 6.10 390
    12.2.6 596 VPLIVGILL 5.80 391
    12.2.7 600 VGILLVLMA 5.80 392
    12.2.8 605 VLMAVVLAS 5.80 393
    12.2.9 604 LVLMAVVLA 5.70 394
    12.2.10   3 LVLKRCLLH 5.40- 395
    7.20
    12.2.11 615 IYRRRLMKQ 5.10- 396
    5.70
    12.2.12 616 YRRRLMKQD 4.50 397
    12.2.13  48 LYPEWTEAQ 4.30  8 398

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0000]
    TABLE 12.3
    Experimentally defined MHC Class II-presented
    epitopes of gp100.
    SEQ ID
    PEPTIDE Pos. Sequence Ii-Key NO:
    12.3.1  44 WNRQLYPEWTEAQRLD 4 399
    12.3.2 615 IYRRRLMKQDFSVPQLPHS 400
    12.3.3 576 SLAVVSTQLIMPG 401
    12.3.4 175 GRAMLGTHTMEVTVY 402
    12.3.5  74 GPTLIGANASFSIALN 403

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the experimentally defined MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope. DR*0401 best presented peptide 12.3.1 (Storkus W. Forum (Genova). 2000 10:256-70) and peptide 12.3.2 (Kierstead L. Brit J Cancer. 2001 85:1738-45). The remaining peptides of this Table were identified by Kobayashi H. (Cancer Res. 2001 61:7577-84).
  • [0000]
    TABLE 12.4
    Ii-Key/gp 100 hybrids containing some of the
    MHC Class II-presented epitopes of Table 12.2
    and 12.3.
    SEQ
    ID
    PEPTIDE Pos. Sequence NO:
    A. Non-overlapping epitopes
    12.4.1 615 Ac-LRMK-ava- 404
    IYRRRLMKQDFSVPQLPHS-NH2
    12.4.2   3 Ac-LRMK-ava-LVLKRCLLH-NH2 405
    12.4.3 150 Ac-LRMK-ava-VYVWKTWGQ-NH2 406
    12.4.5 423 Ac-LRMK-ava-WVETTAREL-NH2 407
    12.4.6 477 Ac-LRMK-ava-LYRYGSFSV-NH2 408
    B. Overlapping epitopes
    12.4.7  44 Ac-LRMK-ava- 409
    WNRQLYPEWTEAQRLD-NH2
    12.4.8  48 Ac-LRMK-ava-LYPEWTEAQ-NH2 410
    12.4.9  44, 48 Ac-LRMK-ava- 411
    WNRQLYPEWTEAQRLD-NH2
    12.4.10 596 Ac-LRMK-ava-VPLIVGILL-NH2 412
    12.4.11 600 Ac-LRMK-ava-VGILLVLMA-NH2 413
    12.4.12 605 Ac-LRMK-ava-VLMAVVLAS-NH2 414
    12.4.13 596, 600, 605 Ac-LRMK-ava- 415
    VPLIVGILLVLMAVVLAS-NH2

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 1.2.
  • [0000]
    TABLE 12.5
    Predicted MHC Class I-presented epitopes of gp
    100.
    PEPTIDE Pos. Sequence Score SEQ ID NO:
    12.5.1 619 RLMKQDFSV 1495 416
    12.5.2 520 LPKEACMEI  629 417
    12.5.3 602 ILLVLMAVV  412 418
    12.5.4 479 RYGSFSVTL  400 419
    12.5.5 154 KTWGQYWQV  315 420
    12.5.6  17 ALLAVGATK   45 421
    12.5.7 614 LIYRRRLMK   20 422

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class I-presented epitope. The MHC Class I-presented epitopes were predicted with the use of the online program at (http://bimas.dcrt.nih.gov/molbio/hla_bind/). Score is the T1/2 of disassociation of a peptide containing this subsequence (Tsang K Y. J Natl Cancer Inst. 1995 87:982-90). Peptides 12.5.1, 12.5.3 and 12.5.5 are presented by HLA-A*0201. Peptide 12.5.2 is presented by HLA-B*5101. Peptides 12.5.4 is presented by HLA-A*24. Peptides 12.5.6 and 12.5.7 are presented by HLA-A3.
  • [0000]
    TABLE 12.6
    Experimentally defined MHC Class I-presented
    epitopes of gp100.
    PEPTIDE Pos. Sequence SEQ ID NO:
    12.6.1 280 YLEPGPVTA 423
    12.6.2  17 ALLAVGATK 424
    12.6.3 209 ITDQVPFSV 425
    12.6.4 614 LIYRRRLMK 426
    12.6.5 619 RLMKQDFSV 427
    12.6.6 639 RLPRIFCSC 428
    12.6.7 154 KTWGQYWQV 429
    12.6.8 177 AMLGTHTMEV 430
    12.6.9 570 SLADTNSLAV 431
    12.6.10  70 VSNDGPTLI 432
    12.6.11  87 ALNFPGSQK 433

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class I-presented epitope. Peptide 12.6.1 is presented by HLA-A2 (Slingluff C. Clin Cancer Res. 2001 7:3012-24). Peptide 12.6.2 is presented by HLA-A3 (Yamshchikov G. Int J Cancer. 2001 92:703-11). Peptides 12.6.3, 12.6.5, 12.6.6 and 12.6.7 are presented by HLA-A*02012 (Kawakami Y. Proc Natl Acad Sci USA 1998). Peptide 12.6.8 and 12.6.9 are presented by HLA-A*0201 (Tsai V. J Immunol. 1997 158:1796-802). Peptide 12.6.10 is presented by HLA-Cw8 (Castelli C. J Immunol. 1999 162:1739-48). Peptide 12.6.11 is presented by HLA-A3 and HLA-A11.
  • [0000]
    TABLE 12.7
    Designed Ii-Key/gp 100 hybrids containing some
    of the MHC Class I- and Class II-presented
    epitopes of Tables 4 and 5.
    SEQ
    ID
    PEPTIDE Pos. Sequence NO:
    A. Non-overlapping epitopes
    12.7.1 II: 520, Ac-LRMK-ava-LPKEACMEI- 434
    I: 552 LVLHQILKG-NH2
    12.7.2 II: 17, Ac-LRMK-ava-ALLAVGATK- 435
    I: 3 LVLKRCLLH-NH2
    B. Overlapping epitopes
    12.7.3 II: 570, Ac-LRMK-ava- 436
    I: 576 SLADTNSLAVVSTQLIMPG-NH2
    12.7.4 II: 177, Ac-LRMK-ava- 437
    I: 175 GRAMLGTHTMEVTVY-NH2
    12.7.5 II: 70, Ac-LRMK-ava- 438
    II87, VSNDGPTLIGANASFSIALNFPGSQK-
    I: 74 NH2
    12.7.6 II: 614 Ac-LRMK-ava- 439
    (619), LIYRRRLMKQDFSVPQLPHS-NH2
    I: 615
    12.7.7 II: 154, Ac-LRMK-ava-VYVKTWGQYWQV-NH2 440
    I: 150
    12.7.8 II: 479, Ac-LRMK-ava-LYRYGSFSVTL-NH2 441
    I: 477

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope, with MHC Class II indicated as I:, and MHC Class II indicated as II:. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 1.2. Peptides 12.7.3, 12.7.4 and 12.7.5 have been already proposed (Kabayashi H. Cancer Res. 2001 61:7577-84). Peptide 12.7.6—amino acid sequence of both MHC Class I- and II-presented gp 100 epitopes are experimentally defined and coincide.
  • Example 13 Ii-Key/Tyrosinase-Related Protein 2 Antigenic Epitope Hybrids
  • [0173]
    The amino acid sequence of tyrosinase-related protein 2 as given in GenBank gi|731026|sp|P40126|TYR2_HUMAN Dopachrome tautomerase precursor (DT) (DCT) (Dopachrome delta-isomerase) (Tyrosinase-related protein 2) (TRP-2) (TRP2) is presented in Table 13.1. Predicted MHC Class II-presented epitopes of TRP-2 are listed in Table 13.2. Designed Ii-Key/TRP-2 antigenic epitope hybrids containing some of the MHC Class II-presented epitopes of Table 13.2 are listed in Table 13.3. Predicted MHC Class-I presented epitopes of TRP-2 are listed in Table 13.4. Experimentally defined MHC Class I-presented TRP-2 epitopes are listed in Table 13.5. Designed Ii-Key/TRP-2 hybrids containing some of the MHC Class I- and II-presented epitopes of Tables 13.2, 13.3, 13.4 and 13.5 are listed in Table 13.6.
  • [0000]
    TABLE 13.1
    Deduced amino acid sequence of melanocyte
    protein Pmel 17. (SEQ ID NO: 442)
      1 msplwwgfll sclgckilpg aqgqfprvcm tvdslvnkec cprlgaesan
     51 vcgsqqgrgq ctevradtrp wsgpyilrnq ddrelwprkf fhrtckctgn
    101 fagyncgdck fgwtgpncer kkppvirqni hslspqereq flgaldlakk
    151 rvhpdyvitt qhwlgllgpn gtqpqfancs vydffvwlhy ysvrdtllgp
    201 grpyraidfs hqgpafvtwh ryhllclerd lqrlignesf alpywnfatg
    251 rnecdvctdq lfgaarpddp tlisrnsrfs swetvcdsld dynhlvtlcn
    301 gtyegllrrn qmgrnsmklp tlkdirdcls lqkfdnppff qnstfsfrna
    351 legfdkadgt ldsqvmslhn lvhsflngtn alphsaandp ifvvlhsftd
    401 aifdewmkrf nppadawpqe lapighnrmy nmvpffppvt neelfltsdq
    451 lgysyaidlp vsveetpgwp ttllvvmgtl valvglfvll aflqyrrlrk
    501 gytplmethl sskryteea
  • [0000]
    TABLE 13.2
    Predicted MHC Class II-presented epitopes of
    TRP-2.
    PEPTIDE Pos. Sequence Score Ii-Key SEQ ID NO:
    13.2.1 156 YVITTQHWL 8.20 9 443
    13.2.2 451 LGYSYAIDL 7.60 444
    13.2.3  64 VRADTRPWSG 6.60 445
    13.2.4 483 LVGLFVLLA 6.10 446
    13.2.5 272 LISRNSRFS 5.70 447
    13.2.6 392 FVVLHSFTD 5.50 448
    13.2.7 219 WHRYHLLCL 5.40 7 449
    13.2.8 498 LRKGYTPLM 5.30 450
    13.2.9 365 VMSLHNLVH 5.20 451
    13.2.10 474 LVVMGTLVA 5.10 452

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0000]
    TABLE 13.3
    Designed Ii-Key/TRP-2 antigenic epitope hybrids
    containing some of the MHC Class II-presented
    epitopes of Table 13.2.
    PEPTIDE Pos. Sequence Score SEQ ID NO:
    13.3.11 156 Ac-LRMK-ava- 453
    YVITTQHWL-NH2
    13.3.12 451 Ac-LRMK-ava- 454
    LGYSYAIDL-NH2
    13.3.13  64 Ac-LRMK-ava- 455
    VRADTRPSG-NH2

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 13.2.
  • [0000]
    TABLE 13.4
    Predicted MHC Class-I presented epitopes of TRP
    2.
    PEPTIDE Pos. Sequence Score SEQ ID NO:
    13.4.1 277 SRFSSWETV 3000 456
    13.4.2 408 KRFNPPADA 3000 457
    13.4.3 325 IRDCLSLQK 2000 458
    13.4.4 150 KRVHPDYVI 1800 459
    13.4.5 427 NRMYNMVPF 1000 460
    13.4.6 485 GLFVLLAFL  999 461
    13.4.7 180 SVYDFFVWL  973 462
    13.4.8 490 LAFLQYRRL  665 463
    13.4.9 431 NMVPFFPPV  363 464
    13.4.10 185 FVWLHYYSV  348 465
    13.4.11 180 SVYDFFVWL  504 466
    13.4.12 199 GPGRPYRAI  440 467
    13.4.13 264 AARPDDPTL  360 468
    13.4.14 353 GFDKADGTL  330 469
    13.4.15 408 KRFNPPADA  300 470
    13.4.16 189 HYYSVRDTL  280 471
    13.4.17 331 LQKFDNPPF  240 472

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. The MHC Class I-presented epitopes of Table 9.4 were predicted with the use of the online program at (http://bimas.dcrt.nih.gov/molbio/hla_bind/). Score is the T1/2 of disassociation of a peptide containing this subsequence (Tsang K Y. J Natl Cancer Inst. 1995 87:982-90). Peptides 13.4.1, 13.4.2, 13.4.3, 13.4.4 and 13.4.5 are presented by HLA-B*2705. Peptides 13.4.6, 13.4.7, 13.4.9 and 13.4.10 are presented by HLA-A*0201. Peptides 13.4.8 is presented by HLA-B*5102. Peptide 13.4.11 is presented by HLA-A*0205. Peptides 13.4.12 is presented by HLA-B5101 and HLA-B*5102. Peptide 13.4.13 is presented by HLA-B7. Peptides 13.4.14 is presented by Cw*0401. Peptide 13.4.15 is presented by HLA-B*2702. Peptide 13.4.16 is presented by HLA-A24. Peptide 13.4.17 is presented by HLA-B62.
  • [0000]
    TABLE 13.5
    Experimentally defined MHC Class I-presented
    TRP-2 epitopes.
    PEPTIDE Pos. Sequence SEQ ID NO:
    13.5.1 180 SVYDFFVWL 473
    13.5.2 360 TLDSQVMSL 474
    13.5.3 288 SLDDYNHLV 475
    13.5.4 455 YAIDLPVSV 476

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the experimentally defined MHC Class I-presented epitope. Peptide 13.5.1 is presented by HLA-A2 (Parkhurst M R. Cancer Res. 1998 58:4895-901). Peptides 13.5.2 and 13.5.3 are presented by HLA-A2.1 (Noppen C. Int J Cancer. 2000 87:241-6). Peptide 13.5.4 is presented by HLA-A2.1 (Harada M. Cancer Res. 2001 61:1089-94).
  • [0000]
    TABLE 13.6
    Designed Ii-Key/TRP-2 hybrids containing some
    of the MHC Class I and II-presented epitopes of
    Tables 13.2, 13.3, 13.4 and 13.5.
    SEQ
    ID
    PEPTIDE Pos. Sequence NO:
    13.6.1 I: 180; Ac-LRMK-ava-YVITTQHWL- 477
    II: 156 SVYDFFVWL-NH2
    13.6.2 I: 455; Ac-LRMK-ava-LGYSYAIDLPVSV-NH2 478
    II: 451
    13.6.3 I: 360; Ac-LRMK-ava-TLDSQVMSLHNLVH-NH2 479
    II: 365

