JP2002516824A - Identification of a Broadly Reactive DR-Restricted Epitope - Google Patents

Abstract

SUMMARY The present invention is based, at least in part, on the discovery and confirmation of specific immunogenic peptide epitopes against various HLA class II DR molecules typical of a global population. Such peptides include epitopes or analogs thereof that bind to HLA class II molecules with an IC ₅₀ of 1,000 nM or less.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This application claims the benefit of priority to US Provisional Patent Application No. 60 / 087,192, filed May 29, 1998. The present application also discloses U.S. patent application Ser.
No. 9953 (filed on January 21, 1998), US Patent Application No. 60 / 036,711.
No. 3 (filed Jan. 23, 1997) and US patent application Ser. No. 60 / 037,432.
No. (filed on Feb. 7, 1997).

BACKGROUND OF THE INVENTION Helper T lymphocytes (HTLs) perform several important functions in immunity to pathogens. First, they provide assistance in inducing both CTL and antibody responses. By both direct contact and the secretion of lymphokines such as IL2 and IL4, HLT promotes and supports the expansion and differentiation of T and B cell precursors into effector cells. In addition, HTLs can also be effectors on their own, and activity is also mediated by direct cell contact and secretion of lymphokines such as IFNγ and TNFα. HTL has been shown to have direct effector activity in tumors, as well as in viral, bacterial, parasitic and fungal infections.

[0003] HTLs are class II MHC molecules and antigenic peptides (typically between 10 and 20 residues in length, and on average 13-16 amino acids in size).
Recognizes the complex formed between The interaction of peptides with class II has been analyzed in detail at both structural and functional levels. And, peptide motifs specific to various human and mouse class II molecules have been proposed.

[0004] In recent years, epitope-based vaccines have received considerable attention as potential tools for the development of new prophylactic vaccines and immunotherapeutic strategies. Selection of appropriate T-cell and B-cell epitopes should allow to focus on the immune system against conserved epitopes of pathogens characterized by high sequence variability (eg, HIV, HCV and malaria) .

[0005] Furthermore, focusing on the immune response to selected determinants may be of value in a variety of chronic viral disease and cancer cases. Here, T cells directed to the immunodominant epitope are inactivated, but subdominant (subdomi).
nant) T cells specific for the epitope can escape T cell tolerance. Use of an epitope-based vaccines are also under conditions TH ₁ response is desirable, to avoid the "suppressive" T cell determinants which induce TH ₂ response, or to allow the reverse.

[0006] Finally, epitope-based vaccines also provide opportunities to be induced in vaccine construct epitopes that have been engineered to regulate their potential, either by increasing MHC binding affinity or Provided either by altering the TCR contact residues, or both. Epitope is genetically unrelated to the non-native or pathogens fully synthetic (e.g., "universal" epitopes from TT) inclusion of also possible to adjust the HTL responses to TH ₁ phenotype or TH ₂ phenotype This is a typical example of the means.

[0007] Once the appropriate epitope determinants are defined, they can be classified or delivered by various means. These include lipopeptides, viral delivery vectors, particles of viral or synthetic origin, naked or particle-adsorbed cDNA.
including.

However, before a suitable epitope can be defined, one major obstacle, in other words a very high degree of polymorphism of the MHC molecules expressed in the human population, must be overcome. In fact, over 200 different types of HLA class I and HLA class II molecules have already been identified. In the case of HLA class I molecules, it has been demonstrated that peptides that can bind to several different HLA class I molecules can be identified. More than 60% of known HLA class I molecules are actually
It can be grouped into four broad HLA supertypes, characterized by similar peptide binding specificities (HLA supermotifs).

[0009] It is also known that in the case of Class II molecules, there are actually peptides that can bind to multiple HLA types and may be immunogenic in the context of different HLA molecules. Certain immunogenic peptides, particularly those that are reactive with multiple allele products, have not been readily identified.

The present invention addresses these and other needs.

SUMMARY OF THE INVENTION The present invention is based, at least in part, on the discovery and confirmation of specific immunogenic peptide epitopes for various HLA class II DR molecules, representative of a global population. . Such peptides include the epitope, or an analog thereof. This epitope, or an analog thereof, binds to an HLA class II molecule with an IC ₅₀ of 1,000 nM or less. The epitopes of the present invention have been identified in a variety of antigens, including carcinoembryonic antigen (CEA), p53,
Tumor-associated antigens such as MAGE-2, MAGE-3, or Her2 / neu;
Viral antigens (eg, from HIV, HBV or HCV); and parasites (eg, Plasmodium falciparum).

The HLA class II binding peptide of the invention further comprises an amino acid that is Y, F, W, L, I, V, or M at the first position from the N-terminus of the epitope, and an N S, T, C, A, P, V, I, L or M at position 6 from the end
May include an epitope having an amino acid that is

The peptide epitope of the present invention or the nucleic acid encoding the peptide of the present invention
In particular, it can be used as a pharmaceutical composition to elicit a helper T cell response in a patient by contacting the epitope with a helper T cell. One or more peptide epitopes of the invention can be included in such compositions. In a preferred embodiment, one or more epitopes are presented to helper T cells by antigen presenting cells pulsed with the peptide ex vivo.