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 13.2 and a MHC Class I epitope of Table 13.4.
  • Example 14 Ii-Key/Melanoma Tyrosinase Antigenic Epitope Hybrids
  • [0174]
    Tyrosinase has many advantages as a target antigen for the immunotherapy of patients with melanoma because it is expressed in nearly all melanoma specimens with a high degree of cellular homogeneity, and its distribution in normal tissues is limited to melanocytes. Several MHC Class I-presented epitopes have been identified and used clinically, and MHC Class II-presented epitopes have been discovered. The following summaries of the current state-of-the-art in identification and use of peptide vaccines, DNA vaccines, and dendritic cell charging with peptide preparations (tumor cell lysates) are presented in part to illustrate the value of the products and methods of this Disclosure to improving these procedures.
  • [0175]
    Rosenberg and colleagues identified a HLA-A2.1-presented restricted melanoma tyrosinase epitope (tyrosinase8-17; CLLWSFQTSA) (SEQ ID NO: 480) (Riley J P. J Immunother. 2001 24:212-20). In this study, the comparative binding to HLA-A2.1 of a series of algorithm-predicted peptides versus that of a standard peptide with an intermediate binding affinity was determined. Twelve peptides with binding affinities within 80% of that of the standard peptide stimulated peripheral blood mononuclear cells (PBMC) in vitro from three HLA-A2.1+ patients with metastatic melanoma. PBMC from 23 HLA-A2.1+ patients were stimulated in vitro with tyrosinase:8-17. Eleven bulk T-cell cultures demonstrated specific peptide recognition, and six of these also recognized HLA-A2.1+ tyrosinase+ melanoma cells. This epitope can be incorporated in an Ii-Key/MHC Class II-presented epitope/MHC Class I-presented epitope hybrid.
  • [0176]
    Weber and colleagues found that patients with resected melanoma mounted an immune response against gp100(209-217)(210M) (IMDQVPSFV) (SEQ ID NO: 481) and tyrosinase(368-376)(370D) (YMDGTMSQV) (SEQ ID NO: 482), emulsified with incomplete Freund's adjuvant (Lee P. J Clin Oncol. 2001 19:3836-47). Patients received peptides/IFA with or without IL-12 (30 ng/kg) to evaluate the toxicities and immune responses. Immunizations were administered every 2 weeks for 8 weeks, then every 4 weeks for 12 weeks, and then once 8 weeks later. Thirty-four of 40 patients developed a positive skin test response to the gp100 peptide but none responded to the tyrosinase peptide. Immune responses were measured by release of gamma-interferon in an enzyme-linked immunosorbent assay (ELISA) by effector cells in the presence of peptide-pulsed antigen-presenting cells or by an antigen-specific tetramer flow cytometry assay. Thirty-three of 38 patients demonstrated an immune response by ELISA after vaccination, as did 37 of 42 patients by tetramer assay. Twenty-four of 48 patients relapsed with a median follow-up of 20 months, and 10 patients in this high-risk group have died.
  • [0177]
    Slingluff and colleagues evaluated peptide vaccine immunogenicity of several peptides restricted to different HLA-A alleles in draining lymph nodes and peripheral blood of melanoma patients because vaccine trials have been limited mostly to those associated with HLA-A2, and immune responses have been detected inconsistently (Yamshchikov G V. Int J Cancer. 2001 92:703-11). They vaccinated stage 1V melanoma patients with a mixture of gp100 and tyrosinase peptides restricted by HLA-A1 (DAEKSDICTDEY) (SEQ ID NO: 483), HLA-A2 (YLEPGPVTA (SEQ ID NO: 484) and YMDGTMSQV (SEQ ID NO: 485)) and HLA-A3 (ALLAVGATK) (SEQ ID NO: 486) in an emulsion with GM-CSF and Montanide ISA-51 adjuvant. CTL responses to vaccinating peptides were found in a lymph node draining a vaccine site (sentinel immunized node, SIN) in 5/5 patients (100%) in PBLs of 2/5 patients (40%). Peptides restricted by HLA-A1 and -A3 and HLA-A2 restricted peptide, YMDGTMSQV (SEQ ID NO: 485), were immunogenic.
  • [0178]
    Cytotoxic T lymphocytes against melanoma-associated antigens were induced by a recombinant vaccinia virus vector expressing multiple immunodominant epitopes and costimulatory molecules in vivo (Oertli D. Hum Gene Ther. 2002 13:569-75). Patients received psoralen-UV-treated and replication-incompetent recombinant vaccinia virus encoding the three immunodominant HLA-A*0201-restricted epitopes Melan-A(27-35), gp100(280-288), and tyrosinase(1-9) together with two costimulatory molecules, B7.1 and B7.2, in the context of systemic granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment. Subsequent boosts used corresponding synthetic nona-peptides and GM-CSF. Within 12 days of injection of the recombinant vector, cytotoxic T cell responses specific for engineered epitopes were detected in three of three patients. During the vaccination treatment, antigen-specific CTL frequencies exceeding 1:10,000 peripheral CD8+ T cells could be observed.
  • [0179]
    Two stage 1V melanoma patients vaccinated with an HLA-A2- or HLA-A24-restricted tyrosinase peptide, and GM-CSF had long-term freedom from recurrence (Scheibenbogen C. Int J Cancer. 2002 99:403-8). While the patients had experienced 9 and 12 relapses (mostly subcutaneous), respectively, during the 3 years before vaccination, they experienced freedom from relapse for more than 2 years after vaccination. T-cell responses to the vaccine peptide were found in the peripheral blood of both patients using an IFN-gamma ELISPOT assay.
  • [0180]
    Mule and colleagues found that addition of keyhole limpet hemocyanin (KLH) augmented the efficacy of both tumor lysate-pulsed dendritic cells and peptide-pulsed dendritic cells immunizations for immune priming and rejection of established metastases of the D5 subline of B16 melanoma in vivo (Shimizu K. Cancer Res. 2001 61:2618-24). Interleukin 2 further augmented the enhancement afforded by KLH, as measured by cure rates and overall survival, in the absence of autoimmune depigmentation. KLH added to dendritic cells immunizations markedly enhances tumor-specific T cell production of IFN-gamma. D5 melanoma exposed to similar levels of IFN-gamma results in substantial expression of MHC Class I molecules. Immunization with dendritic cells pulsed with KLH and mouse tyrosinase-related protein-2 peptide results in enhanced reduction of B16 melanoma metastases; the effect is most pronounced in a setting where tyrosinase-related protein-2 peptide-pulsed dendritic cells alone are completely ineffective.
  • [0181]
    Therapeutic efficacy of a tumor cell-based vaccine against B16 melanoma requires disruption of either of two immunoregulatory mechanisms that control autoreactive T cell responses: the cytotoxic T lymphocyte-associated antigen (CTLA)-4 pathway or the CD25+ regulatory T cells. Combination of CTLA-4 blockade and depletion of CD25+ T cells results in maximal tumor rejection (Sutmuller R P. J Exp Med. 2001 194:823-32). Efficacy of the antitumor therapy correlates with the extent of autoimmune skin depigmentation as well as with the frequency of tyrosinase-related protein 2(180-188)-specific CTLs detected in the periphery. Furthermore, tumor rejection is dependent on the CD8+ T cell subset. The CTL response against melanoma antigens is an important component of the therapeutic antitumor response, and the reactivity of these CTLs can be augmented through interference with immunoregulatory mechanisms. The synergism in the effects of CTLA-4 blockade and depletion of CD25+ T cells indicates that CD25+ T cells and CTLA-4 signaling represent two alternative pathways for suppression of autoreactive T cell immunity. Simultaneous intervention with both regulatory mechanisms is, therefore, a promising concept for the induction of therapeutic antitumor immunity.
  • [0182]
    The amino acid sequence of tyrosinase as given in GenBank 4507753|ref|NP000363.1| tyrosinase (oculocutaneous albinism IA); Tyrosinase [Homo sapiens] is listed in Table 14.1. Predicted MHC Class II-presented epitopes of tyrosinase are listed in Table 14.2. Experimentally defined MHC Class II-presented epitopes of tyrosinase are listed in Table 14.3. Designed Ii-Key/tyrosinase hybrids containing some of the MHC Class II-presented epitopes of Table 14.2 and 14.3 are listed in Table 14.4. Predicted MHC Class I-presented epitopes of tyrosinase are listed in Table 14.5. The experimental identification of MHC Class I-presented epitopes of tyrosinase (Pos. 240, 368, 146) is described in the gp100 example. Experimentally defined MHC Class I-presented epitopes of tyrosinase are listed in Table 14.6. Designed Ii-Key/tyrosinase hybrids containing some of the MHC Class I- and MHC Class II-presented epitopes of Tables 14.2, 14.3, 14.4 and 14.5 are listed in Table 14.7.
  • [0000]
    TABLE 14.1
    Deduced amino acid sequence of tyrosinase.
      1 mllavlycll wsfqtsaghf pracvssknl mekeccppws gdrspcgqls (SEQ ID NO: 487)
     51 grgscqnill snaplgpqfp ftgvddresw psvfynrtcq csgnfmgfnc
    101 gnckfgfwgp ncterrllvr rnifdlsape kdkffayltl akhtissdyv
    151 ipigtygqmk ngstpmfndi niydlfvwmh yyvsmdallg gseiwrdidf
    201 aheapaflpw hrlfllrweq eiqkltgden ftipywdwrd aekcdictde
    251 ymggqhptnp nhlspasffs swqivcsrle eynshqslcn gtpegplrrn
    301 pgnhdksrtp rlpssadvef clsltqyesg smdkaanfsf rntlegfasp
    351 ltgiadasqs smhnalhiym ngtmsqvqgs andpifllhh afvdsifeqw
    401 lrrhrplqev ypeanapigh nresymvpfi plyrngdffi sskdlgydys
    451 ylqdsdpdsf qdyiksyleq asriwswllg aamvgavlta llaglvsllc
    501 rhkrkqlpee kqpllmeked yhslyqshl
  • [0000]
    TABLE 14.2
    Predicted MHC Class II-presented epitopes of
    tyrosinase.
    PEPTIDE Pos. Sequence Score Ii-Key SEQ ID NO:
    14.2.1 401 LRRHRPLQE 6.60 6 488
    14.2.2 179 MHYYVSMDA 6.40 489
    14.2.3 400 WLRRHRPLQ 6.25 5 490
    14.2.4 118 LVRRNIFDL 5.70 9 491
    14.2.5 366 LHIYMNGTM 5.50 492
    14.2.6 368 IYMNGTMSQ 5.40 493
    14.2.7 182 YVSMDALLG 5.50 494
    14.2.8 150 VIPIGTYGQ 5.50 7 495
    14.2.9 338 FSFRNTLEG 5.40 496
    14.2.10 498 LLCRHKRKQ 5.30 497
    14.2.11   1 MLLAVLYCL 5.20 498
    14.2.12 167 FNDINIYDL 5.20 499

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0000]
    TABLE 14.3
    Experimentally defined MHC Class II-presented
    epitopes of tyrosinase.
    SEQ ID
    PEPTIDE Pos. Sequence Ii-Key NO:
    14.3.1  56 QNILLSNAPLGPQFP 500
    14.3.2 365 ALHIYMNGTMSQVQGSA 501
    14.3.3 156 YGQMKNGSTPMFNDINIYDL 502

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the experimentally defined MHC Class II-presented epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope. Peptide 14.3.1 is presented by HLA-DR*0401 (Storkus W. Forum (Genova). 2000 10:256-270). Peptides 14.3.2 and 14.3.3 are presented by HLA-DR*0401 (Kierstead L. Brit J Cancer. 2001 85:1738-45). Peptide 14.3.2 contains an N-glycosylation site.
  • [0000]
    TABLE 14.4
    Designed Ii-Key/tyrosinase hybrids containing
    some of the MHC Class II-presented epitopes of
    Table 14.2 and 14.3.
    SEQ
    ID
    PEPTIDE Pos. Sequence NO:
    Non-overlapping
    14.4.1  56 Ac-LRMK-ava- 503
    QNILLSNAPLGPQFP-NH2
    14.4.2 118 Ac-LRMK-ava- 504
    LVRRNIFDL-NH2
    14.4.3 338 Ac-LRMK-ava- 505
    FSFRNTLEG-NH2
    14.4.4 498 Ac-LRMK-ava- 506
    LLCRHKRKQ-NH2
    Overlapping epitopes
    14.4.5 365 Ac-LRMK-ava- 507
    ALHIYMNGTMSQVQGSA-NH2
    14.4.6 366 Ac-LRMK-ava-LHIYMNGTM-NH2 508
    14.4.7 368 Ac-LRMK-ava-IYMNGTMSQ-NH2 509
    14.4.8 365 366 and Ac-LRMK-ava-ALHIYMNGTMSQ- 510
    368 NH2
    14.4.9 182 Ac-LRMK-ava-YVSMDALLG-NH2 511
    14.4.10 179 Ac-LRMK-ava-MHYYVSMDA-NH2 512
    14.4.11 179 and 182 Ac-LRMK-ava-MHYYVSMDALLG- 513
    NH2
    14.4.12 150 Ac-LRMK-ava-VIPIGTYGQ-NH2 514
    14.4.13 156 Ac-LRMK-ava- 515
    YGQMKNGSTPMFNDINIYDL-NH2
    14.4.14 167 Ac-LRMK-ava-FNDINIYDL-NH2 516
    14.4.15 150 156 and Ac-LRMK-ava- 517
    167 VIPIGTYGQMKNGSTPMFNDINIYDL-
    NH2

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 1.2.
  • [0000]
    TABLE 14.5
    Predicted MHC Class I-presented epitopes of
    tyrosinase.
    PEPTIDE Pos. Sequence Score SEQ ID NO:
    14.5.1 243 KCDICTDEY 25.0 518
    14.5.2 369 YMNGTMSQV 531.4 519
    14.5.3   1 MLLAVLYCL 309.1 520
    14.5.4 207 FLPWHRLFL 540.5 521

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. The MHC Class I-presented epitopes of Table 9.4 were predicted with the use of the online program at (http://bimas.dcrt.nih.gov/molbio/hla_bind/). Score is the T1/2 of disassociation of a peptide containing this subsequence (Tsang K Y. J Natl Cancer Inst. 1995 87:982-90). Peptide 14.4.1 is presented by HLA-A1. Peptides 14.5.2, 14.5.3 and 14.5.4 are presented by HLA*A0201.
  • [0000]
    TABLE 14.6
    Experimentally defined MHC Class I-presented
    epitopes of tyrosinase.
    PEPTIDE Pos. Sequence SEQ ID NO:
    14.6.1 240 DAEKSDICTDEY 522
    14.6.2 368 YMDGTMSQV 523
    14.6.3 146 SSDYVIPIGTY 524

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the experimentally defined MHC Class II-presented epitope. Peptide 14.6.1 is presented by HLA-A1 (Yamshchikov G. Int J Cancer. 2001 92:703-11). Peptide 14.6.2 is presented by HLA-A2 (Yamshchikov G. Int J Cancer. 2001 92:703-11). Peptide 14.6.3 is presented by HLA-A1 (Kawakami Y. J Immunol. 1998 161:6985-92).
  • [0000]
    TABLE 14.7
    Designed Ii-Key/tyrosinase hybrids containing
    some of the MHC Class I- and MHC Class II-
    presented epitopes of Tables 14.2, 14.3, 14.5,
    and 14.6).
    SEQ ID
    PEPTIDE Pos. Sequence NO:
    Non-overlapping epitopes
    14.7.1 240 Ac-LRMK-ava- 525
    and DAEKSDICTDEY-
     56 QNILLSNAPLGPQFP-NH2
    14.7.2 207 Ac-LRMK-ava-FLPWHRLFL- 526
    and LRRHRPLQE-NH2
    401
    Overlapping epitopes
    14.7.3 368 (371D) Ac-LRMK-ava- 527
    and 365 ALHIYMNGTMSQVQGSA-NH2
    (366, 368)
    14.7.4 146 and 156 Ac-LRMK-ava- 528
    SSDYVIPIGTYGQMKNGSTPM
    FNDINIYDL-NH2
    14.7.5   1 and 1 Ac-LRMK-ava- 529
    MLLAVLYCL-NH2

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 14.2. PEPTIDE 14.7.5 includes amino acid sequences of both MHC Class I- and II-presented epitope of tyrosinase, which are experimentally defined and coincide.
  • Example 15 Ii-Key/melanoma Antigen MART-1 Antigenic Epitope Hybrids
  • [0183]
    Rosenberg and colleagues immunized metastatic melanoma patients with autologous dendritic cells presenting epitopes derived from the melanoma-associated antigens MART-1 and gp100 (Panelli M C. J Immunother. 2000 23:487-98). The DCs were generated by 5- to 7-day incubation in interleukin-4 (1,000 U/mL) and granulocyte-macrophage colony-stimulating factor (1,000 U/mL) of peripheral blood monocytes obtained by leukapheresis. Before administration, the DCs were pulsed separately with the HLA-A*0201-associated melanoma epitopes MART-1(27-35) and gp-100-209-2M. The DCs were administered four times at 3-week intervals. A first cohort of patients (n=3) was treated with 6×107 DCs and a second cohort (n=5) with 2×108 DCs (in either case, one half of the DCs were pulsed with MART-1(27-35) and the other half was pulsed with gp-100-209-2M). In a final cohort under accrual (n=2) 2×108 DCs were administered in combination with interleukin-2 (720,000 IU/kg every 8 hours). The recovery of DCs after in vitro culture ranged from 3% to 35% (mean, 15%) of the original peripheral blood monocytes. Administration of DCs caused no symptoms at any of the doses, and the concomitant administration of interleukin-2 did not cause toxicity other than that expected for interleukin-2 alone. Monitoring of patients' cytotoxic T lymphocyte reactivity before and after treatment revealed enhancement of cytotoxic T lymphocyte reactivity only in one of five patients tested. Of seven patients evaluated for response, one had a transient partial response with regression of pulmonary and cutaneous metastases.
  • [0184]
    Ioannides and colleagues demonstrated reduced recognition of metastatic melanoma cells by autologous MART-1 specific CTL correlated to TAP deficiency (Murray J L. J Immunother. 2000 23:28-35). Class I expression in context with T-cell receptor expression is crucial for peptide presentation and induction of CD8+ cytotoxic T lymphocytes (CTL). Presentation of MHC class I bound peptides depends on transporter-associated proteins (TAP) expression and function. Tumor infiltrating lymphocytes from a patient with melanoma were isolated, expanded in vitro in the presence of interleukin-2, and tested for cytotoxicity against HLA-A2 positive, MART-1 positive autologous tumor cells, an HLA-A2-positive, MART-1 positive melanoma cell line (MeI-501), and HLA-A2-negative melanoma cells. Significant killing occurred against both A2-positive cell lines (63% and 65%, respectively), but not against the A2-negative line (18%) or A2-positive autologous tumor (1.5%). These CTL preferentially recognized the MART-1 peptide F119, 27-35, and gp100 peptide F125, 280-288, resulting in a 30% to 60% enhancement of lysis when autologous tumor or major histocompatibility complex class I “empty” T2 cells were pulsed with either peptide. To address whether the deficiency in autologous tumor recognition might be related to a deficiency in Ag presentation, screening for the presence of TAP1 and TAP2 transcripts by polymerase chain reaction, Southern blotting, and scanning densitometry using sequence-specific primers and probes. Both TAP1 and TAP2 expression levels in the autologous tumor were minimal, yet were upregulated 7- to 18-fold, respectively, by interferon-gamma. Despite this increase, a similar increase in cytotoxicity did not occur. In short, deficiencies in TAP presentation may have functional significance for tumor escape from immunosurveillance and with respect to impending vaccine trials.
  • [0185]
    Slingluff and colleagues demonstrated terminal modifications inhibit proteolytic degradation of an immunogenic MART-1(27-35) peptide (Brinckerhoff L H. Int J Cancer 1999 Oct. 29; 83(3):326-34). The stability of the immunogenic peptide MART-1(27-35) in fresh normal human plasma (NHP) was tested to identify modifications protecting against enzymatic destruction without loss of immunogenicity. MART-1(27-35) peptide (AAGIGILTV) (SEQ ID NO: 530) and modified forms were incubated in plasma for varied time intervals and evaluated for their ability to reconstitute the epitope for MART-1(27-35)-reactive CTL. Loss of CTL reactivity signaled loss of immunoreactive peptide. When 1 microM MART-1(27-35) peptide was incubated in plasma prior to pulsing on target cells, CTL reactivity was lost within 3 hr, and the calculated half-life of this peptide was 22 sec. This degradation was mediated by peptidases. The stability of MART-1(27-35) was markedly prolonged by C-terminal amidation and/or N-terminal acetylation (peptide capping), or by polyethylene-glycol modification (PEGylation) of the C-terminus. These modified peptides were recognized by CTL.
  • [0186]
    Romero and colleagues demonstrated that CpG is an efficient adjuvant for specific CTL induction against tumor antigen-derived peptide (Miconnet I. J Immunol. 2002 168:1212-8). Mice transgenic for a chimeric MHC class I molecule were immunized with a peptide analog of MART-1/Melan-A(26-35) in the presence of CpG oligonucleotides alone or emulsified in IFA. The CTL response was monitored ex vivo by tetramer staining of lymphocytes. In blood, spleen, and lymph nodes, peptide mixed with CpG ODN alone was able to elicit a stronger systemic CTL response as compared with peptide emulsified in IFA. Moreover, CpG ODN in combination with IFA further enhanced the CTL response in terms of the frequency of tetramer+CD8+ T cells ex vivo. The CTL induced in vivo against peptide analog in the presence of CpG ODN are functional, as they were able to recognize and kill melanoma cells in vitro.
  • [0187]
    Mitchell and colleagues demonstrated synthetic insertion of signal sequences enhance MHC Class I presentation of a peptide from the melanoma antigen MART-1 (Minev B R. Eur J Immunol. 2000 30:2115-24). Addition of synthetic signal sequences at the N terminus, but not at the C terminus, of an epitope from the human melanoma antigen MART-1 enhanced its presentation in both TAP-deficient and TAP-expressing cells. A peptide construct, composed of the epitope replacing the hydrophobic part of a natural signal sequence, was also very effective. Interestingly, an artificial signal sequence containing the same epitope was the most efficient construct for enhancing its presentation. These peptide constructs facilitated epitope presentation when loaded into the cytosol of TAP-deficient T2 cells, TAP-expressing melanoma cells and human dendritic cells.
  • [0188]
    Zajac and colleagues demonstrated immunogenicity of nonreplicating recombinant vaccinia expressing HLA-A201 targeted or complete MART-1/Melan-A antigen (Schutz A. Cancer Gene Ther. 2001 8:655-61). The first recombinant virus expressed a minigene encoding a fusion product between an endoplasmic reticulum (ER)-targeting signal and the HLA-A201 binding MART-1/Melan-A 27-35 peptide. The second viral construct encoded the complete MART-1/Melan-A protein. The capacity of HLA-A201 cells infected with either viral construct to generate and to stimulate MART-1/Melan-A 27-35 specific cytotoxic T-lymphocytes (CTL), was comparatively characterized. The results obtained confirmed the capacity of vaccinia virus-encoded ER-minigene to generate a very strong antigenic signal. In cytotoxicity assays, recognition of target cells infected with high amounts of both recombinant viruses with activated specific CTL clones, resulted in similar lytic activity. With regard to calcium mobilization, TCR down-regulation, IFN-gamma release, and T cell proliferation assays, the targeted epitope elicited 10- to 1000-fold stronger responses. Remarkably, the immunogenic difference between the two formulations, in their respective capacity to generate CTL from naive HLA-A2 peripheral blood mononuclear cells in vitro as measured by tetramer detection, was lower (2- to 3-fold). Recombinant vectors expressing complete antigens have demonstrated their capacity to generate specific responses and such vaccines might take advantage of a broader potential of presentation. However, as demonstrated for the HLA-A201-restricted MART-1/Melan-A immunodominant epitope, nonreplicative vaccinia virus expressing ER-targeted minigenes appear to represent a significantly more immunogenic epitope vaccine formulation. Enhanced further with Ii-RGC.
  • [0189]
    Falo and colleagues demonstrated direct transfection and activation of human cutaneous dendritic cells (Larregina A T. Gene Ther. 2001 8:608-17). A gene gun was used to transfect human skin organ cultures with a particular goal of expressing transgenic antigens in resident cutaneous dendritic cells. Gold particles delivered to human skin are observed primarily in the epidermis, even when high helium delivery pressures are used. Langerhans cells resident in the basal epidermis can be transfected, and gene gun delivery is sufficient to stimulate the activation and migration of skin dendritic cells. RT-PCR analysis of dendritic cells, which have migrated from transfected skin, demonstrates transgenic mRNA, indicating direct transfection of cutaneous dendritic cells. Transfected epidermal Langerhans cells can efficiently present a peptide derived from the transgenic melanoma antigen MART-1 to a MART-1-specific CTL.
  • [0190]
    Mule and colleagues demonstrated that administration of tumor lysate-pulsed DCs is nontoxic and capable of inducing immunological response to tumor antigen (Chang A E. Clin Cancer Res. 2002 8:1021-32). Fourteen patients with stage 1V solid malignancies were treated in cohorts that received 106, 107, and 108 dendtiric cells i.d. every 2 weeks for three vaccines. Each vaccine was composed of a mixture of half DCs pulsed with autologous tumor lysate and the other half with keyhole limpet hemocyanin (KLH). Local accumulation of CD4+ and CD8+ T cells were found at the vaccination sites. There was a significant proliferative response of PBMCs to KLH induced by the vaccine. In 5 of 6 patients, the vaccine resulted in increased IFN-gamma production by PBMCs to KLH in an ELISPOT assay. Using the same assay, 3 of 7 patients' PBMCs displayed increased IFN-gamma production in response to autologous tumor lysate. One patient with melanoma also was observed to have an increased frequency of MART-1- and gp100-reactive CD8(+) T cells after vaccination. By delayed-type hypersensitivity testing, 8 of 9 and 4 of 10 patients demonstrated reactivity to KLH and autologous tumor, respectively. Ii-Key/antigenic epitope hybrids will improve the efficiency of this immunopriming technology.
  • [0191]
    Kourilsky and colleagues demonstrated cross-presentation by dendritic cells of tumor antigen expressed in apoptotic recombinant canarypox virus-infected dendritic cells (Motta I. J Immunol. 2001 167:1795-802). Recombinant canarypox virus (ALVAC) encoding the melanoma-associated Ag, Melan-A/MART-1 (MART-1), was tested in cancer immunotherapy, using a dendritic cell (DC)-based approach. ALVAC MART-1-infected DC express, and process and present, the antigen encoded by the viral vector. One consistent feature of infection by ALVAC was induction of apoptosis, and cross-presentation of Ag when uninfected DC are cocultured with ALVAC MART-1-infected DC. Uptake of apoptotic virally infected DC by uninfected DC and subsequent expression of tumor antigen in the latter were verified by flow cytometry analysis, image cytometry, and confocal microscopy. Functional activity was monitored in vitro by the stimulation of a MART-1-specific cytotoxic T cell clone. Heightened efficiency in Ag presentation was indicated by 2- to 3-fold increase in IFN-gamma production by the T cell clone, as compared with the ALVAC-infected DC alone. Cocultures of ALVAC MART-1-infected and uninfected DC are able to induce MART-1-specific T cell immune responses, as assessed by HLA class I/peptide tetramer binding, IFN-gamma ELISPOT assays, and cytotoxicity tests.
  • [0192]
    The amino acid sequence of melanoma antigen MART-1 as given in GenBank as 1082589|pir∥A55253 melanoma antigen MART-1—human is presented in Table 15.1. Predicted MHC Class II-presented epitopes of MART-1/Melan-A are listed in Table 15.2. Experimentally defined MHC Class II-presented epitopes of MART-1/Melan-A are listed in Table 15.3. Designed Ii-Key/MART-1/Melan-1 hybrids containing some of the MHC Class II-presented epitopes of Tables 15.2 and 15.3 are listed in Table 15.4. Predicted MHC Class I-presented epitopes of MART-1/Melan-A are listed in Table 15.5. Experimentally defined MHC Class I-presented epitopes of MART-1/Melan-A are listed in Table 15.6. Designed Ii-Key/MART-1 hybrids containing some of the MHC Class I- and Class II-presented epitopes of Tables 15.2, 15.3, 15.5, and 15.6 are listed in Table 15.7.
  • [0000]
    TABLE 15.1
    Deduced amino acid sequence of melanoma antigen
    MART-1. (SEQ ID NO: 531)
      1 mpredahfiy gypkkghghs yttaeeaagi giltvilgvl lligcwycrr
     51 rngyralmdk slhvgtqcal trrcpqegfd hrdskvslqe kncepvvpna
    101 ppayeklsae qspppysp
  • [0000]
    TABLE 15.2
    Predicted MHC Class II-presented epitopes of
    MART-1/Melan-A.
    PEPTIDE Pos. Sequence Score Ii-Key SEQ ID NO:
    15.2.1 33 LTVILGVLL 5.00 532
    15.2.2 35 VILGVLLLI 4.70 533
    15.2.3 96 VVPNAPPAY 4.10 6 534
    15.2.4 30 IGILTVILG 4.28 535
    15.2.5  9 IYGYPKKGH 4.10-5.10 536