Definitions The term “peptide” is used interchangeably herein with “oligopeptide”,
Typically, it refers to a series of residues (typically L-amino acids) linked together by a peptide bond between the α-amino and carboxyl groups of adjacent amino acids. Oligopeptides of the invention are typically less than about 50 residues in length;
And usually consists of between about 10 and about 30 residues, more usually about 1
It is comprised between 2 and about 25, and often between about 15 and about 20 residues.

“Immunogenic peptide” is a peptide that contains an allele-specific motif.
As a result, the peptide binds to the MHC molecule and induces an HTL response. An immunogenic peptide of the invention can bind to the appropriate HLA molecule and induce an HTL response to the antigen from which the immunogenic peptide is derived.

With respect to a particular amino acid sequence, an “epitope” is a set of amino acid residues involved in recognition by a particular immunoglobulin, or, in the case of T cells,
A set of amino acid residues required for recognition by T cell receptor proteins and / or major histocompatibility complex (MHC) receptors. In the context of the immune system, in vivo or in vitro, an epitope is a collective characteristic of a molecule (eg, a primary, secondary or tertiary peptide structure and charge), which together form an immunoglobulin, T cell receptor or HL
Form a site recognized by the A molecule.

“Conserved residues” are typically conserved amino acids that occupy a particular position in the peptide motif, where the MHC structure may provide a point of contact with the immunogenic peptide. One to three, typically two, conserved residues within a peptide of defined length define a motif for an immunogenic peptide. These residues
Typically, they are in close contact with the peptide binding groove, and their side chains are embedded in specific pockets of the groove itself.

The term “motif” usually refers to between about 8 to about 13 amino acids for a class I HLA motif and about 6 to about 2 for a class II HLA motif.
Refers to the pattern of residues in a peptide of defined length between 5 amino acids.
It is recognized by certain HLA molecules. Peptide motifs typically differ for each protein encoded by each human HLA allele and in the pattern of primary and secondary anchor residues.

The term “supermotif”, when present in an immunogenic peptide, refers to a motif that allows the peptide to bind more than one HLA antigen. The supermotif is preferably recognized by at least one HLA allele having a wide distribution in the human population, preferably by at least two alleles, more preferably by at least three alleles. , And most preferably, is recognized by more than three alleles.

A “primary anchor residue” is an amino acid at a particular position along a peptide sequence that is understood to provide a point of contact between the immunogenic peptide and the HLA molecule.
One to three primary anchor residues within a peptide of a given length generally define a "motif" for an immunogenic peptide. It is understood that these residues fit closest to the peptide binding groove of the HLA molecule and that its side chains are embedded in specific pockets in the binding groove itself. For example, analog peptides can be made by altering the presence or absence of certain residues at these primary anchor positions. Such analogs are used to modulate the binding affinity of a peptide containing a particular motif or supermotif.

“Secondary anchor residues” are amino acids at positions other than the primary anchor position in a peptide, which can affect peptide binding. The secondary anchor residue is 1
It occurs at a significantly higher frequency among the bound peptides predicted by the random distribution of amino acids at one position. This secondary anchor residue is said to occur at the “secondary anchor position”. The secondary anchor residue can be identified as a residue that is frequently present in the high or moderate affinity binding peptide, or otherwise has a high affinity binding or moderate affinity binding peptide. Can be identified as residues that associate with the affinity binding of For example, analog peptides can be made by altering the presence or absence of certain residues at these secondary anchor positions. Such analogs are used to fine-tune the binding affinity of peptides containing a particular motif or supermotif.

A “negative binding residue” is an amino acid that, when present, results in a reduced binding affinity of the peptide for the corresponding HLA molecule of the peptide at a particular position (typically the primary anchor position) of the peptide epitope. .

Throughout this disclosure, the results are represented by “IC ₅₀ ”. The IC ₅₀ is the concentration of the peptide in the binding assay where 50% inhibition of the binding of the reference peptide is observed. Given the conditions under which the assay is performed (ie, limiting HLA protein and labeled peptide concentration), these values approximate K _D values. It should be noted that IC ₅₀ values can often vary dramatically if the assay conditions vary, and depending on the particular reagents used (eg, HLA preparations, etc.). For example, HLA molecules excessive concentration increases the measured IC ₅₀ of the apparent IC ₅₀ of a given ligand. Assays for determining binding are described in detail in PCT Publication Nos. WO94 / 20127 and WO94 / 03205. Alternatively, the binding is expressed relative to the reference peptide. As certain assays are more sensitive or less sensitive, the IC ₅₀ of the peptide being tested may vary somewhat. However, binding to the reference peptide does not vary significantly. For example, in an assay run under conditions where the IC _{50 of} the reference peptide is increased by a factor of 10, the IC ₅₀ value of the test peptide will also shift by approximately a factor of 10. Therefore, in order to avoid ambiguity, the peptide is satisfactory, moderate, whether a binder of weak or negative evaluation is generally based on the IC ₅₀ of the peptide for IC ₅₀ of a standard peptide.