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for the relative likelihood of being presented by many common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0000]
    TABLE 15.3
    Experimentally defined MHC Class II-presented
    epitopes of MART-1/Melan-A.
    PEPTIDE Pos. Sequence Ii-Key SEQ ID NO:
    15.3.1 51 RNGYRALMDKSLHVGTQ 537
    CALTRR

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope. Peptide 15.3.1 is presented by HLA-DR4 (Zarour H. Proc Natl Acad Sci USA. 2000 97:400-5).
  • [0000]
    TABLE 15.4
    Designed Ii-Key/MART-1/Melan-1 hybrids
    containing some of the MHC Class II-presented
    epitopes of Table 1.
    SEQ
    ID
    PEPTIDE Pos. Sequence NO:
    A. Non-overlapping
    15.4.1 95 Ac-LRMK-ava- 538
    VVPNAPPAY-NH2
    15.4.2  8 Ac-LRMK-ava- 539
    IYGYPKKGH-NH2
    15.4.3 51 Ac-LRMK-ava- 540
    RNGYRALMDKSLHVGTQCAL
    TRR-NH2
    B. Overlapping
    15.4.4 32 Ac-LRMK-ava-LTVILGVLL-NH2 541
    15.4.5 34 Ac-LRMK-ava-VILGVLLLI-NH2 542
    15.4.6 29 Ac-LRMK-ava-IGILTVILG-NH2 543
    15.4.7 29/32/34 Ac-LRMK-ava-IGILTVILGVLLLI-NH2 544

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 15.2.
  • [0000]
    TABLE 15.5
    Predicted MHC Class I-presented epitopes of
    MART-1/Melan-A.
    PEPTIDE Pos. Sequence Score SEQ ID NO:
    15.5.1  40 LLLIGCWYC 1289.01 545
    15.5.2  56 ALMDKSLHV 1055.10 546
    15.5.3  25 EEAAGIGIL 40.0 547
    15.5.4 109 AEQSPPPYS 12.0 548

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 1.2. The MHC Class I-presented epitopes of Table 9.4 were predicted with the use of the online program at (http://bimas.dcrt.nih.gov/molbio/hla_bind/). Score is the T1/2 of disassociation of a peptide containing this subsequence (Tsang K Y. J Natl Cancer Inst. 1995 87:982-90). Peptide 15.5.1 is presented by HLA-A*0201, HLA-A3, and HLA-A31. Peptide 15.5.2 is presented by HLA-A*0201. Peptides 15.5.3 and 15.5.4 are presented by HLA-B40.
  • [0000]
    TABLE 15.6
    Experimentally defined MHC Class I-presented
    epitopes of MART-1/Melan-A.
    PEPTIDE Pos. Sequence SEQ ID NO:
    15.6.1 27 AAGIGILTV 549
    15.6.2 32 ILTVILGVL 550
    15.6.3 24 AEEAAGIGILT 551

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the experimentally defined MHC Class II-presented epitope. Peptide 15.6.1 is presented by HLA-A*0201 (Kawakami, Y. J Exp Med. 1994 180:347-52). Peptide 15.6.2 is presented by HLA-A*0201 (Castelli C. J Exp Med. 1995 181:63-8). Peptide 15.6.3 is presented by HLA-B*4501 (Schneider J. Intl J Cancer. 1998 75:451-8).
  • [0000]
    TABLE 15.7
    Designed Ii-Key/MART-1 hybrids containing some
    of the MHC Class I- and Class II-presented
    epitopes of 15.2, 15.3, 15.5, and 15.6).
    SEQ ID NO: Pos. Sequence SEQ ID NO:
    15.7.1 27 Ac-LRMK-ava- 552
    and AAGIGILTV-
    51 RNGYRALMDKSLHVGTQCALT
    RR-NH2