[0024] As used herein, "high affinity" for HLA class II molecules is defined as binding with an IC ₅₀ or K _D of less than 100 nM. By "moderate affinity" is binding with an IC ₅₀ or K _D of between about 100nM and about 1000 nM.

“Cross-reactive bond” refers to the binding of a peptide by more than one HLA molecule and is synonymous with degenerate bond.

The phrase “isolated” or “biologically pure” refers to a substance that is substantially or essentially free of components normally associated with it, as found in its natural state. Thus, the peptides of the invention are free of substances that are normally associated with their in situ environment (eg, MHC I molecules on antigen presenting cells). Even when proteins are isolated to homogenous or dominant bands, trace contaminants of 5-10% of the native protein are present. It is co-purified with the desired protein. The isolated peptides of the present invention do not include such endogenous co-purified proteins.

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

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is directed to compositions and methods for preventing, treating or diagnosing a number of pathological conditions, such as viral, fungal, bacterial and parasitic diseases and cancer. In particular, the present invention provides novel peptides that can bind to selected major histocompatibility complex (MHC) class II molecules with an IC ₅₀ of 1000 nM or less and induce an immune response.

The peptides that bind to the MHC molecule are determined by the allele type of the MHC molecule and the amino acid sequence of the peptide. MHC class II binding peptides are usually
Within their sequence are two conserved ("anchor") residues that interact with the corresponding binding pocket in the MHC molecule. Anchor residues required for binding by several allelic forms of human MHC (HLA, histocompatible leukocyte antigen) (
Certain combinations (often referred to as "MHC motifs") are described in International Application WO
94/03205 and WO94 / 20127. According to the definition of a specific MHC motif, from the amino acid sequence of each protein,
It is possible to predict if a TL has the potential to be immunogenic. These applications describe methods for the preparation and use of immunogenic peptides in the treatment of disease.

[0030] The affinity threshold that strongly correlates with immunogenicity in the context of HLA class II DR molecules was estimated as disclosed herein. To define a biologically significant threshold for DR binding affinity, a database of 32 binding affinities of DR restricted epitopes was compiled for those restriction elements (ie, HLA molecules that bind to the motif). In nearly half of these cases (15 out of 32 epitopes), DR binding was high for binding affinity (ie, 1
Binding affinity of less than 00 nM). In the other half of these cases (3
16 out of 2), DR restriction was associated with moderate affinity (binding affinity in the range of 100-1000 nM). In only one of the 32 cases, the DR constraint is 100
Associated with an IC _{50 of} 0 nM or more. Therefore, 1000 nM, preferably 100 nM
May be defined as the affinity threshold associated with immunogenicity in the context of a DR molecule.

[0031] The peptide epitopes described herein can also be used in combination with peptide epitopes that induce a CTL response. See also WO 95/07077.

[0032] The peptide epitopes of the invention may also include analogs of the epitope. Although the peptide epitope may exhibit cross-reactive binding with multiple DR alleles, the cross-reactivity is not always perfect, and in such cases, a procedure to further increase the cross-reactivity of the peptide Can be useful; such procedures can be used to modify other properties of the peptide epitope. Achieve broader (or otherwise modified) HLA binding capacity, since general rules governing peptide cross-reactivity for HLA alleles in a given motif or supermotif have been established In order to alter the structure of a particular peptide of interest (ie, make analogs). More specifically, peptides exhibiting the broadest pattern of cross-reactivity (both in known T cell epitopes and in a more expanded set of peptides containing appropriate supermotifs) are described in the teachings herein. According to the following.

The strategy used utilizes motifs or supermotifs that correlate with binding to a particular HLA molecule. This motif or supermotif is defined by having a primary anchor, but secondary anchors can also be modified. Analog peptides can be made by substituting amino acid residues at primary, secondary, or primary and secondary anchor positions. Generally, analogs are made for peptides that already have a motif or supermotif. The supermotifs and preferred secondary anchor residues of the motifs, defined for HLA class II binding peptides, are shown in Table IX.

For a number of motifs or supermotifs according to the present invention, residues that are detrimental to binding to an allele-specific HLA molecule or a member of the HLA supertype that binds to the respective motif or supermotif are defined. Therefore,
Removal of residues detrimental to binding may be performed according to the present invention. For example, in the case of the A3 supertype, the frequency of cross-reactivity increases from 22% to 37% when all peptides with such deleterious residues are removed from the population of peptides analyzed (eg, Sidney, J. et al., Hu.
996). Thus, one strategy for improving the cross-reactivity of a peptide within a given supermotif is simply to delete one or more harmful residues present in the peptide and to use small ""Neutral" residues (T
(Cannot affect cell recognition). If residues that associate with high affinity binding to multiple alleles within the superfamily are inserted, along with elimination of deleterious residues in the peptide, an increased probability of cross-reactivity is expected.

An alteration in a natural or native epitope recognized by a T cell is
To confirm that it does not lead to failure to induce helper T cells that cross-react with the wild-type peptide, the modified peptides can be used to immunize T cells from individuals with the appropriate HLA allele in vitro, The ability of the cells to induce lysis of the target cells sensitized to the wild-type peptide increases. Class I and Class II
In both systems, cells that are either infected or transfected with the appropriate gene to confirm whether the endogenously produced antigen is also recognized by the relevant T cells are targeted. It is desired to use.

Another embodiment of the invention to identify a sufficient number of cross-reactive cell binders is to make analogs of weakly binding peptides. Class II peptides that exhibit a binding affinity of greater than 1000 nM and carry an acceptable but sub-optimal primary anchor residue at one or both positions can be obtained by substituting preferred anchor residues according to the respective supertype. Can be "fixed". The analog peptide can then be tested for cross-linking activity.

Another embodiment for producing effective peptide analogs involves substitution of residues that have a deleterious effect on the stability or solubility of the peptide in a liquid environment. This substitution can occur at any position in the peptide epitope. For example, cysteine (C)
May be substituted for α-aminobutyric acid. Due to its chemical characteristics, cysteine has a tendency to form disulfide bonds and alters the peptide structurally enough to reduce its binding capacity. Substitution of C with α-aminobutyric acid not only alleviates this problem, but in certain cases does indeed improve binding and cross-linking abilities (review: A. Sette et al., In: Persistent Viral Inf.
actions, R.M. Ahmed and I.S. Edited by Chen, John Wiley
& Sons, England, (during printing), 1988). Substitution of cysteine with α-aminobutyric acid can occur at any residue of the peptide epitope (ie, at either an anchor or non-anchor position).

The DR-binding peptides of the present invention or nucleic acids encoding them can be administered to mammals (particularly humans) for prophylactic and / or therapeutic purposes. DR peptide epitopes can be used to enhance the immune response to other immunogens administered with the peptide. For example, a CTL epitope / DR epitope mixture
It can be used to treat and / or prevent viral infections and cancer. Alternatively, an immunogen that induces an antibody response can be used. Examples of diseases that can be treated using immunogenic mixtures of DR peptides with other immunogens include prostate cancer, hepatitis B, hepatitis C, AIDS, kidney cancer, cervical cancer, lymphoma, CMV and apical. Kei Condyloma.

[0039] DR binding peptides or nucleic acids encoding them can also be used to treat various conditions associated with unwanted T cell reactivity. Examples of diseases that can be treated using DR binding peptides include autoimmune diseases (eg, rheumatoid arthritis, multiple sclerosis, and myasthenia), allograft rejection, allergies (eg, pollen allergy), lime Disease, hepatitis, LCMV, after endocarditis due to streptococci,
Or glomerulonephritis, and food hypersensitivity.

In therapeutic applications, the immunogenic compositions or DR-binding peptides or nucleic acids of the invention are administered to an individual already suffering from a cancer, an autoimmune disease, or an infection with the virus of interest. During the incubation or acute phase of the disease, they can be treated, where appropriate, using the DR-binding peptide or immunogenic conjugate separately or in combination with other treatments.

In therapeutic applications, the composition containing the immunogenic composition is administered in an amount sufficient to treat or at least partially arrest the symptoms of the disease and its complications. An amount adequate to accomplish this is defined as "therapeutically effective dose." Effective amounts for this use will depend, for example, on the peptide composition, the mode of administration, the stage and severity of the disease being treated, the weight and general state of the patient, and the judgment of the prescribing physician.

[0042] Therapeutically effective amounts of the immunogenic compositions of the present invention are within the general range of immunogenically effective doses described below. These doses may be followed by booster doses, depending on the patient's response and condition by measuring specific HTL activity in the patient's blood, according to booster dosing regimes over weeks to months.

It must be kept in mind that the compositions of the present invention can generally be used in severe disease states (ie, life-threatening or potentially life-threatening situations). . In such cases, a substantial excess of these compositions by the treating physician is possible, given the minimization of foreign substances and the relatively non-toxic nature of the conjugate, and is not desirable. You can be convinced.

For prophylactic use, administration should be to the at-risk group. For example,
Protection against malaria, hepatitis, or AIDS may be achieved by administering the compositions of the present invention prophylactically, thereby increasing immune competence. Therapeutic administration can be initiated shortly after the first signs of disease, or detection or surgical resection of a tumor, or diagnosis in the case of acute infection. This is followed by a booster dose at least until the symptoms are substantially reduced, and thereafter. In chronic infections, loading doses followed by booster doses may be required.

Treatment of an infected individual with a composition of the invention may hasten resolution of the infection in acutely infected individuals. For those individuals susceptible to (or predisposed to) developing a chronic infection, the compositions are particularly useful in methods for preventing the progression of an acute to chronic infection. A susceptible individual is identified before or during infection, for example, as described herein. The compositions can be targeted to them, minimizing the need for administration to a larger population.

The peptide mixtures or conjugates can also be used for treating chronic infections and for stimulating the immune system to eliminate virally infected cells of the carrier. It is important to provide the amount of the immunopotentiating peptide in the dosage regimen and embodiments sufficient to effectively stimulate a helper T narrow response. For example, immunizing doses at established intervals of one to four weeks, followed by boosting doses, may be required for an extended period of time to effectively immunize the individual. For chronic infections,
Dosing should be continued at least until clinical symptoms or laboratory tests indicate that the viral infection has been eliminated or substantially reduced, and thereafter.