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II-presented epitope of Table 15.2 and a MHC Class I-presented epitope of Table 15.6.
  • Example 16 Ii-Key/Her-2 Neu Antigenic Epitope Hybrids
  • [0193]
    Immunotherapy directed against the epidermal growth factor receptor which is overexpressed on some cancer cells can control the growth of those tumors. HER-2/neu is over-expressed on tumors in up to 30% of patients with invasive breast cancer and that over-expression is associated with poor clinical outcome. Carr et al. demonstrated in a retrospective consecutive series from 1995 to 1999 that the HER-2/neu gene was amplified in invasive breast carcinomas of 40 of 90 patients (43%) (Carr J A. Arch Surg. 2000 135:1469-7420). Following initial therapy, patients with HER-2/neu amplification had a shorter median disease-free interval (22 months) than did patients with breast cancers not amplifying that gene (40 months; p=0.003). Disease recurred in seventy-two (72%) patients, with 18 (25%) recurring locally. HER-2/neu gene amplification is an independent prognostic indicator for a subset of breast cancer patients who are at high risk for early recurrence regardless of tumor grade, estrogen/progesterone receptor status, and lymph node status. In both early stage, lymph node-negative and node-positive disease, as well as in women with metastatic disease, HER-2/neu overexpression is associated with worse survival. Women with tumors that overexpress HER-2/neu have a less favorable outcome despite adjuvant treatment with either hormonal therapy or chemotherapy. Among HER-2/neu-negative, early stage patients in the Naples GUN trial, tamoxifen benefited overall survival. However, among patients with HER-2/neu-gene amplification, tamoxifen did not improve survival (De Placido S. Br J Cancer. 1990 62:643-6). HER-2/neu over-expression is an independent predictor for tamoxifen failure. Over-expression of HER-2/neu is selective for tumor cells and is observed early in the course of malignant transformation. More importantly, the cytological characteristics of HER-2/neu over-expression (32%) in primary and metastatic lesions is nearly identical (Masood S. Ann Clin Lab Sci. 2000 30:259-65). Inasmuch as micrometastases are the primary source of relapse following primary therapy and HER-2/neu is over-expressed in metastases, HER-2/neu is an excellent target for immunotherapy of patients with early disease, both to consolidate the anti-tumor response locally and to eradicate micrometastases. Likewise, HER-2/neu should be targeted in conjunction with other major treatment regimens in patients who have relapsed following initial therapy.
  • [0194]
    Of many approaches to targeting HER-2/neu, the clinically most advanced approach is passive immunotherapy with trastuzumab (Herceptin®), an FDA-approved humanized monoclonal antibody that binds to the extracellular domain of the HER-2/neu receptor for epidermal growth factor (EGF). This monoclonal antibody is indicated both as a single agent and in combination with classical chemotherapies. Slamon et al. evaluated Herceptin® in combination with doxorubicin and cyclophosphamide (AC), or paclitaxel in 496 women with metastatic breast carcinomas that over expressed HER-2/neu (Vogel C L. J Clin Oncol. 2002 20:719-26; Slamon D J. N Engl J Med. 2001 344:783-92). Patients receiving Herceptin®, as compared to patients randomized to chemotherapy alone (either paclitaxel or AC), had a significantly longer time to disease progression (7.4 mo vs. 4.6 mo; p<0.0001), a higher rate of objective response (50% vs. 32%; p<0.001), a longer duration of response (median 9.1 vs. 6.1; p<0.001), a higher 1 year survival rate (78% vs. 67%; p=0.008), longer survival (median survival 25.1 mo vs. 20.3 mo; p=0.046), and a 20% reduction in the risk of death.
  • [0195]
    While clinical trials might proceed to alternate trastuzumab dosing regimens and combination therapies, one can suggest that the mechanism of action of trastuzumab will not lead to significantly increased efficacy. Specifically, Trastuzumab blocks the HER-2/neu EGF receptor and induces antibody dependent cellular cytotoxicity (Sliwkowski M X. Semin Oncol. 1999 4 Suppl 12:60-70). ADCC does not lead to antigen-specific memory of T- or B-lymphocytes, nor does it induce proliferation of antigen-specific cytotoxic T-lymphocytes.
  • [0196]
    HER-2/neu is also the target for several vaccine trials to induce an active specific immune response. In the NCI PDQ, three current clinical trials use HER-2/neu protein, antigen-pulsed dendritic cells, liposome-encapsulated HER-2/neu MHC peptide epitopes, and a DNA vaccine (http://www.cancer.gov/cancer_information/doc.aspx?viewid=F2 AFAEA4-64BD-4E44-B421-56026E252389). The rationale, of course, is to enhance therapeutic efficacy and clinical ease of administration by inducing: (1) antigen-specific CD8+ and CD4+ lymphocytes; (2) autoantibodies against HER-2/neu with memory B-cells; and (3) memory helper T cells.
  • [0197]
    Compared to cell-based vaccines, DNA vaccines, and gene therapy approaches, peptide vaccination is preferred for several reasons. Specifically, peptide vaccines are: (1) easily constructed and manufactured; (2) chemically stable; (3) free of adventitious agents and other pathogens; and, (4) devoid of oncogenic potential. Until recently, most groups have focused on the use of MHC Class I peptide vaccines, which have triggered low-intensity CD8+ cytotoxic T cell responses. Shiku and colleagues have identified a novel human Her-2/neu2-derived peptide which is homologous to a mouse H-2Kd-restricted tumor antigen induces HLA-A24-restricted cytotoxic T lymphocytes in ovarian cancer patients and healthy individuals (Okugawa T. Eur J Immunol. 2000 30:3338-46; Ikuta Y. Int J Cancer. 2000 87: 553-8; Nagata Y. J Immunol. 1997 159:1336-43). In addition they have demonstrated presentation of a MHC Class I-binding peptide by monocyte-derived dendritic cells incorporating a hydrophobized polysaccharide-truncated Her-2/neu protein complex (Ikuta Y. Blood. 2002 99:3717-24; Araki H. Br J Haematol. 2001 114:681-9).
  • [0198]
    Peptide vaccines do enhance responses by CTL cells recognizing MHC Class I-presented peptides, but can be augmented by also immunizing T helper cells with MHC Class II-presented peptides. HER-2/neu-derived, MHC Class II-presented peptides are expressed by human breast, colorectal and pancreatic adenocarcinomas and are recognized by in vitro-induced, specific CD4+ T cell clones (Perez S. Cancer Immunol Immunother. 2002 50:615-24; Sotiriadou R. Br J Cancer. 2001 85:1527-34). Murray et al. showed that the Her-2/neu(777-789) peptide induced peripheral blood mononuclear cells from patients with metastatic breast cancer to secrete IFN-γ (Murray J L. Semin Oncol. 2000 27 Suppl:71-5). This group also showed that Her-2/neu(369-377) induced strong CTL response in peripheral blood mononuclear cells from healthy donors (Anderson B W. Clin Cancer Res. 2000 6:4192-200; Anderson B W. Cancer Immunol Immunother. 2000 49:459-68), as well as the secretion of CXC chemokine IP-10 from peripheral blood mononuclear cells from breast cancer patients and healthy donors (Lee T V. J Interferon Cytokine Res. 2000 20:391-401). However, in a clinical trial with that MHC Class I peptide only 3/9 patients had lymphocyte proliferative responses that were above baseline following vaccination (Murray J L. Semin Oncol. 2000 27 Suppl:71-5). Increased CTL proliferation and IFN-ã levels were seen in stimulated cultures of peripheral blood mononuclear cells of only one vaccinated patient. In 3 of 5 patients, IFN-ã and CTL activity were increased significantly by IL-12 addition, indicating that weak antigen presentation leads to weak CTL induction, which is reversed partially in vitro with pro-inflammatory cytokines. However, MHC Class I peptide immunization does not induce helper CD4+ T cell responses. For this reason, peptide vaccines are sought with either only a MHC Class II presented, CD4+ T-helper cell stimulating epitope or with a peptide in which a MHC Class II-presented, CD4+ T-helper cell stimulating epitope overlays a MHC Class I-presented, CD8+ T-cytotoxic cell stimulating epitope.
  • [0199]
    Peripheral blood mononuclear cells from healthy donors and ovarian cancer patients do respond to Her-2/neu peptides (Fisk B. Anticancer Res. 1997 17:45-53). Peptide sequences from Her-2/neu containing anchors for major human MHC-class II molecules induced proliferative and cytokine responses at a higher frequency in healthy donors than in ovarian cancer patients. Four Her-2/neu peptides of sequences: 396-406, 474-487, 777-789, and 884-899 stimulated proliferation of a larger number of healthy donors than three other distinct HER-2 peptides 449-464, 975-987 and 1086-1098. The pattern of responses of twenty-five ovarian cancer patients was different from that of healthy donors. T cell lines were developed by stimulation with peptides of peripheral blood mononuclear cells of an ovarian cancer patient who showed a stable response to all four Her-2/neu peptides over six months. Each T cell line differed in secretion of IFN-gamma and IL-10. These results demonstrate (a) that Her-2/neu peptides can stimulate expansion of T cells in both healthy donors and ovarian cancer patients, and (b) different peptides induce different cytokine secretion patterns. (J Interferon Cytokine Res. 2002 May; 22(5):583-92)
  • [0200]
    Ioannides and colleagues demonstrated axillary lymph nodes from patients with breast carcinoma respond to HER-2/neu peptides (Kuerer H M. J Interferon Cytokine Res. 2002 22:583-92). Freshly isolated lymphocytes from lymph nodes of 7 women undergoing surgery for invasive breast cancer were stimulated with HER-2/neu peptides at 50 μm/ml and with control antigens. IFN-γ, IL-4, and IL-10 levels were determined at priming and at restimulation with HER-2/neu peptides. Lymphocytes isolated from the axillary lymph nodes of the patients responded to HER-2/neu peptides, proliferating and specific cytokine production. Proliferative responses to HER-2/neu peptides were seen in lymphocytes of patients with and without overexpression of HER-2/neu in the primary tumor. In some patients, the proliferative response to HER-2/neu peptides in lymphocytes from lymph nodes with metastases was absent or decreased compared to response in lymphocytes from lymph nodes without metastases from the same patient (p<0.05). HER-2/neu peptides induced a predominantly T helper type 1 (Th1) pattern of cytokine response in nodal lymphocytes isolated from breast cancer patients. A Th1-specific cytokine production pattern was maintained at priming and restimulation with HER-2/neu peptides and was amplified with IL-12 costimulation. These results indicate that HER-2/neu peptides can activate T cells in draining lymph nodes from women with invasive breast cancer.
  • [0201]
    Patients immunized with an HLA-A2-presented, Her-2/neu peptide developed only a low level and short-lived CTL response, in the absence of concurrent vaccination with a MHC Class II-presented epitope (Ward R L. Hum Immunol. 1999 60:510-5). Six HLA-A2 patients with Her-2/neu-overexpressing cancers received 6 monthly vaccinations with a vaccine preparation consisting of 500 μg of Her-2/neu(369-377) peptide, admixed with 100 μg of GM-CSF. The patients had either stage III or IV breast or ovarian cancer. Immune responses to the Her-2/neu(369-377) peptide were examined using an IFN-γ enzyme-linked immunosorbent spot assay. Although HER-2/neu MHC class I epitopes induced HER-2/neu peptide-specific IFN-γ-producing CD8+ T cells, the magnitudes of the responses were low, as well as short-lived, indicating that CD4+ T-cell help is required for robust and lasting immunity to this epitope.
  • [0202]
    Disis and colleagues immunized with breast cancer patients a HER-2/neu helper peptide vaccine generating HER-2/neu CD8 T-cell immunity (Knutson K L. J Clin Invest. 2001 107:477-84). Nineteen HLA-A2 patients with HER-2/neu-overexpressing cancers received a vaccine preparation consisting of Her-2/neu(369-384), Her-2/neu(688-703), and Her-2/neu(971-984). Contained within these sequences are HLA-A2-binding motifs Her-2/neu(369-377), Her-2/neu(689-697), and Her-2/neu(971-979). After vaccination, the mean peptide-specific T-cell precursor frequency to the HLA-A2 peptides increased in the majority of patients. In addition, the peptide-specific T cells were able to lyse tumors. The responses were long-lived and detected for more than 1 year after the final vaccination in some patients. These results demonstrate that Her-2/neu MHC class II epitopes containing overlaying MHC Class I epitopes induce long-lasting Her-2/neu-specific IFN-γ-producing CD8+ T cells.
  • [0203]
    Disis and colleagues immunized sixty-four patients with HER-2/neu-overexpressing breast, ovarian, or non-small-cell lung cancers with vaccines composed of peptides derived from potential T-helper epitopes of the HER-2/neu protein mixed with granulocyte-macrophage colony-stimulating factor and administered intradermally (Disis M L. J Clin Oncol. 2002 20:2624-32). Nine different epitopes were used: 3 derived from the intracellular domain of her-2/neu (p776-790, p927-941, and p1166-1180), 3 derived from the extracellular domain of her-2/neu (p42-56, p98-114, and p328-345), and 3 with helper epitopes that encompass in their natural sequence HLA-A2 binding motifs (p369-384, p688-703, and p971-984). Ninety-two percent of patients developed T-cell immunity to HER-2/neu peptides and 68% to a HER-2/neu protein domain. Epitope spreading was observed in 84% of patients and correlated with the generation of a HER-2/neu protein-specific T-cell immunity (P=0.03). At 1-year follow-up, immunity to the HER-2/neu protein persisted in 38% of patients. No patient developed any detected autoimmune toxicity, particularly in organs known to express basal levels of her-2/neu protein including the liver, digestive tract, and skin. The incorporation of MHC Class II epitopes used in this study in Ii-Key hybrid molecules might lead to more rapid anti-her-2/neu immune responses with lower and fewer doses, greater epitope spreading, induction of higher affinity T-cells against tumor, more prolonged immune responses against epitopes and her-2/neu protein, and greater clinical efficacy.
  • [0204]
    Finding tumor-reactive CTLs in tumor infiltrates and in the peripheral blood of cancer patients, raises the question that any anti-tumor immune response does not control disease spread (Anderson B W. Clin Cancer Res. 2000 6:4192-200). One might then question whether amplification of this response by peptide vaccines is useful during disease progression. Induction of tumor-reactive CTLs in healthy donors at risk, as well as in patients free of disease, has been proposed on the hypothesis that CTLs that recognize tumors early are more effective in containing their progression than CTLs that expand only when the disease progresses. Priming of cytolytic T cell activity in 10 healthy donors was tested with Her-2/neu(369-377) peptide as an immunogen and autologous peripheral blood mononuclear cell-derived dendritic cells as antigen presenting cells. Of those two responded at priming with Her-2/neu(369-377) peptide presented on autologous dendritic cells by induction of Her-2/neu(369-377) peptide-specific CTL activity. Three other responders were identified after two additional restimulations. Induction of cytolytic activity at priming was enhanced in responders by tumor necrosis factor-alpha and IL-12 but not in the non-responders.
  • [0205]
    Determinant spreading and Th1 responses were induced by in vitro stimulation with Her-2/neu peptides (Anderson B W. Cancer Immunol Immunother 2000 49:459-68). The induction of a response to Her-2/neu(776-789) induced reactivity to other Her-2/neu peptides. Her-2/neu(776-789) expanded a response to Her-2/neu (884-899) in both an ovarian cancer patient with progressive disease and a healthy donor who shared HLA-DR11. This response was characterized mainly by increased IFN-γ secretion, and proliferation, but did not occur with another donor who shared only HLA-DR14 and HLA-DQ5 with the patient. Epitope spreading can also be enhanced by the coordinated use of Ii-Key/antigenic epitope hybrids immunizations with Ii reverse gene construct, Her-2/neu gene immunizations.
  • [0206]
    Hess and colleagues found that a chimeric construct of an MHC class II binding peptide from Her-2/neu and the N-terminal flanking region of CLIP elicited potent antitumor activity against a Her-2/neu-positive tumor in a rat model system (Hess A D. Clin Immunol 2001 101:67-76). Induction of effective antitumor immunity required presentation of the chimeric peptide on irradiated tumor cells or in concert with a Her-2/neu MHC class I-restricted peptide from Her-2/neu. Adoptive transfer studies showed the need for CD4 T helper cells for protective antitumor immunity. Immunization with the epitope-only peptide caused a weak immune response to the unmodified peptide in vitro of both type 1 (IL-2, IFN-γ) and type 2 (IL-4, IL-10) cytokine-producing cells analyzed by RT-PCR (qualitative and quantitative) and by limiting dilution assay. Comparatively, immunization with the chimeric construct elicited a potent immune response to the parent epitope with predominantly type 1 cytokine-producing cells.
  • [0207]
    Accelerated Her-2/neu degradation enhanced ovarian tumor recognition by CTL (Castilleja A. Mol Cell Biochem. 2001 217:21-33). In those studies, Her-2/neu degradation was enhanced in the ovarian tumor line, SKOV3.A2, that constitutively overexpressed Her-2/neu by the addition of geldanamycin, which down-modulated Her-2/neu from the cell surface and promoted its polyubiquitinylation and degradation. Presentation of the immunodominant cytotoxic T lymphocyte (CTL) epitope, Her-2/neu(369-377) from SKOV.A2 was inhibited by proteosome inhibitors, such as LLnL. Additional experiments indicated that the newly synthesized Her-2/neu in the presence of GA was the main source of epitopes recognized by CTL. Twenty-hour GA-treated SKOV3.A2 cells were better inducers of CTL activity directed to a number of Her-2/neu CTL epitopes, in peripheral blood mononuclear cells compared with control untreated SKOV3.A2 cells thereby promoting immunogenecity. Similarly geldanamycin and other compounds acting by a similar mechanism, are expected to enhance binding of MHC Class II epitopes in the ER in the absence of Ii protein.
  • [0208]
    Ward and colleagues used phage-displayed ErbB-2 gene fragment libraries and synthetic peptides to epitope-map a panel of anti-Her-2/neu monoclonal antibodies (Yip Y L. Cancer Immunol Immunother. 2002 50:569-87; Yip Y L. J Immunol. 2001 166:5271-8). The epitopes of three monoclonal antibodies, N12, N28, and L87, were successfully located to Her-2/neu(C531-A586), Her-2/neu(T216-C235), and Her-2/neu(C220-C235) of Her-2/neu, respectively. It was found that while N12 inhibited tumor cell proliferation, N28 stimulated the proliferation of a subset of breast cancer cell lines over-expressing Her-2/neu. The peptide region recognized by N12, Her-2/neu(C531-A586), was used as an immunogen to selectively induce an inhibitory immune response in mice. Mice immunized with the GST fusion peptide, GST-Her-2/neu(C531-A586), recognized native Her-2/neu, the peptide Her-2/neu(531-586), three 15-amino acid peptides of Her-2/neu(533-548), Her-2/neu(545-5560), and Her-2/neu(571-586). More importantly, immunoglobulins purified from mouse sera were able to inhibit up to 85% of tumor cell proliferation. This study supports the use of some of the potential antibody recognized determinants in the construction of Ii-Key/Her-2/neu MHC Class II-presented antigenic epitope/antibody-recognized determinant hybrids. The antibody recognized determinants are presented in Table 16.8 and hybrids containing those epitopes are presented in Table 16.9. Such hybrids containing antibody-recognized determinants might be preferred can be used for the development of both passive and active immunotherapies of Her-2/neu over-expressing tumors.
  • [0209]
    Given the experimentally identified MHC Class II-presented epitopes (above) such epitope can be synthesized within Ii-Key/Her-2/neu antigenic epitope hybrids for stimulation of a diagnostic or therapeutic immune response.
  • [0210]
    The amino acid sequence of human Her-2/neu protein [Homo sapiens](gi|19575768|) was obtained from GenBank (Table 16.1). An important consideration in the selection of peptides for cancer immunotherapy is the high degree of sequence homology between Her-2/neu and another member of the subclass I family of growth factor receptor (EGF-r) (Lustgarten J. Hum Immunol. 1997 52:109-18). Unlike Her-2/neu, the EGF-r is widely expressed in the body. Peptide sequences identical between Her-2/neu and the mouse or human EGF-r were not selected for two reasons. First, it is likely that T-cell tolerance to such sequences would have eliminated from the repertoire high affinity T cells with specificity for such epitopes. Second, it would be undesirable to target CTL against normal cell expressing EGF-r peptides. Predicted MHC Class II-presented epitopes of Her-2/neu protein are presented in Table 16.2. Experimentally determined MHC Class II-restricted epitope of human Her-2/neu protein are listed in Table 16.3. Designed Ii-Key/Her-2/neu hybrids using some of the MHC Class II-presented epitopes of Tables 2 and 3 are listed in Table 16.4. Predicted MHC Class I-presented epitopes of Her-2/neu protein are listed in Table 16.5. Experimentally determined MHC Class I-presented epitopes of Her-2/neu protein are listed in Table 16.6. Designed Ii-key/MHC Class II epitope/MHC Class I epitope hybrids are listed in Table 16.7. Antibody-recognized determinants on Her-2/neu are listed in Table 16.8 Designed Ii-Key/Her-2/neu hybrids using some of the antibody-recognized determinants of Table 16.8 and MHC Class II-presented epitopes of Tables 2 and 3 are presented in Table 16.9.
  • [0000]
    TABLE 16.1
    Deduced amino acid sequence of Her-2/neu.
       1 melaalcrwg lllallppga astqvctgtd mklrlpaspe thldmlrhly (SEQ ID NO: 553)
      51 qgcqvvqgnl eltylptnas lsflqdiqev qgyvliahnq vrqvplqrlr
     101 ivrgtqlfed nyalavldng dplnnttpvt gaspgglrel qlrslteilk
     151 ggvliqrnpq lcyqdtilwk difhknnqla ltlidtnrsr achpcspmck
     201 gsrcwgesse dcqsltrtvc aggcarckgp lptdccheqc aagctgpkhs
     251 dclaclhfnh sgicelhcpa lvtyntdtfe smpnpegryt fgascvtacp
     301 ynylstdvgs ctlvcplhnq evtaedgtqr cekcskpcar vcyglgmehl
     351 revravtsan iqefagckki fgslaflpes fdgdpasnta plqpeqlqvf
     401 etleeitgyl yisawpdslp dlsvfqnlqv irgrilhnga ysltlqglgi
     451 swlglrslre lgsglalihh nthlcfvhtv pwdqlfrnph qallhtanrp
     501 edecvgegla chqlcarghc wgpgptqcvn csqflrgqec veecrvlqgl
     551 preyvnarhc lpchpecqpq ngsvtcfgpe adqcvacahy kdppfcvarc
     601 psgvkpdlsy mpiwkfpdee gacqpcpinc thscvdlddk gcpaeqrasp
     651 ltsiisavvg illvvvlgvv fgilikrrqq kirkytmrrl iqetelvepl
     701 tpsgampnqa qmrilketel rkvkvlgsga fgtvykgiwi pdgenvkipv
     751 aikvlrents pkankeilde ayvmagvgsp yvsrllgicl tstvqlvtql
     801 mpygclldhv renrgrlgsq dllnwcmqia kgmsyledvr lvhrdlaarn
     851 vlvkspnhvk itdfglarll dideteyhad ggkvpikwma lesilrrrft
     901 hqsdvwsygv tvwelmtfga kpydgipare ipdllekger lpqppictid
     951 vymimvkcwm idsecrprfr elvsefsrma rdpqrfvviq nedlgpaspl
    1001 dstfyrslle dddmgdlvda eeylvpqqgf fcpdpapgag gmvhhrhrss
    1051 strsgggdlt lglepseeea prsplapseg agsdvfdgdl gmgaakglqs
    1101 lpthdpsplq rysedptvpl psetdgyvap ltcspqpeyv nqpdvrpqpp
    1151 spregplpaa rpagatlerp ktlspgkngv vkdvfafgga venpeyltpq
    1201 ggaapqphpp pafspafdnl yywdqdpper gappstfkgt ptaenpeylg
    1251 ldvpv
  • [0000]
    TABLE 16.2
    Predicted MHC Class II-presented epitopes of
    Her-2/neu protein.
    Ii- SEQ. ID.
    PEPTIDE Pos. Sequence Score Key NO.
    16.2.1  985 FVVIQNEDL 7.40  6 554
    16.2.2   98 LRIVRGTQL 7.30  4 555
    16.2.3  952 MIMVKCWMI 7.20 556
    16.2.4  894 LRRRFTHQS 7.00  6 557
    16.2.5  684 YTMRRLLQE 6.70  6 558
    16.2.6  664 VVLGVVFGI 5.90 559
    16.2.7 1041 MVHHRHRSS 5.60 560
    16.2.8  421 LSVFQNLQV 5.50 561
    16.2.9  180 LTLIDTNRS 5.40  4 562
    16.2.10  670 FGILIKRRQ 5.40 563
    16.2.11  396 LQVFETLEE 5.20 564
    16.2.12   61 LTYLPTNAS 5.10 11 565
    16.2.13  951 YMIMVKCWM 5.00 566
    16.2.14  719 LRKVKVLGS 5.00  4 567
    16.2.15  424 FQNLQVIRG 5.20 568

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. Score is the score reported by the ProPred program, for high scoring selections with multiple common HLA-DR alleles. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0000]
    TABLE 16.3
    Experimentally determined MHC Class II-
    restricted epitope of human Her-2/neu protein.
    SEQ. ID.
    PEPTIDE Pos. Sequence NO.
    16.3.1  884 VPIKWMALESILRRR 569
    16.3.2  776 GSPYVSRLLGICL 570
    16.3.3  396 QLQVFETLEEI 571
    16.3.4  474 LCFVHTVPWDQLF 572
    16.3.5  450 GISWLGLRSLRE 573
    16.3.6  975 EFSRMARDPQRF 574
    16.3.7 1086 FDGDLGMAAKGL 575
    16.3.8   42 HLDMLRHLYQGCQVV 576
    16.3.9   98 LRIVRGTQLFEDNYAL 577
    16.3.10  328 TQRCEKCSKPCARVCYGL 578
    16.3.11  776 LGSGAFGTVYKGIWI 579
    16.3.12  927 PAREIPDLLEKGERL 580
    16.3.13 1166 TLERPKTLSPGKNGV 581
    16.3.14  369 KKIFGSLAFLPESFDGD 582
    16.3.15  688 RQQKIRKYTMRRLLQE 583
    16.3.16  971 ELVSEFSRMARDPQ 584

    Pos. is the residue position in the primary sequence of the first amino acid in the peptide. Sequence is the amino acid sequence of the experimentally determined MHC Class II-presented epitope. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope. Peptide 16.3.1 was reported by Perez S. et al. (Cancer Immunol Immunother. 2002 50:615-24). Peptide 16.3.2 was reported by Sotiriadou R. et al. (Br J Cancer. 2001 85:1527-34). Peptide 16.3.3 was reported by Fisk B. et al. (Anticancer Res. 1997 17:45-53). Peptides 16.3.8-16.3.16 are those reported in a Phase I clinical trial by Disis and colleagues (Disis M L. J Clin Oncol 2002 20:2624-32). Peptide 16.3.9 contains a predicted HLA-DRB1-0101-presented motif LRIVRTGTQL (SEQ ID NO: 585) and PEPTIDE 16.3.16 contains a DRB1-0101-presented motif LVSEFSRMA (SEQ ID NO: 586); both stimulated lymphocytes from an immunized patients. Additional peptides in the series studied by Disis et al. might be found to containing MHC Class II-presented motifs when tested for additional HLA-DB alleles and to lower indices for scoring. Such epitopes are subject to being incorporated in Ii-Key/Her-2 antigenic epitope hybrids.
  • [0000]
    TABLE 16.4
    Designed Ii-Key/Her-2/neu hybrids using some of
    the MHC Class II-presented epitopes of Tables 2
    and 3.
    SEQ.
    ID.
    PEPTIDE Pos. Sequence NO.
    A. Non-overlapping
    16.4.1  776 Ac-LRMK-ava-GSPYVSRLLGICL-NH2 587
    16.4.2  396 Ac-LRMK-ava-QLQVFETLEEI-NH2 588
    16.4.3  985 Ac-LRMK-ava-FVVIQNEDL-NH2 589
    16.4.4   98 Ac-LRMK-ava-LRIVRGTQL-NH2 590
    16.4.5  894 Ac-LRMK-ava-LRRRFTHQS-NH2 591
    16.4.6  684 Ac-LRNK-ava-YTMRRLLQE-NH2 592
    16.4.7 1041 Ac-LRMK-ava-MVHHRHRSS-NH2 593
    16.4.8  972 Ac-LRMK-ava-LVSEFSRMA-NH2 594
    B. Overlapping
    16.4.8  884, Ac-LRMK-ava- 595
     894 VPIKWMALESILRRRFTHQS-NH2
    16.4.9  664, Ac-LRMK-ava-VVLGVVFGILIKRRQ-NH2 596
     670
    16.4.10  951, Ac-LRMK-ava-YMIMVKCWMI-NH2 597
     952
    16.4.11  421, Ac-LRMK-ava-LSVFQNLQVIRG-NH2 598
     424

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 16.2 and 16.3.
  • [0000]
    TABLE 16.5
    Predicted MHC Class I-presented epitopes of
    Her-2/neu protein.
    SEQ. ID.
    PEPTIDE Pos. Sequence Score NO.
    16.5.1  661 ILLVVVLGV 1006.2 599
    16.5.1  369 KIFGSLAFL 481.2 600
    16.5.1  167 ILWKDIFHK 450.0 601
    16.5.1   63 TYLPTNASL 360.0 602
    16.5.2  106 QLFEDNYAL 324.1 603
    16.5.3  553 EYVNARHCL 300.0 604
    16.5.4  440 AYSLTLQGL 240.0 605
    16.5.5  907 SYGVTVWEL 220.0 606
    16.5.6 1022 EYLVPQQGF 180.0 607
    16.5.7  689 RLLQETELV 126.1 608
    16.5.8  714 ILKETELRK 60.0 609
    16.5.9  754 VLRENTSPK 30.0 610
    16.5.10  673 ILIKRRQQK 30.0 611

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class I-presented epitope. The MHC Class I-presented epitopes were predicted with the use of the online program at (http://bimas.dcrt.nih.gov/molbio/hla_bind/). Score is the T1/2 of disassociation of a peptide containing this subsequence (Tsang K Y. J Natl Cancer Inst. 1995 87:982-90).
  • [0000]
    TABLE 16.6
    Experimentally determined MHC Class I-presented
    epitopes of Her-2/neu protein.
    SEQ. ID.
    PEPTIDE Pos. Sequence NO.
    16.6.1 106 QLFEDNYAL 612
    16.6.2 369 KIFGSLAFL 613
    16.6.3 689 RLLQETELV 614
    16.6.4 435 ILHNGAYSL 615
    16.6.5 665 VVLGVVFGI 616
    16.6.6 952 YMIMVKCWM 617
    16.6.7 654 IISAVVGIL 618
    16.6.8 654 FLSAVVGILV 619
    16.6.9 773 VMAGVGSPYV 620
    16.6.10 754 VLRENTSPK 621

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the experimentally defined MHC Class I-presented epitope. Peptide 16.6.1 is presented by HLA-A2.1 (Kono K. Int j Cancer. 1998 78:202-8). Peptide 16.6.2 is presented by HLA-A2.1 (Kono K. Int J Cancer. 1998 78:202-8), as confirmed by Rongcun Y., et al. (J Immunol. 1999 163:1037-44). It was also shown to be immunogenic in double transgenic mice expressing HLA-A2.1 and human CD8 (Lustgarten J. Hum Immunol. 1997 52:109-18). Peptide 16.6.3 is presented by HLA-A2.1 (Kono, K. Int J Cancer. 1998 78:202-8; Rongcun Y. J Immunol. 1999 163:1037-44). It was nonimmunogenic in the study of Lustgarten J. et al. (Hum Immunol. 1997 52:109-18). Peptides 16.6.4, 16.6.5 and 16.6.6 are presented by HLA-A2.1 (Rongcun Y. J Immunol. 1999 163:1037-44). Peptide 16.6.7 is presented by HLA-A2 (Peoples G. Proc Natl Acad Sci USA. 1995 92:432-6) and is nonimmunogenic in the study of Lustgarten, J. et al. (Hum Immunol. 1997 52:109-18). Peptide 16.6.8 is presented by HLA-A2 (Tanaka Y. Int J Cancer. 2001 94:540-4). Peptide 16.6.9 is presented by HLA-A2.1 (Lustgarten J. Hum Immunol. 1997 52:109-18). Peptide 16.6.10 is presented by HLA-A3 (Kawashima I. Cancer Res. 1999 59:431-5).
  • [0000]
    TABLE 16.7
    Designed Ii-key/MHC Class II epitope/MHC Class
    I epitope hybrids.
    SEQ.
    ID.
    PEPTIDE Pos. Sequence NO.
    16.7.1 II: 76, Ac-LRMK-ava- 622
    I: 73 VMAGVGSPYVSRLLGICL-NH2
    16.7.2 II: 396, Ac-LRMK-ava-QLQVFETLEEI- 623
    I: 369 KIFGSLAFL-NH2
    16.7.3 II: 670, Ac-LRMK-ava-FGILIKRRQQK-NH2 624
    I: 673

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope, with MHC Class II indicated as I: and MHC Class II indicated as II:. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 1.2.
  • [0000]
    TABLE 16.8
    Antibody-recognized determinants on Her-2/neu.
    Peptide Pos. Sequence SEQ ID NO:
    16.8.1 216 TRTVCAGGCARCKGP 625
    16.8.2 220 CAGGCARCKGPLPTD 626
    16.8.3 533 QFLRQECVEECRVLQ 627
    16.8.4 545 VLQGLPREYVNARHC 628
    16.8.5 571 NGSVTCFGPEADQCV 629

    These peptides are reported to react with serums of mice which were immunized with a GST fusion protein containing the Her-2/neu(C220-C235) sequence (Yip Y L. Cancer Immunol Immunother. 2002 50:569-87; Yip Y L. J Immunol. 2001 166:5271-8).
  • [0000]
    TABLE 16.9
    Designed Ii-Key/Her-2/neu hybrids using some of
    the antibody-recognized determinants of Table
    16.8 and MHC Class II-presented epitopes of
    Tables 2 and 3.
    SEQ.
    ID.
    PEPTIDE Pos. Sequence NO.
    A. Non-overlapping (MHC Class II and antibody-
    recognized epitopes)
    16.9.1 776; Ac-LRMK-ava-GSPYVSRLLGICL- 630
    216, TRTVCAGGCARCKGPLPTD-NH2
    220
    16.9.2 396; Ac-LRMK-ava-QLQVFETLEEI- 631
    571 NGSVTCFGPEADQCV-NH2
    B. Overlapping
    16.9.3 534; Ac-LRMK-ava-SQFLRGQECVEECRVLQ- 632
    533 NH2
    16.9.4 555; Ac-LRMK-ava-RVLQGLPREYVNARHC- 633
    556 NH2