[0048] Pharmaceutical compositions for therapeutic or prophylactic treatment are contemplated for parenteral, topical, oral, or topical administration. Typically, the pharmaceutical compositions are administered parenterally (eg, intravenously, intrathecally, subcutaneously, intradermally, or intramuscularly).
Due to the ease of administration, the vaccine compositions of the invention are particularly suitable for oral administration. Accordingly, the present invention provides a composition for oral administration comprising a solution of a peptide or conjugate dissolved or suspended in an acceptable carrier, preferably a water-soluble carrier. Various water-soluble carriers (eg, water, buffer, 0.9
% Saline, 0.3% glycine, hyaluronic acid, etc.). These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions can be packaged for use or lyophilized, and the lyophilized preparation combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances required for appropriate physiological conditions, such as pH adjusting agents, buffering agents, tonicity adjusting agents, wetting agents, such as sodium acetate, sodium lactate, Sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like).

The concentration of the DR and / or CTL stimulating peptide of the invention in a pharmaceutical formulation
Can vary very widely. That is, by weight, less than about 0.1% to usually about 2% or at least about 2%, on the order of 20% to 50% or more, and according to the particular mode of administration chosen, the volume of liquid, First selected by viscosity etc.

[0049] The peptides or conjugates of the present invention may also be used in certain tissues (eg, lymphoid tissues).
Or can be administered via liposomes that act to be selectively targeted to infected cells and to increase the half-life of the peptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, lipid crystals, phospholipid dispersions, lamellar layers, and the like. In these preparations, the delivered peptide can be used as part of a liposome, alone or in combination with a molecule that binds to, for example, a common receptor among lymphoid cells (eg, a monoclonal antibody that binds to the CD45 antigen). Or with other therapeutic or immunogenic compositions. Thus, liposomes filled with the desired peptides or conjugates of the invention can be directed against the site of lymphoid cells, where the liposomes can be used to transform the selected therapeutic / immunogenic peptide composition. Deliver. Liposomes for use in the present invention are generally formed from standard vesicle-forming lipids, including neutral and negatively charged phospholipids and sterols (eg, cholesterol). The choice of lipid is generally guided by, for example, considerations of liposome size, acid instability, and liposome stability in the bloodstream. Various methods (e.g., incorporated herein by reference)
Szoka et al., Ann. Rev .. Biophys. Bioeng. 9,
467 (1980), U.S. Pat. Nos. 4,235,871 and 4,501,7
No. 28, 4,837,028, and 5,019,369) are available for preparing liposomes.

Ligands to be incorporated into liposomes to target immune cells can include, for example, antibodies or fragments thereof specific for cell surface determinants of cells of the desired immune system. Liposomal suspensions containing peptides or conjugates can be administered intravenously, topically, topically, etc.
And may be administered in a volume that varies according to the stage of the disease being treated.

Alternatively, the DNA or RNA encoding one or more DR peptides (and, optionally, a polypeptide comprising one or more CTL epitopes or antibody-derived epitopes) may cause an immune response to the polypeptide encoded by the nucleic acid. May be introduced to the patient to obtain This approach is described, for example, in Wolff et al., Science.
e 247: 1465 (1990) and U.S. Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118.
No. 5,736,524; No. 5,679,647; WO 98/047
No. 20; and described in more detail below. Examples of DNA-based delivery techniques include "naked DNA", (bupivacaine, polymer, peptide mediated) facilitated delivery, cationic lipid complex delivery, and particle mediated "gene gun" (Gene gun) delivery.

A preferred means of administering a nucleic acid encoding a peptide of the invention uses a minigene construct that encodes one or more epitopes of the invention. The use of multiple epitope minigenes is described below and in, for example, An, L. et al. And Whiton, J .;
L. , J. et al. Virol. 71: 2292, 1997; Thomson, S .; A.
J. et al. Immunol. 157: 822, 1996; L
. J. et al. Virol. 67: 348, 1993; Hanke, R et al., Vacc.
ine 16: 426, 1998. For example, HBV and HI
PADRE ^™ universal helper T cells (H)
The (TL) epitope as well as the ER transfer signal sequence were engineered. Immunization of HLA transgenic mice with this plasmid construct is similar to the response observed with known immunogenic lipopeptides in humans against the nine epitopes tested, and with an oil-based adjuvant. Produced a strong CTL-induced response that was significantly greater than Furthermore, the immunogenicity of the DNA-encoded epitope in vivo correlated with the in vitro response of certain CTL lines to target cells transfected with the DNA plasmid.

For example, D encoding an epitope selected for expression in human cells
To make the NA sequence (minigene), the amino acid sequence of the epitope can be back-translated. A table of human codon usage can be used to guide codon selection for each amino acid. These epitope-encoding DNA sequences can be immediately flanked so that when translated, a continuous polypeptide sequence is created. Additional elements to optimize expression and / or immunogenicity
Can be incorporated into minigene design. Examples of amino acid sequences that can be back-translated and included in the minigene sequence include: HLA class I epitope, HLA class II epitope, ubiquitination signal sequence, leader sequence and / or ER targeting signal. Furthermore, the CTL and HTL epitope H
LA presentation can be improved by including synthetic (eg, polyalanine) or naturally occurring flanking sequences adjacent to the CTL or HTL epitope.