    Pos. is the residue position in the primary sequence of the first amino acid in the MHC Class II-presented epitope and after the semicolon is the first residue in the peptide reported to contain an antibody-recognized epitope. Sequence is the amino acid sequence of a hybrid peptide.
  • Example 17 Ii-Key/Anthrax MHC Class II Antigenic Epitope Hybrids
  • [0211]
    Ii-Key/antigenic epitope hybrids can be applied as vaccines against anthrax and other bioterrorism agents. In order to understand well the applications of Ii-Key/antigenic epitope hybrids as stand-alone vaccines or as components of a multivaccine protocol against anthrax, a review of the biology and pathogenesis of bacillus anthracis is useful. Likewise, the currently available vaccines against anthrax are considered in light of improvements offered by the products and methods of this disclosure. Specifically, the Ii-Key/antigenic epitope hybrid technology provides for enhanced antigen-specific T-helper cell responses, which enable existing vaccines and independently offer a significant degree of protection against anthrax infection. The Ii-Key/anthrax epitope peptide vaccine offers safety and effectiveness for use by both military and civilian populations.
  • [0212]
    Anthrax is an infectious disease caused by the spores of the bacterium, Bacillus anthracis, a large gram-positive, non-motile, bacterial rod. Human anthrax disease has three major forms: cutaneous, inhalational, and gastrointestinal. If untreated, anthrax in all forms can lead to septicemia and death. Early treatment of cutaneous anthrax is usually curative. Patients with gastrointestinal anthrax have reported case fatalities of 25% to 75%. Case fatality rates for inhalation anthrax are 90% to 100%. Early treatment of all forms of anthrax with antibiotics is essential because antibiotics are ineffective once the bacteria grow densely enough to secrete anthrax toxin (Leppla S H. Nature Medicine. 2001 7:659-660). Inhalational anthrax has two phases. During the first phase, which occurs within one to five days following exposure, the patient has flu-like symptoms (cough, malaise, fatigue and mild fever). The following phase includes sudden onset of severe respiratory distress, chest pain, and fever. Within a day, septic shock and death will likely occur. In the case of inhalational anthrax, antibiotic therapy is of limited benefit except when given immediately following exposure.
  • [0213]
    Anthrax toxin, the major virulence factor produced by B. anthracis, consists of three proteins. PA binds to human cells and forms a channel through which LF, the dominant virulence factor, enters the cytosol (Leppla S H. Nature Medicine. 2001 7:659-660). LF is a metalloproteinase that cleaves mitogen-activated protein kinases (MEKs), resulting in cell death and a clinical picture resembling septic shock. It is the binding of LF to PA63 (an area on PA that is made available following cellular binding and furin catalysis of PA) that triggers LF-PA63 binding, oligomerization, heptamer formation, and cytosolic transport of LN (Leppla S H. Bacterial protein toxins (eds. Fehrenbach F. et al.) 111-112 Gustav Fischer, N.Y., 1988).
  • [0214]
    Anthrax lethal toxin comprises two proteins: protective antigen (PA; MW 83 kDa) and lethal factor (LF; MW 87 kDa). The crystal structure of PA was determined in monomeric and heptameric forms (Liddington R. J Appl Microbiol. 1999 87:282-290). It bears no resemblance to other bacterial toxins of known three-dimensional structure, and defines a new structural class, which includes homologous toxins from other Gram-positive bacteria. Membrane insertion involves the water-soluble heptamer undergoing a substantial pH-induced conformational change thereby creating a 14-stranded beta-barrel. Recent work by Collier's group lends support to this model of membrane insertion (Benson E L. Biochemistry. 1998 37:3941-8). Lethal factor is the catalytic component of anthrax lethal toxin. It binds to the surface of the cell-bound PA heptamer and, following endocytosis and acidification of the endosome, translocates to the cytosol.
  • [0215]
    Liddington and colleagues determined the crystal structure of the anthrax lethal factor (Pannifer A D. Nature 2001 414:229-33). Lethal factor (LF) is highly specific protease that cleaves members of the mitogen-activated protein kinase kinase (MAPKK) family near their amino termini, leading to the inhibition of one or more signaling pathways. The crystal structure of LF and its complex with the N terminus of MAPKK-2 was determined. LF comprises four domains: domain I binds the membrane-translocating component of anthrax toxin, the protective antigen (PA); domains II, III and IV together create a long deep groove that holds the 16-residue N-terminal tail of MAPKK-2 before cleavage. Domain II resembles the ADP-ribosylating toxin from Bacillus cereus, but the active site has been mutated and recruited to augment substrate recognition. Domain III is inserted into domain II, and seems to have arisen from a repeated duplication of a structural element of domain II. Domain IV is distantly related to the zinc metalloprotease family, and contains the catalytic center; it also resembles domain I. The structure thus reveals a protein that has evolved through a process of gene duplication, mutation and fusion, into an enzyme with high and unusual specificity.
  • [0216]
    Proteasome activity is required for anthrax lethal toxin to kill macrophages (Tang G. Infect Immun. 1999 67:3055-60). Anthrax lethal toxin (LeTx), consisting of protective antigen (PA) and lethal factor (LF), rapidly kills primary mouse macrophages and macrophage-like cell lines. LF is translocated by PA into the cytosol of target cells, where it cleaves mitogen-activated protein kinase kinase 1 (MEK1) and possibly other proteins. Proteasome inhibitors such as acetyl-Leu-Leu-norleucinal, MG132, and lactacystin efficiently block LeTx cytotoxicity, whereas other protease inhibitors do not. Various data indicate that the proteasome mediates a toxic process initiated by LF in the cell cytosol. This process probably involves degradation of unidentified molecules that are essential for macrophage homeostasis. Moreover, this proteasome-dependent process is an early step in LeTx intoxication, but it is downstream of the cleavage by LF of MEK1 or other putative substrates.
  • [0217]
    Leppla and colleagues found oligomerization of anthrax toxin protective antigen and binding of lethal factor during endocytic uptake into mammalian cells (Singh Y. Infect Immun. 1999 67:1853-9). The protective antigen (PA) protein of anthrax toxin binds to a cellular receptor and is cleaved by cell surface furin to produce a 63-kDa fragment (PA63). The receptor-bound PA63 oligomerizes to a heptamer and acts to translocate the catalytic moieties of the toxin, lethal factor (LF) and edema factor (EF), from endosomes to the cytosol. The essential role of PA oligomerization in LF translocation was shown with PA protein cleaved at residues 313-314. The structure of the toxin proteins and the kinetics of proteolytic activation, LF binding, and internalization are balanced in a way that allows each PA63 subunit to internalize an LF molecule.
  • [0218]
    Leppla and colleagues identified three advances which is point to possible therapies by inhibiting the toxin (Chaudry G J. Trends Microbiol. 2002 10:58-62). Identification of the cell surface toxin receptor could lead to the design of binding competitors and receptor decoys. Determination of the crystal structure of the lethal factor protease will facilitate ongoing efforts to develop protease inhibitors as therapies. Finally, the susceptibility of certain inbred mice to anthrax lethal toxin was associated with mutations in the kinesin-like protein Kif1C, a discovery that could help to explain how anthrax toxin kills animals.
  • [0219]
    Various vaccine strategies have been developed to protect humans against the pathological effects of PA and LF, which are released by Bacillus anthracis. In order to appreciate the usefulness of Ii-Key(MHC Class II and Ii-Key/MHC Class II epitope/ARD hybrids in augmenting those vaccines, it is useful to review the current state of vaccination against and treatment of anthrax infections.
  • [0220]
    During the bioterrorism attacks of late 2001 in which thousands of people were potentially exposed to anthrax spores contained in letters to elected officials and employees of media outlets, more than 30,000 individuals received prophylactic antibiotic therapy (principally ciprofloxacin and doxycycline) for 60 days. Because anthrax spores can persist in the lungs of animals for more than 60 days, these potentially exposed individuals were then offered another 40-day course of antibiotics and therapeutic vaccination with the Anthrax Vaccine Adsorbed upon completion of the initial antibiotic regimen. However, since there is only a narrow time-window for effective antibiotic therapy following exposure to anthrax spores, using antibiotics on a mass scale is not a realistic option. Prophylactic and therapeutic vaccination against anthrax and anthrax toxin is thus the most promising form of mass intervention in case of an anthrax-bioterrorism event.
  • [0221]
    Prior to its emergence as a potentially preferred bioterrorism weapon, anthrax infection was limited to animals and humans with occupations involving direct and extensive handling of animals or animal products. Approval of the current anthrax vaccine, Anthrax Vaccine Adsorbed was based on a clinical trial conducted by Philip S. Brachman in the 1950's involving U.S. mill workers who processed animal hides. Prior to the availability of this vaccine, the yearly average number of human anthrax cases was 1.2 per 100 employees in these mills. The Brachman study provided evidence for the efficacy of anthrax vaccination: (a) 26 patients developed anthrax during the study—5 inhalation and 21 cutaneous; (b) of the 5 inhalation anthrax cases, 2 patients received placebo and 3 were in the observation group; (c) four of the 5 patients with inhalation anthrax died; (d) of the 21 cases of cutaneous anthrax, 15 individuals received placebo, three were in the observation group, and two individuals were partially immunized, and one individual was fully immunized; (e) the authors calculated vaccine efficacy level of 92.5% for fully vaccinated individuals (Brachman P S. American Journal of Public Health. 1962 52:432-440).
  • [0222]
    In 1966 the CDC initiated a clinical study of a vaccine that was a modification of the vaccine used in the Brachman trial. Although both vaccines were based on immunity induced by protective antigen (PA), their methods of preparation differed. The IND trial used three lots of material produced by the Michigan Department of Public Health (MDPH). The data submitted to the Division of Biologics Standards described the CDC's experience with 16,000 doses of the anthrax vaccine administered to 7,000 study participants. Mild local reactions ranged between 3 to 36%, moderate reactions between 1 to 3%, and severe local reactions in less than 1%. Systemic reactions were reported in 4 cases over the 5-year period; these reactions included transient fever, chills, nausea, and general body aches. The vaccine was approved in 1970 for individuals who might contact animal products that might be contaminated with B. anthracis spores, individuals at high risk (including veterinarians), and those engaged in diagnostic or investigational activities that might bring them in contact with the spores.
  • [0223]
    In 1985 an Advisory Panel Review under the Public Health Service Act designated the anthrax vaccine produced by MDPH as a Category I product, that is safe, effective and not misbranded (Federal Register 1985 50:51002). The efficacy data from the Brachman study and the safety data from the CDC study were the basis for these findings. In May 1988, the Department of Defense (DOD) approved the prophylactic vaccination of US military personnel. In December 2001, therapeutic vaccination was also initiated in individuals previously exposed to anthrax spores (as a result of acts of bioterrorism in Florida, New York, and Washington, D.C.), and who were receiving prophylactic antibiotic therapy.
  • [0224]
    The current AVA vaccine produced by BioPort (the successor to MDPH in anthrax vaccine manufacturing) is derived from a strain of B. anthracis that does not cause anthrax disease. It is a cell-free filtrate containing no whole bacteria. The vaccination protocol includes an initial dose of 0.5 ml s.c., followed by 0.5 ml s.c. booster doses at 2 and 4 weeks, and 6, 12 and 18 months, with yearly boosters thereafter. The manufacturing process is difficult, costly, time consuming, limited in scale, and laden with many biologics controls. Development of a nontoxinogenic and nonencapsulated recombinant B. anthracis spore vaccine and lethal factor DNA vaccine have been initiated recently (Cohen S. Infect Immun. 2000 68:4549-58; Price B M. Infect Immun. 2002 69:4509-15). Also three new anthrax vaccines based on the PA protein are being studied (Friedlander A M. JAMA 1999 282:2104-6; Thomas L J. 4th International Conference on Anthrax. Abstracts Book. Jun. 10-13, 2001, Annapolis, Md., USA; Turnbull P C B. Curr Opin Infect Dis. 2001 13:11). Being products of biologic manufacturing, the process and controls are much more involved and wrought with regulatory issues than for simple peptides.
  • [0225]
    The Anthrax Vaccine Expert Committee (AVEC) reviewed adverse events reported to the Vaccine Adverse Event Reporting System (VAERS) (Sever J L. Pharmacoepidemiol Drug Saf. 2002 11:189-202; Geier D A. Clin Exp Rheumatol. 2002 20:217-20). Nearly half the reports noted a local injection-site adverse effect, with more than one-third of these involving a moderate to large degree of inflammation. Six events qualified as serious adverse effects, and all were judged to be certain consequences of vaccination. Three-quarters of the reports cited a systemic adverse effect (most common: flu-like symptoms, malaise, rash, arthralgia, headache), but only six individual medically important events were judged possibly or probably due to vaccine (aggravation of spondyloarthropathy (2), anaphylactoid reaction, arthritis (2), bronchiolitis obliterans organizing pneumonia). They concluded, since some cases of local inflammation involved distal paresthesia, AVEC recommends giving subcutaneous injections of AVA over the inferior deltoid instead of the triceps to avoid compression injury to the ulnar nerve.
  • [0226]
    Ii-Key/LF(MHC Class II epitope) hybrids will induce strong Th1 immune responses that will in turn augment CTL activity, macrophage-mediated bacteria lysis, and B cell-mediated antibody production. The resulting immune responses will mediate destruction of the bacteria via enhanced macrophage activation. In addition, the hybrid will provide for augmented B cell activation, which, in the setting of concomitant or subsequent exposure to LF, will hasten and enhance the production of antibodies that block binding of LF to PA, thereby preventing internalization of anthrax toxin.
  • [0227]
    Prophylactic vaccination with the Ii-Key/LF(MHC II epitope) hybrid peptide vaccine will induce memory T-helper cells that, upon subsequent exposure to B. anthracis, will activate macrophages more potently and more rapidly, thereby resulting in efficient lysis and clearance of bacteria. Priming with the Ii-Key/LF(MHC II epitope) hybrid peptide vaccine will lead to an expanded population of specific T-helper cells that will more quickly and efficiently activate B cells for antibody production upon vaccination with LF vaccine or exposure to B. anthracis. Boosting with the Ii-Key/LF(MHC Class II epitope) hybrid peptide vaccine in patients previously vaccinated or previously exposed to the disease will create a robust and rapid anamnestic response involving efficient activation of macrophages and B cells. Prior vaccination with the Ii-Key/LF(MHC II epitope) hybrid will result in more rapid stimulation of T-helper cells and activation of B-cells providing for augmented and more rapid antibody production, which is critical in the neutralization of anthrax toxin, upon exposure to a classical anthrax vaccine or the infection itself.
  • [0228]
    In another aspect Ii-Key/anthrax MHCC lass II epitope/anthrax ARD hybrids can be used to create an effective blocking antibody eliciting vaccine. Compound peptide constructs consisting of ARDs from PA binding sites on LF, are designed with covalently linkage to the Ii-Key/antigenic epitope hybrids. In some instances the sequences of the MHC Class II epitope and an ARD overlap. These double hybrid constructs [Ii-Key/LF(MHC II epitope)/LF1-255(ARD)] trigger robust production of antibodies to LF1-255 via concomitant antigen-specific activation of T-helper cells and B-cells. The double hybrid construct focus and magnify the immune response on the most critical area, the PA63 binding site for LF. The antibodies produced following vaccination disrupt LF binding to PA63 and anthrax toxin internalization, thereby obviating the virulence of the disease. Methods of the process of developing immunization procedures with these Ii-Key/antigenic epitope hybrids for protection against anthrax and anthrax toxins include the following. 1. The most effective double hybrid(s) (in terms of inducing the most potent CD4+ T cell immunity and blocking the binding of LF1-255 to PA63 and entry of LF1-255 into cells) are tested in vivo in animal infection models to evaluate inhibition of bacteria growth and the virulence of the lethal toxin. 2. Immunization formulations (different doses with or without adjuvants), roots of immunization (s.c. or i.v.), and immunization schedules (with or without boosts) are evaluated in animal models. Toward application in a human trial, dose, dosage schedule, formulation, cytokine adjuvant, and basic local and systemic toxicities are evaluated in a murine protective model. 3. Activation of Th memory cells is tested in groups of immunized mice at 3, 6, 9 and 12 months for potency of CD4+ cell responses on a secondary challenge with the peptide, recombinant protein, or cDNA LF vaccine. 4. The most potent human HLA-DR restricted LF epitopes are determined for human clinical application. The most potent epitope for certain HLA-DR alleles are predicted using the Rammensee program. In as much as LF MHC Class II epitope aa576-591 might be presented by both HLA-DR1 and HLA-DR4, efforts to identify other pan-DR allele binding epitopes are made. The predicted Ii-Key/LF(HLA-DR epitope) constructs are tested for activity in ex vivo human PBMC stimulation and re-stimulation studies. Th1 and Th2 responses (double staining for CD4 and IFN-γ or CD4 and IL-4) are evaluated. 5. Double hybrids of the structure Ii-Key/LF(HLA-DR)/LF1-255(ARD) are synthesized using the most active Ii-Key/LF(HLA-DR epitope) and the most active antibody determinant (ARD). These are tested in animal toxicology and pharmacokinetics studies. 6. Clinical in vivo immunization and ex vivo PBMC re-stimulation studies in volunteers are performed with double hybrids to evaluate Th1 and Th2 responses. The several most promising double hybrids are evaluated in a subsequent clinical trial in which the induction of CD4+ T cell activation (double staining of PBMC for CD4 and IFN-γ or IL-4) and blocking antibodies are evaluated. ex vivo studies of the induced antibodies are performed to evaluate inhibition of the binding of LF1-255 to PA63 and LF1-255 entry into cells. The optimal hybrid(s) are further developed as an anthrax vaccine in clinical trials involving greater numbers of individuals. Appropriate efficacy endpoints and immunological surrogates are selected based on extensive discussion with appropriate regulatory agencies.
  • [0229]
    Ii-Key hybrid anthrax vaccines have significant advantages. (1) Safety. Since the Ii-Key hybrid vaccines are small peptides, as opposed to the full-length LF or PA protein, there is less risk of inducing unwanted immune responses against extraneous regions of the protein(s) which may be cross-reactive with normal host molecules, thereby resulting in autoimmune mediated toxicity. Peptide vaccine does not have reverse affect and thus can be safely used for large military and civilian populations; (2) Efficacy. To date, vaccines based on MHC Class II epitopes have not induced robust antigen specific immune responses primarily due to low binding efficiency. The Ii-Key hybrid technology enhances the charging efficiency of MHC Class II epitopes such that strong antigen-specific immune responses that are usually seen only in the context of concomitant IL-12 administration are observed. (3) Precise-targeting. Although current vaccines may induce high titers of polyclonal antibodies. However, these antibodies are not always against critical target, the LF binding site for PA. The Ii-Key double hybrid, Ii-Key/LF(MHC II epitope/LF1-255(ARD), will result in the production of antibodies specifically and precisely targeted to the LF binding sites for PA, thereby making efficient use of the resources brought to bear by the immune system. (4) Dual-action—the Ii-Key double hybrid will induce T-helper memory cells that will activate macrophages to effect cell-mediated bacterial lysis and clearing, as well as strong antibodies to the PA63 binding sites that will obviate the virulence of the anthrax toxin. Even in the setting of dense bacterial growth, the antibodies to PA binding sites on LF will protect from the virulent effects of the anthrax toxin. (5) Platform technology—once shown to be effective in the anthrax system, this approach is readily adaptable for use in other Category A (i.e., botulism, plague and smallpox), Category B, and Category C bioterrorism threats.
  • [0230]
    Ii-Key/LF(MHC II epitope) hybrids are designed to induce of LF-specific CD4+ T cell activation, which forms a major defense line to inhibit the growth of B. anthracis. Then the most potent Ii-Key/LF(MHC II epitope) hybrid are linked to putative ARDs of the PA63 binding site on LF to form double hybrids of the structure Ii-Key/LF(MHC II epitope)LF1-255(ARD). The ARDs are chosen from the published mapping of the sites on LF for binding to PA by mutation/binding assay (Lacy D B. J Biol Chem. 2002 277:3005-10). The linkage of Ii-Key/LF(MHC Class II epitope) hybrid to ARDs will offer strong CD4+ T cell help for the induction of antibodies to the covalently linked ARDs (Golvano J. Eur J Immunol. 1990 20:2363-6. These antibodies will bind to the surface of the PA binding sites on LF and block the binding of LF to PA63. The induction of high-titered antibodies against precisely targeted binding sites creates another line of defense, which abrogates the toxicity of B. anthracis LF, although the bacterial infection can be ongoing. MHC Class II-presented LF epitopes predicted with the SYFPEITHI program identifies three epitopes match perfectly the consensus sequence of the H-2Ek motif: LF(91-106; HISLEALSDKKKIK) (SEQ ID NO: 634) LF(249-264; EQEINLSLEELKDQR) (SEQ ID NO: 635); LF(305-320; DDIIHSLSQEEKELL) (SEQ ID NO: 636). The activity of all hybrids in T cell activation studies will be compared with epitopes unlinked to Ii-Key. T cell activation is measured by two-color staining (anti-CD4 plus anti-IFN-γ for Th1 and anti-CD4 plus anti-IL-4 for Th2). AKR or C3H mice (H-2Kk) are immunized (3 mice/group) with varying doses (0.8, 4, and 20 nmol) of the Ii-Key/LF(MHC II epitope) hybrids. The concentration of 20 nmol, used by Berzofsky and colleagues (Berzofsky, J. A. J Clin Invest. 1991 88: 876-84), induced optimal T cell proliferation. A much lower concentration of hybrids will induce the same or higher levels of T cell response. In the first experiment, the adjuvant emulsion consists of equal volumes of CFA containing 1 mg/ml of Mycobacterium tuberculosis and hybrid peptides dissolved in PBS. Mice are immunized s.c. on the left side at the base of the tail. The same amount of hybrid peptides in incomplete Freund's adjuvant (IFA) are injected into the right side at the base of the tail 9 days later. Hybrids are injected in saline intravenously according to the same schedule to test the requirement for CFA in the efficacy of hybrids. It should be noted that Ii-Key hybrids will interact directly with MHC Class II molecules on the cell surface of APCS, thereby bypassing classical MHC Class II epitope processing and rendering the adjuvant superfluous. Four days following the second injection, the activation of lymphocytes from spleen, popliteal, inguinal, and para-aortic nodes of immunized mice are determined by established two color staining for CD4 and either IFN-γ or IL-4 (Varga S M. J. Immunol. 2001 166:1554-61).
  • [0231]
    Ii-Key/LF(MHC II epitope)/LF1-255(ARD) double hybrids will produce antibodies which inhibit binding of LF to PA. The binding of LF to PA and subsequent entry of LF into cells are essential for the principal toxicity of B. anthracis infections. Blocking the binding of LF to PA is thus an effective way to control the virulence of B. anthracis. Lacy et al. have identified the PA binding sites on the surface of LF 1-255 by mutation/binding assays. Nine overlapping ARDs from these sites are synthesized in Ii-Key/MHC Class II antigenic epitope/ARD hybrids. Coupling to either a carrier or a MHC Class II epitope is required in order to induce antibodies against these short peptides (Golvano J. Eur J Immunol. 1990 20:2363-6). LF has been crystallized and its functional domains have been defined (Pannifer A D. Nature 2001 414:229-33; Lacy D B. J Biol. Chem. 2002 277:3005-10). By LF mutation and PA/LF binding experiments, Lacy et al. have mapped the PA63 binding sites on LF. Mutations clustered at two locations greatly abolish the binding of LF to PA63: aa182-188 and aa223-236. Because these two clusters are located on the surface of LF, at that exposed binding site (Lacy D B. J Biol Chem. 2002 277:3005-10), they are logically good targets for developing antibodies to block the binding of LF to PA63.
  • [0232]
    In another aspect this disclosure relates to augmenting the immune response to DNA vaccines for PA or LF. The Ii-Key/anthrax antigenic epitope hybrids of this disclosure can be applied as a prevaccine given in advance of a DNA vaccine for an anthrax-coded protein. Several examples of such vaccines follow.
  • [0233]
    Galloway and colleagues developed protection against anthrax lethal toxin challenge by immunization with plasmids encoding LF(10-254) or PA(175-764) or both (Price B M. Infect Immun. 2001 69:4509-15). Gold particles coated with either or both plasmids were gene-gun injected into mice three times at 2-week intervals. Antibody titers both PA and LF were five times greater than titers from mice immunized with either gene alone. All mice immunized with either or both plasmids survived an i.v. challenge with a lethal dose of PA+LF.
  • [0234]
    Gu and colleagues also studied comparable PA DNA vaccines (GU ML. Vaccine 1999 17:340-4). A 1:100 dilution of serum from mice immunized with PA DNA protected cells in vitro against cytotoxic concentrations of PA. 7 of 8 mice immunized three times with the PA DNA vaccine were protected against lethal challenge with a combination of anthrax protective antigen plus lethal factor. The augmentation of such immunizations with DNA vaccines for PA might be further augmented by a later boost with recombinant protective antigen. Such protein antigens will further enhance antibody production to PA because although Ii-Key hybrids augment the MHC-Class II restricted response to antigen expressed from a DNA vaccine, there is presumably not enough PA protein available extracellularly to bind to B cells for internalization and processing of MHC Class Ii epitopes to activate those B cells to progress to plasma cells and soluble immunoglobulin production.
  • [0235]
    The efficacy of Ii-Key/anthrax antigenic epitope hybrids in potentiating DNA and protein vaccines can be tested in guinea pigs, rabbits, and rhesus macaques against spore challenge by Bacillus anthracis isolates of diverse geographical origin (Fellows P F. Vaccine 2001 19:3241-7).
  • [0236]
    In another aspect the Ii-Key/anthrax MHC Class II epitope/anthrax ARD hybrids can be used to elicit antibodies which block the interaction of LF with PA required for the internalization of LF into cells. Examples of the creation and use of antibodies with such protective blocking effects follow.
  • [0237]
    Georgiou and colleagues found protection against anthrax toxin by recombinant antibody fragments correlates with antigen affinity (Maynard J A. Nat Biotechnol. 2002 20:597-601). The tripartite toxin produced by Bacillus anthracis is the key determinant in the etiology of anthrax. They engineered a panel of toxin-neutralizing antibodies, including single-chain variable fragments (scFvs) and scFvs fused to a human constant kappa domain (scAbs), that bind to the protective antigen subunit of the toxin with equilibrium dissociation constants (K(d)) between 63 nM and 0.25 mM. The entire antibody panel showed high serum, thermal, and denaturant stability. in vitro, post-challenge protection of macrophages from the action of the holotoxin correlated with the Kd of the scFv variants. Strong correlations among antibody construct affinity, serum half-life, and protection were also observed in a rat model of toxin challenge. High-affinity toxin-neutralizing antibodies can be of therapeutic value for alleviating the symptoms of anthrax toxin in infected individuals and for medium-term prophylaxis to infection.
  • [0238]
    In another aspect, this disclosure relates to Ii-Key/anthrax MHC Class II epitope/ARD hybrids to generate protective antibodies to a segment of PA binding LF for internalization into cells. Varughese and colleagues identified two such potential sites in solvent-exposed loops of domain 4 of PA (aa 679 to 693 and 704 to 723) by mutagenesis and testing of the purified proteins for toxicity in the presence of LF (Varughese M. Infect Immun. 1999 67:1860-5). Mutations were designed in these loops and were introduced by errors occurring during PCR. Substitutions within the large loop (aa 704 to 723) had no effect on PA activity. Comparisons among 28 mutant proteins showed that the large loop (aa 704 to 722) is not involved in receptor binding, whereas residues in and near the small loop (aa 679 to 693) are relevant to receptor interaction. Peptides through that small loop are good candidates for incorporation in Ii-Key/LF MHC Class II epitope/LF ARD hybrids.
  • [0239]
    Anthrax lethal factor can be used either to draw other proteins into a cell or for its toxic activity to inactivate MAP-kinase-kinase (Duesbery N S. Science 1998 280:734-7; Liu S. Cancer Res. 2000 60:6061-7; Liu S, J Biol Chem. 2001 276:17976-84).
  • [0240]
    The hybrids of this disclosure will enhance responses to subsequently administered anthrax toxoid vaccine adsorbed to alum (Pittman P R. Vaccine 2002 20:1412-20). The IM route of administering this is safe and has comparable peak anti-PA IgG antibody levels when two doses are administered 4 weeks apart compared to the licensed initial dose schedule of three doses administered 2 weeks apart.
  • [0241]
    The Ii-Key/antigenic epitope hybrids of this disclosure can be assayed in a rabbit model of inhalational anthrax (Pitt M L. Vaccine 2001 19:4768-73). A serological correlate of vaccine-induced immunity was identified in the rabbit model of inhalational anthrax. Animals are inoculated intramuscularly at 0 and 4 weeks with varying doses of Anthrax Vaccine Adsorbed ranging from a human dose to a 1:256 dilution in phosphate-buffered saline. At 6 and 10 weeks, both the quantitative anti-PA IgG ELISA and the toxin-neutralizing antibody assays were used to measure antibody levels to PA. Rabbits were aerosol-challenged at 10 weeks with a lethal dose of Bacillus anthracis spores. All the rabbits that received the undiluted and 1:4 dilution of vaccine survived, whereas those receiving the higher dilutions of vaccine (1:16, 1:64 and 1:256) had deaths in their groups. Results showed that antibody levels to PA at both 6 and 10 weeks were significant (P<0.0001) predictors of survival. In addition non-invasive nasal immunization can be used to vaccinate against anthrax (Gaur R. Vaccine 2002 20:2836-9). Mice were inoculated intranasally, subcutaneously or through the skin on days 0, 15 and 28 with purified PA. Intranasal and subcutaneous immunization with PA resulted in high IgG ELISA titers. High titers of IgA were observed only in intranasally immunized mice. In a cytotoxicity assay these sera protected J774A.1 cells from lethal toxin challenge.
  • [0242]
    Table 17.1 presents the deduced amino acid sequence of anthrax toxin lethal factor (GenBank gi|16974824; Pannifer A D. Nature 2001 414:229-233. (2001)). Table 17.2 presents predicted MHC Class II-presented epitopes of anthrax toxin lethal factor. Table 17.3 presents predicted MHC Class I-presented epitopes of anthrax toxin lethal factor. Designed Ii-Key/MHC Class II epitope hybrids for anthrax lethal factor are presented in Table 17.4. Table 17.5 presents designed Ii-Key/MHC Class II epitope/ARD hybrids for anthrax lethal factor. Table 17.6 presents the deduced amino acid sequence of anthrax protective antigen (GenBank gi:9280533; Cohen, S. Infect Immun. 2000 68:4549-4558). Table 17.7 presents predicted MHC Class II-presented epitopes of anthrax protective antigen. Table 17.8 presents predicted MHC Class I-presented epitopes of anthrax protective antigen. Designed Ii-Key/MHC Class II epitope hybrids for anthrax protective antigen are presented in Table 17.9. Table 17.10 presents designed Ii-Key/anthrax protective antigen MHC Class II epitope/anthrax protective antigen ARD hybrids.
  • [0000]
    TABLE 17.1
    Deduced amino acid sequence of anthrax toxin
    lethal factor (SEQ ID NO: 637)
      1 agghgdvgmh vkekeknkde nkrkdeernk tqeehlkeim khivkievkg
     51 eeavkkeaae kllekvpsdv lemykaiggk iyivdgditk hislealsed
    101 kkkikdiygk dallhehyvy akegyepvlv iqssedyven tekalnvyye
    151 igkilsrdil skinqpyqkf ldvlntikna sdsdgqdllf tnqlkehptd
    201 fsvefleqns nevqevfaka fayyiepqhr dvlqlyapea fnymdkfneq
    251 einlsleelk dqrmlsryek wekikqhyqh wsdslseegr gllkklqipi
    301 epkkddiihs lsqeekellk riqidssdfl steekeflkk lqidirdsls
    351 eeekellnri qvdssnplse kekeflkklk ldiqpydinq rlqdtgglid
    401 spsinldvrk qykrdiqnid allhqsigst lynkiylyen mninnltatl
    451 gadlvdstdn tkinrgifne fkknfkysis snymivdine rpaldnerlk
    501 wriqlspdtr agylengkli lqrnigleik dvqiikqsek eyiridakvv
    551 pkskidtkiq eaqlninqew nkalglpkyt klitfnvhnr yasnivesay
    601 lilnewknni qsdlikkvtn ylvdgngrfv ftditlpnia eqythqdeiy
    651 eqvhskglyv pesrsillhg pskgvelrnd segfihefgh avddyagyll
    701 dknqsdlvtn skkfidifke egsnltsygr tneaeffaea frlmhstdha
    751 erlkvqknap ktfqfindqi kfiins
  • [0000]
    TABLE 17.2
    Predicted MHC Class II-presented epitopes of
    anthrax toxin lethal factor.
    SEQ.
    Ii- ID.
    Peptide Pos. Sequence Score Allele Key NO:
    17.2.1 501 WRIQLSPDT 3.1  1, 4 0 638
    17.2.2 542 YIRIDAKVV 2.4  1 4 639
    17.2.3 741 FRLMHSTDH 2.4  1, 3, 4 0 640
    17.2.4 521 LQRNIGLEI 1.6  1, 8(519), 0 641
    15, 15(518)
    17.2.5 341 LQIDIRDSL 5.4  3 0 642
    17.2.6 404 INLDVRKQY 4.5  3, 13(407) 0 643
    17.2.7 677 LRNDSEGFI 4.3  3 7 644
    17.2.8 129 LVIQSSEDY 3.6  4, 11(124) 0 645
    17.2.9 698 YLLDKNQSD 3.0  4 8 646
    17.2.10 477 YSISSNYMI 7.2  7 4 647
    17.2.11 398 LIDSPSINL 6.7  7 5 648
    17.2.12 595 IVESAYLIL 6.0  7 9 649
    17.2.13 475 FKYSISSNY 5.5  7 2 650
    17.2.14 241 FNYMDKFNE 4.7  8 6 651
    17.2.15 375 FLKKLKLDI 4.2  8, 11 0 652
    17.2.16 549 VVPKSKIDT 3.9  8 3 653
    17.2.17 148 YYEIGKILS 3.4 11 0 654
    17.2.18 416 IQNIDALLH 3.2 11 5 655
    17.2.19 707 LVTNSKKFI 4.1 13 4 656
    17.2.20 582 LITFNVHNR 3.9 13 5 657
    17.2.21 527 LEIKDVQII 3.8 13 4 658
    17.2.22 435 IYLYENMNI 7.5 15 7 659
    17.2.23  71 LEMYKAIGG 4.7 15 3 660

    Pos. is the first amino acid of the predicted MHC Class II-presented epitope of the specified sequence. Score is the score calculated by the ProPred program for the first of the given HLA-DRB*01 alleles which were examined. The second listed allele is for exactly the same epitope or for an overlapping epitope for which the first amino acid position is given in parentheses.
  • [0000]
    TABLE 17.3
    Predicted MHC Class I-presented epitopes of
    anthrax toxin lethal factor.
    PEPTIDE Pos. Sequence Score SEQ. ID. NO:
    17.3.1 684 FIHEFGHAV 685.4 661
    17.3.2 765 FINDQIKFI 342.2 662
    17.3.3 147 VYYEIGKIL 336.0 663
    17.3.4 277 HYQHWSDSL 300.0 664
    17.3.5 113 LLHEHYVYA 285.7 665
    17.3.6 331 STEEKEFLK 225.0 666
    17.3.7 295 KLQIPIEPK 135.0 667
    17.3.8 659 YVPESRSIL 126.0 668

    Pos. is the first amino acid of the epitope of the listed sequence. The score is calculated with the SPEYETHEI program for HLA-A2.
  • [0000]
    TABLE 17.4
    Designed Ii-Key/MHC Class II epitope hybrids
    for anthrax lethal factor.
    SEQ. ID.
    PEPTIDE Pos. Sequence NO:
    17.4.1 501 Ac-LRMK-WRIQLSPDT-NH2 669
    17.4.2 542 Ac-LRMK-YIRIDAKVV-NH2 670
    17.4.3 741 Ac-LRMK-FRLMHSTDH-NH2 671
    17.4.4 519 Ac-LRMK-LIQRNIGLEI-NH2 672
    17.4.5 341 Ac-LRMK-LQIDIRDSL-NH2 673
    17.4.6 404 Ac-LRMK-INLDVRKQYKRDI-NH2 674
    17.4.7 677 Ac-LRMK-LRNDSEGFI-NH2 675
    17.4.8 125 Ac-LRMK-YEPVQSSEDY-NH2 676

    These hybrids incorporate some for the predicted MHC Class II epitopes of Table 17.3.
  • [0000]
    TABLE 17.5
    Designed Ii-Key/anthrax lethal factor MHC Class
    II epitope/ARD hybrids.
    SEQ ID
    PEPTIDE Pos. Sequence NO:
    17.5.1 166-184 Ac-LRMK- 677
    II: 170 PYQKFLDVLNTIKNASDSD-NH2
    17.5.2 190-213 Ac-LRMK- 678
    II: 191; TNQLKEHPTDFSVEFLEQNSNEVQ-
    203 NH2
    17.5.3 200-224 Ac-LRMK- 679
    II: 203, DFSVEFLEQNSNEVQEVFAKAFAYYI-
    215 NH2
    17.5.4 228-243 Ac-LRMK-QHRDVLQLYAPEAFN-NH2 680
    II: 230

    Pos. is the first and last amino acids of the LF sequence, which is incorporated into the hybrid. The first amino acid of the predicted MHC Class II epitopes are listed after II:. The MHC Class II alleles predicted with high scores to present individual epitopes are the following: 170: HLA-DRB*1301. 191: HLA-DRB*0401. 203: HLA-DRB*0401. 215: HLA-DRB*0101. 230: HLA-DRB*0101. 239:HLA-DRB*0801. Only the 10 alleles were scored with the ProPred predicting program. These peptides were chose from the segment of LF(182-236) containing interaction sites for binding to PA as indicated by loss of activity upon alanine substitutions at D182, D187, Y223, H229, L235 and Y236 (Lacy D B. J Biol Chem. 2002 277:3005-10). In these hybrids the intervening sequence is supplied by the natural sequence of LF, potentially contributing to the ARD structure. Upon identification of biological activity with any of these hybrids, additional hybrids would be tested with systematic deletions/extensions of the epitope-containing peptide sequence.
  • [0000]
    TABLE 17.6
    Deduced amino acid sequence of anthrax
    protective antigen. (SEQ ID NO: 681)
      1 mkkrkvlipl malstilvss tgnleviqae vkqenrllne sesssqgllg
     51 yyfsdlnfqa pmvvtssttg dlsipssele nipsenqyfq saiwsgfikv
    101 kksdeytfat sadnhvtmwv ddqevinkas nsnkirlekg rlyqikiqyq
    151 renptekgld fklywtdsqn kkevissdnl qlpelkqkss nsrkkrstsa
    201 gptvpdrdnd gipdsleveg ytvdvknkrt flspwisnih ekkgltkyks
    251 spekwstasd pysdfekvtg ridknvspea rhplvaaypi vhvdmeniil
    301 sknedqstqn tdsqtrtisk ntstsrthts evhgnaevha sffdiggsvs
    351 agfsnsnsst vaidhslsla gertwaetmg lntadtarln aniryvntgt
    401 apiynvlptt slvlgknqtl atikakenql sqilapnnyy psknlapial
    351 naqddfsstp itmnynqfle lektkqlrld tdqvygniat ynfengrvrv
    501 dtgsnwsevl pqiqettari ifngkdlnlv erriaavnps dplettkpdm
    551 tlkealkiaf gfnepngnlq yqgkditefd fnfdqqtsqn iknqlaelnv
    601 tniytvldki klnakmnili rdkrfhydrn niavgadesv vkeahrevin
    651 ssteglllni dkdirkilsg yiveiedteg lkevindryd mlnisslrqd
    701 gktfidfkky ndklplyisn pnykvnvyav tkentiinps engdtstngi
    751 kkilifskkg yeig
  • [0000]
    TABLE 17.7
    Predicted MHC Class II-presented epitopes of
    anthrax protective antigen. (SEQ ID NOS 682-699
    respectively, in order of appearance)
    PEPTIDE Pos. SEQUENCE Allele Score
    17.7.1 404 YNVLPTTSL B1, B7(405) 1.6
    17.7.2   7 LIPLMALST B1 1.4
    17.7.3 395 YVNTGTAPI B1, B3(392), B4, 1.1, 3.9,
    B7, B13(392) 4.9, 7.3,
    2.8,
    17.7.4 717 YISNPNYKV B1 1.0
    17.7.5 697 LRQDGKTFI B3, B13(690) 6.3, 2.8
    17.7.6 619 LIRDKRFHY B3, B8(617), 5.9, 5.8,
    B13(617) 4.7
    17.7.7 610 IKLNAKMNI B3, B11(603), B13 5.3, 2.7,
    4.1
    17.7.8 625 FHYDRNNIA B4 4.9
    17.7.9 298 IILSKNEDQ B4 3.9
    17.7.10 174 VISSDNLQL B7, B15 6.8, 4.1
    17.7.11 648 VINSSTEGL B7 6.8
    17.7.12 161 FKLYWTDSQ B8 4.0
    17.7.13 225 VKNKRTFLS B8, B13 3.5
    17.7.14  96 FIKVKKSDE B8 2.7
    17.7.15 752 ILIFSKKGY B13 4.9
    17.7.16  47 LLGYYFSDL B15 4.2
    17.7.17 663 IRKILSGYI B15 4.1
    17.7.18 360 VAIDHSLSL B15 4.1

    Pos. is the first amino acid of the predicted epitope. Allele is the HLA-DRB*01 allele with a high score for presentation of the epitope. When a second allele is listed it predicts either exactly the same sequence or an overlaying sequence, the first amino acid residue position of which is given in parentheses. The score is the prediction score in the ProPred program for the given epitope and allele.
  • [0000]
    TABLE 17.8
    Predicted MHC Class I epitopes of anthrax
    protective antigen. (SEQ ID NOS 700-709
    respectively, in order of appearance)
    PEPTIDE Pos. Sequence
    17.8.1 607 ILSGYIVEI
    17.8.2  32 AIWSGFIKV
    17.8.3 171 FLSPWISNI
    17.8.4 328 RLNANIRYV
    17.8.5 530 NIKNQLAEL
    17.8.6 155 SLEVEGYTV
    17.8.7 551 KLNAKMNIL
    17.8.8 657 YISNPNYKV
    17.8.9 225 VAAYPIVHV
    17.8.10 352 LVLGKNQTL

    Pos. is the first amino acid of the epitope of the listed sequence. The score is calculated with the SYFPEITHI program for HLA-A2.
  • [0000]
    TABLE 17.9
    Designed Ii-Key/anthrax protective antigen MHC
    Class II epitope hybrids.
    PEPTIDE Pos. Sequence SEQ. ID. NO:
    17.9.1 404 Ac-LRMK-NVLPTTSL-NH2 710
    17.9.2   7 Ac-LRMK-LIPLMALST-NH2 711
    17.9.3 395 Ac-LRMK-VNTGTAPI-NH2 712
    17.9.4 717 Ac-LRMK-YISNPNYKV-NH2 713
    17.9.5 697 Ac-LRMK-LRQDGKTFI-NH2 714
    17.9.6 619 Ac-LRMK-LIRDKRFHY-NH2 715
    17.9.7 610 Ac-LRMK-IKLNAKMNI-NH2 716
    17.9.8 625 Ac-LRMK-FHYDRNNIA-NH2 717
    17.9.9 298 Ac-LRMK-IILSKNEDQ-NH2 718
    17.9.10 174 Ac-LRMK-VISSDNLQL-NH2 719
  • [0000]
    TABLE 17.10
    Designed Ii-Key/anthrax protective antigen MHC
    Class II epitope/anthrax protective antigen ARD
    hybrids.
    SEQ.
    ID.
    PEPTIDE Pos. Sequence NO:
    17.10.1 173-200 Ac-LRMK-ava- 720
    II: 173 VISSDNLQLPELKQKSSNSRKKRSTSA
    G-NH2
    17.10.2 212-232 Ac-LRMK- 721
    II: 221, PDSLEVEGYTVDVKNKRTFLS-NH2
    223, 225
    17.10.3 203-232 Ac-LRMK- 722
    II: 221, VPDRDNDGIPDSLEVEGYTVDVKNKRT
    223, 225 FLS-NH2
    17.10.4 664-684 Ac-LRMK-ava- 723
    II: 664, IRKILSGYIVEIEDTEGLKEV-NH2
    667, 672
    17.10.5 685-705 Ac-LRMK- 724
    II: 690, INDRYDMLNISSLRQDGKTFI-NH2
    696

    Pos. is the first and last amino acids of the PA sequence, which is incorporated into the hybrid. The first amino acid of the predicted MHC Class II epitopes are listed after II:. The MHC Lass II alleles predicted with high scores to present individual epitopes are the following: 173: HLA-DRB0401, 0701, 1501. 221 HLA-DRB0301. 223: HLA-DRB1101. 225: HLA-DRB0301, 801, 1101, 1301. 664: HLA-DRB0101, 0301. 690:HLA-DRB0401, 1101. 696:HLA-DRB0301. Only the **01 alleles were scored with the ProPred predicting program. In hybrids 17.10.2, 0.3, and 0.5 the intervening sequence is supplied by the natural sequence of PA, potentially contributing to the ARD structure. Upon identification of biological activity with any of these hybrids, additional hybrids would be tested with systematic deletions/extensions of the epitope-containing peptide sequence. Peptides 17.10.1 and 17.10.2 were chosen from the region PA(197-222) shown by Collier and colleagues to be sensitive to LF binding with alanine substitutions at K197, R200, P205, I207, I210 and K214 (Cunningham K. Proc Natl Acad Sci USA 2002 99:7049-53). Peptides 17.10.4 and 17.10.5 were chosen from the smaller loop of PA(679-693) shown by Leppla and colleagues to contain interaction sites for binding to PA (Varughese M. Infect Immun. 1999 67:1860-5). Upon identification of biological activity with any of these hybrids, additional hybrids would be tested with systematic deletions/extensions of the epitope-containing peptide sequence. Additional Ii-Key/PA MHC Class II epitope/ARD hybrids can be constructed with the peptides derived by phage display analyses to bind with PA-neutralizing antibodies. In these peptides the MHC Class II epitopes would be chosen from the best experimentally determined MHC class II-presented epitopes. Examples are presented in Table 17.11 for such constructs, using only a single MHC Class II-presented epitope.
  • [0000]
    TABLE 17.11
    Designed Ii-Key/anthrax protective antigen MHC
    Class II epitope/anthrax protective antigen ARD
    hybrids.
    SEQ.
    ID.
    PEPTIDE Pos. Sequence NO:
    17.11.1 II: 163 Ac-LRMK-YVNTGTAPI-NH2 725
    17.11.2 209-230 Ac-LRMK-YVNTGTAPI-NH2 726
    II: 222
    17.11.3 655-675 Ac-LRMK-ava-YVNTGTAPI- 727
    II: 655, 664, NH2
    667
    17.11.4 655-680 Ac-LRMK-ava-YVNTGTAPI- 728
    II: 664, 667 NH2
    17.11.5 666-680 Ac-LRMK-ava-YVNTGTAPI- 729
    II: 667 NH2
    17.11.6 693-706 Ac-LRMK-ava-YVNTGTAPI- 730
    II: 693 NH2
    17.11.7 688-706 Ac-LRMK-YVNTGTAPI-NH2 731
    II: 693
    17.11.8 686-706 Ac-LRMK- 732
    II: 693 NGIKKILIFSKKGYEIG-NH2