The minigene sequence is converted to DNA by assembling oligonucleotides encoding the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases in length) can be synthesized, phosphorylated, purified, and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotide are ligated using, for example, T4 DNA ligase. This synthetic minigene encoding the epitope polypeptide can then be cloned into the desired expression vector.

[0055] Standard regulatory sequences, well known to those skilled in the art, are preferably included in the vector to ensure expression in the target cell. Several vector elements are required: a promoter with a downstream cloning site for insertion of the minigene;
A polyadenylation signal for efficient transcription termination; E. coli origin of replication; coli selectable markers (eg, ampicillin or kanamycin resistance). Numerous promoters (eg, human cytomegalovirus (hCMV)
) Promoter) can be used for this purpose. For other suitable promoter sequences, see US Patent Nos. 5,580,859 and 5,589,46.
See No. 6.

Further vector modifications may be desired to optimize minigene expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally occurring introns can be incorporated into the transcribed region of the minigene. The inclusion of mRNA stabilizing sequences and sequences for replication in mammalian cells can also be considered to increase minigene expression.

[0057] Once the expression vector is selected, the minigene is cloned into the polylinker region downstream of the promoter. This plasmid contains the appropriate E. coli. col
i strain is transformed and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene and other elements contained in the vector are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells with the correct plasmid can be stored as a master cell bank and a working cell bank.

In addition, immunostimulatory sequences (ISS or CpG) appear to play a role in the immunogenicity of DNA vaccines. These sequences can be included in the vector outside of the minigene coding sequence if it is desired to enhance immunogenicity.

In some embodiments, a dual gene to allow for the production of both the epitope encoded by the minigene and a second protein (included to enhance or reduce immunogenicity). A bi-cistronic expression vector may be used. Examples of proteins or polypeptides that can advantageously enhance an immune response when co-expressed include cytokines (eg, IL-2
, IL-12, GM-CSF), cytokine-inducible molecules (eg, LeIF)
Or costimulatory molecules. A helper (HTL) epitope can be linked to the intracellular targeting signal and expressed separately from the CTL epitope;
This allows the HTL epitope to be directed to a different cell compartment than the CTL epitope. If required, this could be the HTL to the HLA class II pathway.
It may facilitate more efficient entry of epitopes, thereby improving CTL induction. In contrast to HTL or CTL induction, immunosuppressive molecules (eg, TG
Specific reduction of the immune response by co-expression of F-β) may be advantageous in certain diseases.

[0060] Therapeutic amounts of plasmid DNA can be found, for example, in E. coli. It can be produced by fermentation in E. coli followed by purification. Aliquots from the working cell bank are used to inoculate the growth medium and are grown to saturation in shake flasks or bioreactors according to well-known techniques. Plasmid DNA is QI
AGEN, Inc. (Valencia, California) can be purified using standard biological separation techniques, such as a solid phase anion exchange resin. If required, supercoiled DNA can be isolated from the open and linear forms using gel electrophoresis or other methods.

[0061] Purified plasmid DNA can be prepared for injection using various formulations. The simplest of these is sterile phosphate buffered saline (PB
Reconstitution of the lyophilized DNA in S). This approach, known as "naked DNA", is currently used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of the minigene DNA vaccine, alternative methods for formulating purified plasmid DNA may be desired. Various methods have been described and new techniques may become available. Cationic lipids also
It can be used in formulations (see, for example, WO 93/24640).
Mannino and Gould-Fogerite, BioTechniq;
Ues 6 (7): 682 (1998); U.S. Pat. No. 5,279,833; W
O 91/06309; and Felgner et al. (1987) Proc. N
atl. Acad. Sci. USA 84: 7413 (1987)). In addition, peptides and compounds, collectively referred to as glycolipids, fusogenic liposomes, protective, interactive, non-condensing (PINC), are also complexed and purified to purified plasmid DNA. It can affect variables such as gender, intramuscular dispersion, or transport to a particular organ or cell type.

[0062] In vitro assays can be used as functional assays for expression of HTL epitopes. For example, plasmid DNA is introduced into a mammalian cell line that is a suitable presenter of the HTL epitope. The transfection method used will depend on the final formulation. Electroporation can be used for "naked" DNA, while cationic lipids allow for direct in vitro transfection. Green fluorescent protein (GFP)
May be co-transfected to allow enrichment of the transfected cells by using fluorescent cell analysis separation (FACS). The cells can then be isolated from the HT using methods known in the art.
It can be assayed for its ability to elicit an L response (see, for example, Alxander et al., Immunity 1: 751-761, 1994).

In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations (including both CTL and HTL epitopes). A transgenic mouse expressing the appropriate human HLA protein was constructed using DNA
Immunized with the product. The dose and route of administration will depend on the formulation (eg, P
IM for DNA in BS, IP for lipid-complexed DNA). Twenty-one days after immunization, splenocytes are harvested and restimulated for one week in the presence of the peptide encoding each epitope to be tested. For CTL effector cells, assays are performed using standard techniques for cell lysis of chromium-51 labeled target cells loaded with peptide. Lysis of target cells sensitized by HLA loading with a peptide corresponding to the epitope encoded by the minigene demonstrates the function of a DNA vaccine for in vivo induction of CTL.