    Pos. is the first amino acid of the MHC Class II-presented epitope, for which only one example is given. The best epitopes determined experimentally are favored. The sequences following that epitope are the ARD sequences discovered by Collier and colleagues by selection and sequencing of phages which interact with PA binding antibodies. Some of those antibodies inhibit internalization of LF.
  • Example 18 Ii-Key/Variola B5R Protein Antigenic Epitope Hybrids
  • [0243]
    Ii-Key/smallpox antigenic epitope vaccines offer robust and relatively safe protection against smallpox, when used either alone or in combination with other vaccination methods. The potency and safety of certain other vaccines such as vaccinia virus are enhanced substantially, when preceded by one or more immunizations with an Ii-Key/smallpox antigenic epitope vaccine. Protection of a large population can be achieved with solely the use of the Ii-Key/smallpox antigenic epitope hybrid vaccine or preferably with such a vaccine in which the MHC Class II epitope is joined or overlapped in sequence with a MHC Class I-presented (cytotoxic T lymphocyte inducing) epitope and/or an antibody-recognized (virus neutralizing) epitope. Immunization with Ii-Key/smallpox antigenic epitope vaccines also improves clinical outlook for individuals infected with smallpox virus without prior vaccinia immunizations. The Ii-Key/antigenic epitope hybrid vaccines will enhance the protective responses of persons receiving a preventative vaccine with either vaccinia virus or a DNA for a smallpox or vaccinia viral protein. The efficacy of vaccinia virus vaccines given to individuals immediately upon exposure or potentially exposure to smallpox (“ring vaccination”), will be accelerated in terms of the speed and potency of the protective response. The biology and clinical course of smallpox infections is reviewed in order to understand the substantial benefits brought to the prevention of smallpox by the products and methods of this Disclosure.
  • [0244]
    Variola major, the smallpox virus, belongs to the family Poxyiridae, subfamily Chordopoxyirinae, and genus orthopoxvirus, which includes vaccinia (the smallpox vaccine), monkey poxvirus, and several others animal poxviruses that cross-react serologically (Breman J G. N Engl J Med. 2002 346:1300-8; Moss B. in Fields B N. Fields Virology. 1996: 2637-71; Fenner F. in Fields B N. Virology. 1996: 2673-83). The poxviruses are among the largest viruses known, containing one linear, double-stranded DNA molecule of 130 to 375 kb and replicating inn the cytoplasm.
  • [0245]
    There are five patterns of smallpox infections. Variola major (ordinary smallpox) was responsible for 90% of cases in the pre-eradication era and is associated with an overall case-fatality rate of 30% (15% to 45%) in unvaccinated patients. Flat-type or malignant smallpox and hemorrhagic smallpox typically occur in patients with a defective immune system, and case fatality rates are 97% and 96% respectively. Smallpox in children is generally similar to smallpox in adults except the case fatality rate in infants is over 40%. Variola minor is the mildest form that predominated in outbreaks in the U.S. and Great Britain, with case fatality rates <1% (Fenner F. Bull WHO. 1988 1-68, 121-208; Henderson D A. JAMA. 1999 281:2127-39).
  • [0246]
    The smallpox virus enters through the respiratory tract, passing rapidly to lymph nodes to multiply in the reticuloendothelial system over 14 days. Mucous membranes in the oropharynx become infected, as well as the capillary epithelium of the dermis leading to skin lesions. Oropharyngx and skin lesions contain abundant viral particles; virus is also present in the urine and conjunctival secretions. Cytotoxic T-cells and B-cells arise to limit the infection; neutralizing antibodies appear in the first week of infection but are delayed if infection is severe (Fenner F. in Fields B N. Virology. 1996: 2673-831996; Roberts J A. Br J Exp Pathol. 1962 43:451-61; Bedson H S. J Pathol Bacteriol. 1963 85:1-20; Buller R M. Microbiol Rev. 1991 55:80-122; Zaucha G M. Lab Invest. 2001 81:1581-600; Sarkar J K. Bull World Health Organ. 1973 48:517-22). The incubation period is 7 to 17 days (mean 10 to 12). The prodromal phase, which lasts for two to three days, is characterized by severe headache, backache, and fever, all beginning abruptly (Dixon C W. Smallpox. London, 1962). Enanthema of the tongue, mouth, and oropharynx precede the rash by a day. The rash begins as small, reddish macules, which become papules with a diameter of 2 to 3 mm. The papules become vesicles with a diameter of 2 to 5 mm. Pustules of 4 to 6 mm diameter develop four to seven days after the rash. Smallpox lesions with a peripheral distribution, generally are all at the same stage of development (in contrast to chicken pox lesions). Lesions on the palms and soles persist the longest. Death from smallpox is ascribed to toxemia, associated with immune complexes, and hypotension secondary to fluid and protein loss.
  • [0247]
    Variola is transmitted predominantly from person to person by droplet inhalation, most commonly among those with close face-to-face contact (Fenner F. Bull WHO. 1988 1-68, 121-208). Airborne and fomite (laundry, bedding) transmission occurs (Dixon C W. Smallpox. London, 1962). Patients are infectious from the time of fever onset, immediately prior to rash development. Secondary attack rates range from 37% to >70% (Rao A R. Indian J Med Res. 1968 56:1826-54; Arnt N. Am J Epidemiol. 1972 94:363-70; Heiner G G. Am J Epidemiol. 1971 94:316-26), with a primary case infecting 3.6 to 6 others (Gani R. Nature. 2001 414:748-51). In the 1970s outbreaks in Yugoslavia and Germany, there were 11 to 38 infected contacts per index case (Fenner F. Bull WHO. 1988 1-68, 121-208). Thus in populations with low herd immunity, transmission rapidly creates outbreak cases before control measures take hold. Infectivity lasts until all lesions have scabbed over and the scabs have fallen off.
  • [0248]
    Patients with smallpox are treated supportively—adequate fluid intake (which is difficult due to oropharyngeal enanthema), alleviation of pain and fever, keeping skin lesions clean to prevent bacterial superinfection. Although no antivirals are approved for smallpox by the U.S. FDA, many compounds have been screened for therapeutic activity. Cidofivir (Vistide®, approved for CMV retinitis) shows activity against orthopoxviruses, including variola (CIDRAP/IDSA. 2002).
  • [0249]
    Smallpox vaccination began in China in 1000 AD with “variolation”, administration of infectious material from an infected patient to uninfected individuals. Edward Jenner discovered in the late 1700s that cowpox protected against smallpox. Vaccinia virus, genetically distinct from cowpox, has replaced cowpox as a vaccine (CIDRAP/IDSA. 2002). Protection is afforded for 5-10 years after primary vaccination; neutralizing antibodies are detected up to 10 years in 75% of individuals receiving 2 doses of vaccine, and up to 30 years in those vaccinated with 3 doses (Henderson D A. JAMA. 1999:281:2127-39). After an intensive worldwide campaign initiated in earnest in 1967, smallpox eradication was declared in 1980. With no natural reservoirs, variola has since existed only in laboratories. The WHO has sanctioned two depositories—The Center for Disease Control and Prevention (Atlanta, Ga.) and the State Research Center of Virology and Biotechnology (the Vektor Institute) in Novosibirsk, Russia. Inappropriately available variola virus could be a weapon of terrorists. Since less than 20% of 157 million individuals vaccinated before the early 1970s (when routine vaccination was discontinued in the US) are protected today and 119 million Americans have never been vaccinated, the need and problems of vaccinating against smallpox are being considered most carefully.
  • [0250]
    The Working Group on Civilian Biodefense has identified a number of widely known organisms that could cause disease and deaths in sufficient numbers to cripple a city or region. Smallpox used as a biological weapon, is perhaps the most serious threat to civilian populations due to its ease of transmission, case-fatality rate of 30% or more among unvaccinated persons, and the absence of a specific therapy. Although smallpox has long been feared as the most terrible of all infectious diseases, its potential for devastation today is much greater than at any previous time. Routine vaccination throughout the US ceased 25 years ago. In a now highly susceptible, mobile population, smallpox would spread widely and rapidly throughout this country and the world (Henderson D A JAMA. 1999 281:2127-39; Fenner F. Bull WHO. 1988 1-68, 121-208).
  • [0251]
    The U.S. vaccinia vaccine since the 1970s, Dryvax, is a lyophilized live vaccinia virus preparation manufactured by Wyeth. The vaccine is administered on a bifurcated needle containing a droplet of the reconstituted product; the skin of the upper arm is poked approximately 15 times creating a wound producing a drop of blood. To elicit a protective response, a “Jennerian pustule” must be induced. In an effort to expand current supplies in light of bioterrorism threats, recent clinical trials have tested the protective effects of Dryvax at dilutions of 1:1, 1:5, 1:10, and 1:100 (Frey S E. N Engl J Med. 2002 346:1265-75; Frey S E. N Engl J Med. 2002 346:1275-80). A major response was observed in 95% with undiluted product, 70% with 1:10 diluted vaccine, and 15% with 1:100 diluted vaccine. One month after vaccination, 34 of the 36 subjects with major reactions developed antibody responses compared to 1 of 24 patients who did not develop Jennerian pustules (Frey S E. N Engl J Med. 2002 346:1275-80). Vigorous cytotoxic T-cell and IFN-ã responses occurred in 94% of subjects with major reactions and only 1 of 24 patients who did not develop Jennerian pustules.
  • [0252]
    Routine vaccination was discontinued in 1979 because the risk of complications from the vaccine outweighed the threat of endemic smallpox (Fenner F. Bull WHO. 1988 1-68, 121-208). A 10 state study indicated that there were 1254 complications per 1 million primary vaccinations including encephalitis, progressive vaccinia, eczema vaccinatum, generalized vaccinia, and erythema multiforme (Lane J M. J Infect Dis. 1970 122:303-9). A nationwide survey showed that the case fatality rate was 1 per 1 million primary vaccinations (Lane J M. N Engl J Med. 1969 281:1201-8). Certain groups of individuals are contraindicated to be vaccinated—those with conditions causing immunodeficiency (i.e., HIV infection, leukemia, lymphoma, generalized malignancy, agammaglobulinemia, organ transplant recipients, or therapy with alkylating agents, antimetabolites, radiation, or large doses of corticosteroids), persons with eczema, persons with household contacts who are immunodeficient or who have a history of eczema, and pregnant women.
  • [0253]
    Based on the observed morbidity and mortality associated with vaccinia vaccination in the US from 1967 to 1979, a mass smallpox preventative vaccination campaign in the U.S. general public aged 1 to 65 could result in as many as 4,600 serious adverse events and 285 deaths (excluding high-risk persons and their immediate contacts) (Kemper A R. Eff Clin Pract. 2002 5:84-6). Indeed, dictating that everyone receives the Dryvax vaccine would sentence as many as 400 people to death and many others to seriously debilitating side effects (Grand Rapids Press Apr. 10, 2002). Therefore, the CDC has recommended a “ring vaccination” or containment strategy. In this approach, the following individuals receive the vaccine following actual or potential release of variola virus: persons directly exposed to the release; persons with face-to-face or household contact with an infected patient or in close proximity (within 2m); personnel directly involved in the evaluation, care, or transport of infected patients; laboratory personnel involved in processing specimens; and others likely to have contact with infectious materials (CDC Interim Smallpox Response Plan CDC November 2001; Vaccinia ACIP Morb Mortal Wkly Rep. 2001 50:1-25).
  • [0254]
    Compared to mass vaccination, ring vaccination is clearly not optimal the following reasons. (1) Pre-emptive voluntary vaccination eliminates the value of smallpox as a weapon, serving as an effective deterrent. (2) Ring vaccination is effective only for the eradication of small, localized outbreaks in a population with widespread immunity. In a largely non-immune mobile population, epidemic control after multiple simultaneous exposures is a vastly different challenge. (3) Ring vaccination requires prompt identification and vaccination of infected individuals within the 3-day post exposure period when the vaccination might be effective. A person might be infective for several days before smallpox is clinically obvious, therefore, identification of cases of exposure to an infected terrorist, for example, within a four-day period is logistically impossible. (4) The CDC is assuming that each infected person will infect only 2 to 3 others, however, as many as 38 secondary infections have been observed. (5) The logistical complexity of administering millions of vaccine doses in an acute emergency is daunting and likely to induce panic and collapse of the medical and public health service as was observed in the Dark Winter simulation exercise conducted by Johns Hopkins University in June 2001 (Bicknell W J. N Engl J Med. 2002 346: 1323-25; Henderson D A. JAMA. 1999 281:2127-39; Millar J D. Public Health Policy Advisory Board. 2000; Fenner F. Bull WHO. 1988:1-68, 121-208; O'Toole T. Johns Hopkins Center for Civilian Biodefense Strategies. 2001). In contrast, pre-exposure vaccination does not pose the logistical difficulties of vaccination during an outbreak and is less expensive. In addition, pre-exposure vaccination reduces the risk of infection among immunocompromised persons (Rosenthal S R. Emerg Infect Dis. 2001 7:920-6).
  • [0255]
    Improved vaccines capable of safely and rapidly eliciting long-lasting immunity against smallpox in all persons are clearly needed. Whether used in mass or ring vaccination strategies, greater safety and efficacy relative to Dryvax is required. The Ii-Key/antigenic epitope hybrid used alone or in combination with DNA vaccines will have the following preferred characteristics relative to Dryvax: (1) significantly reduced complication rate including death and debilitating side effects, (2) more rapid induction of protective antibodies and viral-specific cytotoxic T-cells (3) simpler vaccination method, (4) greater period of protection following primary vaccination, and (5) broader target population including use in immunocompromised individuals and in pregnancy.
  • [0256]
    One preferred approach to protecting large populations is administration of one or more immunizations with an Ii-Key/smallpox antigenic epitope hybrid of this Disclosure, followed according to the ring immunization concept by vaccinia or similar viral vaccines in the population subset of exposed or potentially exposed individuals. However, in addition, when individuals who were not in the immunized ring, contract smallpox, significant protection is afforded by prior expansion and memory of CD4+ T helper cell clones, CD8+ cytotoxic T lymphocyte clones, and B cell immunoglobulin producing clones as the case might be. Such responses create a more rapid time frame for development of clinically protective responses frame to presentation of those same and other epitopes by the smallpox virus, than would be the case in individuals not immunized with the hybrids. The process of inducing responses to viral epitopes other than that in the immunizing Ii-Key/smallpox antigenic epitope hybrid, is referred to as epitope spreading.
  • [0257]
    Although vaccination is generally regarded to be the best defense against smallpox virus, the approved vaccines and some in development are not optimally safe or potent. The Ii-Key/smallpox MHC Class II epitope hybrid vaccines can be used either alone or together with other approaches, including whole virus preparations, DNA and RNA vaccines, inactivated whole virus, and virus-like particles. The Ii-Key/antigenic epitope hybrid vaccines revealed in this Disclosure can be used in conjunction with diluted whole virus preparations, e.g., Dryvax, in order to improve the major reaction rate typically observed with diluted preparations and allow for decreased rates of complications (Frey S E. N Engl J Med 2002 346:1265-75; Frey S E. N Engl J Med 2002 346:1275-80). In addition, Ii-Key/smallpox MHC Class II epitope hybrid vaccines can be used with attenuated virus strains that have been developed (Ankara MVA and Japanese strain LC16m8) in order to augment their efficacy (Rosenthal S R. Emerg Infect Dis 2001 7:920-6; Henderson D A JAMA. 1999:281:2127-39). Ii-Key/smallpox MHC Class II epitope hybrid vaccines can be used with DNA or RNA vaccines targeting gene products that are critical for viral pathogenicity and infectivity, for example, B5R and others (Phillpotts R J. Acta Virol 2000 44:151-6; Mathew E C. J Gen Virol 2001 82:1199-213).
  • [0258]
    Ii-Key/smallpox antigenic epitope hybrids offer potent and safe vaccines against smallpox. One favored example uses Ii-Key/antigenic epitope hybrids containing the Ii-Key LRMK (SEQ ID NO: 3) motif and an MHC Class II epitope of the smallpox B5R gene product gp42. Such a construct can be further enhanced with a linked or overlapping MHC Class I epitope(s) and/or antibody-determined epitope(s). By boosting the Th response >200 times to the MHC Class II epitope, Th1 cells are recruited to elicit potent CTL and humoral responses with immunological memory. Addition of a MHC Class I epitope to the hybrid affords antigenic epitope-specific enhancement of the cytotoxic T lymphocyte response. Addition of an antibody-recognized epitope to the hybrid affords antigenic epitope-specific enhancement of the antibody-determined response.
  • [0259]
    Smallpox gp42 is selected for several reasons. (1) Gene B5R encodes a 42 kD glycoprotein that is expressed throughout the course of infection and forms part of the envelope of the extracellular virus. (2) gp42 is required for the envelopment and egress of extracellular virus and virus virulence. (3) gp42-specific IgG neutralizing antibodies are correlated with protection against orthopox infection in humans (Phillpotts R J. Acta Virol 2000 44:151-6; Englestad M. Virology. 194:627-37; Mathew E C. J Gen Virol 2001 82:1199-213). In the course of routine experimentation to identify the biologically function and vaccine potential of additional proteins coded for or induced by the smallpox virus, additional candidates for the design, synthesis and use of Ii-Key/smallpox antigenic epitope hybrids will be targeted. The methods of this Disclosure can be applied without undue experimentation toward the development of additional Ii-Key/smallpox antigenic epitope hybrid vaccines. Other extracellular envelope proteins such as A33R, A34R, A36R, and A56R, can be used to produce Ii-Key/antigenic epitope hybrids.
  • [0260]
    In addition to the above vaccine methods, the Ii-Key/smallpox antigenic epitope hybrids can be used to enhance responses to DNA vaccines encoding B5R gp42. Such DNA vaccines can also be enhanced further by incorporating the Ii reverse gene construct in the same plasmid or delivery construct. Suppression of Ii protein expression allows for the presentation of endogenous gp42 epitopes. In the context of B5R DNA vaccination, targeted Ii-suppressed antigen presenting cells will present an increased repertoire of novel, perhaps cryptic, B5R epitopes.
  • [0261]
    This invention relates in part to the design of Ii-Key/Variola B5R protein antigenic epitope hybrids. The genes of the variola virus have been identified and sequenced principally by investigators in Russia (Shchelkunov S N. FEBS Lett. 1993 319:80-83; Shchelkunov S N. Virus Res. 1994 34:207-236; Shchelkunov S N. Virus Genes 1995: 9:231-245; Shchelkunov S N. Virus Res. 1996 40:169-183). The sequence of Variola virus B5R protein (g510228) is presented in Table 18.1. Predicted MHC Class II-presented epitopes of the B5R protein are presented in Table 18.2. Table 18.3 lists Ii-Key/variola B5R protein epitope hybrids containing some of the MHC Class II-presented epitopes of Table 18.2. Predicted MHC Class I-presented epitopes of variola B5R protein are presented in Table 18.4. Table 18.5 lists Ii-Key/MHC Class II-presented/MHC Class I-presented B5R hybrids.
  • [0000]
    TABLE 18.1
    Deduced amino acid sequence of the B5R protein
    of the variola virus. (SEQ ID NO: 733)
      1 mktisvvtll cvlpavvyst ctvptmnnak ltstetsfnd kqkvtftcds
     51 gyysldpnav cetdkwkyen pckkmctvsd yvselynkpl yevnaiitli
    101 ckdetkyfrc eekngntswn dtvtcpnaec qslqldhgsc qpvkekysfg
    151 ehitincdvg yevigasyit ctanswnvip scqqkcdips lsnglisgst
    201 fsiggvihls cksgfiltgs psstcidgkw npvlpicirs neefdpvedg
    251 pddetdlskl skdvvqyeqe iesleatyhi iivaltimgv iflisvivlv
    301 cscnknndqy kfhklll
  • [0000]
    TABLE 18.2
    Predicted MHC Class II-presented epitopes of
    the B5R protein.
    SEQ
    Ii- ID
    PEPTIDE Pos. Sequence Key Score Allele NO:
    18.2.1 289 VIFLISVIV  6 2.2 01 734
    18.2.2 290 IFLISVIVL  7 3.8 03, 07, 15 735
    18.2.3 291 FLISVIVLV  8 6.3 07 736
    18.2.4  51 YYSLDPNAV  4 2.2 01 737
    18.2.5 229 WNPVLPICI 13 2.1 01, 07 738
    18.2.6 206 IHLSCKSGF  4 4.8 03 739
    18.2.7 281 IVALTIMGV  0 4.2 03, 11, 13, 15 740
    18.2.8 279 IIIVALTIM  0 3.8 03 741
    18.2.9 214 FILTGSPSS  0 3.8 04 742
    18.2.10 175 WNVIPSCQQ  0 3.6 04 743
    18.2.11  52 YSLDPNAVC  5 3.4 04, 08, 11 744
    18.2.12 277 YHIIIVALT 11 3.5 08, 11 745
    18.2.13 284 LTIMGVIFL  0 2.3 08, 07, 13, 15 746
    18.2.14   6 VTLLCVLPA  0 3.8 06, 01, 13 747
    18.2.15  84 LYNKPLYEV  6 2.5 14 748
    18.2.16 289 VIFLISVIV  6 3.6 15 749

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. Sequence is the amino acid sequence of the predicted MHC Class II-presented epitope. When a given sequence is predicted to be presented by multiple HLA-DR alleles, the first residue position of each sequence is indicated. Score is the score reported by the ProPred program, for the relative likelihood of being presented by the first HLA-DR allele listed. The respective alleles are in each case the HLA-DRB*01 allele. Ii-Key is the number of residue positions intervening between an Ii-Key motif and the first residue of the antigenic epitope.
  • [0000]
    TABLE 18.3
    Ii-Key/variola B5R epitope hybrids containing
    some of the MHC Class II-presented epitopes of
    Table 18.2.
    SEQ
    ID
    PEPTIDE Pos. Sequence NO:
    18.3.1 289, 290, Ac-LRMK-ava-VIFLISVIVLV-NH2 750
    291
    18.3.2  16 Ac-LRMK-ava-YYSLDPNAV-NH2 751
    18.3.3 229 Ac-LRMK-ava-WNPVLPICI-NH2 752
    18.3.4 206 Ac-LRMK-ava-IHLSCKSGF-NH2 753
    18.3.5 279, 281 Ac-LRMK-ava-IIIVALTIMGV-NH2 754
    18.3.6 214 Ac-LRMK-ava-FILTGSPSS-NH2 755
    18.3.7 175 Ac-LRMK-ava-WNVIPSCQQ-NH2 756
    18.3.8  52 Ac-LRMK-ava-YHIIIVALT-NH2 757
    18.3.9 277 Ac-LRMK-ava-YHIIIVALT-NH2 758
    18.3.10 284 Ac-LRMK-ava-LTIMGVIFL-NH2 759
    18.3.11   6 Ac-LRMK-ava-VTLLCVLPA-NH2 760
    18.3.12  84 Ac-LRMK-ava-LYNKPLYEV-NH2 761
    18.3.13 289 Ac-LRMK-ava-VIFLISVIV-NH2 762

    Pos. is the residue position in the primary sequence of the first amino acid in the antigenic epitope. In cases of closely overlapping predictions, the first residue position is given for each predicted epitope. Sequence is the amino acid sequence of a hybrid peptide containing a MHC Class II epitope of Table 18.2.
  • [0000]
    TABLE 18.4
    Predicted MHC Class I-presented epitopes of
    variola B5R protein.
    PEPTIDE Pos. Sequence Score SEQ ID NO:
    18.4.1 292 FLISVIVLV 736 763
    18.4.2   8 TLLCVLPAV 592 764
    18.4.3  74 KMCTVSDYV 474 765
    18.4.4 286 TIMGVIFLI  71 766