Alternatively, the nucleic acid can be administered using ballistic delivery, for example, as described in US Pat. No. 5,204,253. By using this technique, particles containing a single DNA are administered. In a further alternative embodiment, the DNA may be attached to a particle (eg, a gold particle).

For solid compositions, conventional non-toxic solid carriers can be used. This includes, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, magnesium carbonate and the like. For oral administration, the non-toxic pharmaceutically acceptable compositions can be prepared using any of the commonly used excipients (e.g., the carriers listed above and typically 10-95% of the active ingredient, i.e., 1 of the present invention
One or more conjugates, and more preferably at a concentration of 25% to 75%).

For aerosol administration, the peptide is preferably supplied in finely divided form with a surfactant and a propellant. A typical ratio of conjugate is 0.0
It is 1% to 20% by weight, preferably 1% to 10%. The surfactant must, of course, be non-toxic and, preferably, soluble in the propellant. Representative of such agents are esters or partial esters of fatty acids containing 6 to 22 carbon atoms (e.g., caproic acid, octanoic acid with aliphatic polyhydric alcohols or cyclic anhydrides thereof). , Lauric acid, palmitic acid,
Stearic acid, linoleic acid, linolenic acid, olesteric acid (olesteric
acid), and oleic acid). Mixed esters, such as mixed or natural glycerides, can be used. Surfactants comprise 0.1% of the composition.
% To 20% by weight, preferably 0.25 to 5%. The balance of the composition is usually propellant. For example, a carrier may also be included, if desired, to use lecithin for intranasal delivery.

In another aspect, the invention relates to an immunogenic DR, as described herein.
The present invention relates to a vaccine comprising, as an active ingredient, an immunogenic effective amount of a peptide or a CTL / DR peptide conjugate or a nucleic acid encoding them. The conjugate (s) can be linked to its own carrier or introduced into a host, including a human, as a homopolymer or heteropolymer of active peptide units. Such polymers have the advantage of increased immunological response, and if different peptides are used to make the polymer, antibodies and / or that react with different antigenic determinants of the virus or tumor cells It has the additional ability to induce CTL. Useful carriers are also well known in the art and include, for example, thyroglobulin, albumin (eg, bovine serum albumin), tetanus toxin, polyamino acids (eg, poly (lysine: glutamic acid)), hepatitis B virus core protein, B Hepatitis virus recombinant vaccine and the like. Vaccines also include physiologically tolerable (acceptable) diluents, such as, for example, water, phosphate buffered saline, or saline, and more typically include an adjuvant.
Adjuvants such as incomplete Freud's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are well-known materials in the art. And
As described above, a CTL response can be sensitized by conjugating a peptide of the invention to a lipid (eg, P ₃ CSS). Upon immunization via injection, aerosol, oral, transdermal, or other routes, with a peptide composition as described herein, the host immune system will be specific for the desired antigen. Large amount of CT
Responds to the vaccine by producing L and / or HTL. The host is then at least partially immunized against subsequent infection or becomes resistant to the development of a chronic infection.

A vaccine composition comprising a DR peptide of the invention is susceptible to or otherwise susceptible to a disease (eg, a viral infection or cancer) in an amount that elicits an immune response to the antigen. Administered to a patient with sexual activity, thus increasing the patient's own immune response capacity.

For a vaccine of the peptides disclosed herein, a therapeutically effective amount and the amount used is defined as an “immunogenically effective dose”. In this use, the precise amounts again depend on the condition and weight of the patient, the mode of administration, the nature of the formulation, and the like, but generally will be at lower levels of about 1, 5, 50, 500, or 1000 μg, and at higher levels about 10,000; 20,000; 3
Or at unit doses ranging from 50,000 μg. Dosage values for a human are typically from about 500 μg to about 50,000 μg / 70 kg patient.
g.

In some cases, it may be desirable to combine the peptide vaccines of the invention with a vaccine that induces a neutralizing antibody response against the virus of interest, especially the viral envelope antigen. For example, a PADRE peptide can be combined with a herpes vaccine to increase the likelihood or broaden the population. A suitable herpes vaccine that can be used in this manner is Recomvivax H
B (Merck) and Engerix-B (Smith-Kli)
ne).

For therapeutic or immunization purposes, the peptides of the invention can also be expressed by an attenuated viral host such as vaccinia or fowlpox. This approach involves the use of vaccinia virus as a vector to express a nucleotide sequence encoding a peptide of the invention. Upon introduction into an infected host, acute or chronic, or into an uninfected host, the recombinant vaccinia virus expresses an immunogenic peptide, thereby eliciting a CTL response in the host. Vaccinia vectors and methods useful in immunization protocols are described, for example, in US Pat. No. 4,722,848, incorporated herein by reference. Another vector is BCG (Bacille Calmette Guerin). The BCG vector is described in Stover et al., Na, incorporated herein by reference.
351, 456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the present invention (eg, Salmonella typhi vectors, etc.) will be apparent to those skilled in the art from the description herein.

The peptide epitopes of the invention can be administered to antigen presenting cells (APCs), preferably dendritic cells, also ex vivo. In a preferred embodiment,
Response to a particular pathogen (pathogen, or tumor antigen) is induced by pulsed APC with a peptide epitope, followed by administration of the pulsed APC, and the cells are then expressed in vivo. Will present this peptide. The pulsed APC can be administered in vivo, as described above for the peptide.

The peptide epitopes of the invention can also be used ex vivo, in combination with CTL epitopes, to induce CTL as well. The resulting C
TL can be used to treat infections or tumors. Ex vivo CTL responses to certain pathogens are induced by incubating patient CTL precursor cells in tissue culture with antigen presenting cells and a source of the appropriate immunogenic peptide epitope. After an appropriate incubation time for CTL precursor cells to be activated and expanded to effector CTL ( ^* typically 1
４4 weeks), the cells are infused back into the patient, where they destroy their specific target cells (infected or tumor cells).

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

This peptide may also find use as a diagnostic reagent. For example, the peptides of the invention can be used to determine the susceptibility of a particular individual to a treatment regimen using the peptide or related peptides. Thus, this may be useful in modifying existing treatment protocols for affected individuals, or in determining prognosis. In addition, peptides can also be used to predict which individuals have a substantial risk of developing a chronic infection.

EXAMPLES Materials and Methods Cells The following Epstein-Barr virus (EBV) transformed homozygous cell lines were used as a source of human HLA class II molecules: LG2 [
DRB1c0101 (DR1) 1; GM3107 [DRB50101 (DR2w
2a)]; MAT (DRB10301 (DR3) 1; PREISS [DRB10
401 (DR4w4) 1; BIN40 [DRB10404 (DR4w14) 1;
SWEIG [DRB11101 (DR5w11)]; PITOUT [DRB10
701 (DR7)] (a); KT3 [DRB10405 (DR4w15)];
Herluf [DRB11201 (DR5w12)]; HO301 [DRB11
302 (DR6w19)]; OLL [DRB10802 (DR8w2)]; and HTC9074 [DRB10901 (DR9), courtesy of Dr. Paul Harris, Columbia University]. In some cases, transfected fibroblasts were used: L466.
1 [DRB11501 (DR2w2b)]; TR81.19 [DRB30101
(DR52a)]; and L257.6 [DRB40101 (DRw53)].
(Villi et al., J. Clin. Invest. 91: 616 (1993). Cells were treated with 2 mM L-glutamine [GIBCO, Grand Island, NY].
], 50 μM 2-ME, and 10% heat inactivated FCS [Irvine Sc
RPMI 1640 supplemented with C. identific, Santa Ana, CA]
Maintained in vitro by culturing in medium. Cells were also harvested at 100 μg /
ml of streptomycin and 100 U / ml penicillin [Irvine
Scientific]. Large amounts of cells were grown in stirred cultures.

[0077] Cells were harvested with 1% NP-40 [Fluka Biochemika, Buchs.
, Switcherland], 1 mM PMSF [CalBioChem, L
a Jolla, CA], 5 mM Na-orthovanadate (orthovan
adate), and 25 mM iodoacetamide [Sigma Chemi
cal, St .; [Louis, Mo] at a concentration of 10 ⁸ cells / ml. Lysates were freed of debris and nuclei by centrifugation at 10,000 xg for 20 minutes.

Affinity Purification of HLA-DR Molecules Class II molecules were prepared as previously described (Sette et al., J. Immunol. 142: 35 (1989) and Gorga et al., J. Biol. Chem. 262: 16087 (1987)
), Purified by affinity chromatography using mAb LB3.1 conjugated to Sepharose 4B beads. Lysates were filtered through 0.8 and 0.4 μM filters and then passed over an anti-DR column. This was then combined with 15% of 1% NP-40, 10 mM TRIS in PBS.
PB containing 0.4% n-octyl glucoside, washed with column volume
Washed with 2 column volumes of S. Finally, DR was prepared using 50 mM diethylamine (pH 11) in 0.15 M NaCl containing 0.4% n-octylglucoside.
. Eluted in 5). 1/25 volume of 2.0 M Tris (pH 6.8) was added to about 8
. Add to the eluate until the pH drops to 0, then Centriprep 3
0 by centrifugation at 2000 rpm in a concentrator (Amicon, Be.
very, MA).

Class II Peptide Binding Assay A panel of 13 different specific DR peptide assays was utilized in this study. These assays are almost universal DR
It was selected as being representative of the allele. Table I lists, for each DR antigen, the representative allele product utilized, the cell line utilized as a source of DR, and the radiolabeled probe utilized in the assay. Purified human class II molecules [5-500 nM] were combined with various unlabeled peptide inhibitors and 1-10 nM ¹²⁵ I radiolabeled probe peptide in 5%
Were incubated for 48 hours in the presence of a protease inhibitor cocktail in PBS containing DMSO. The radiolabeled probe used was HA Y30
7-319 (DR1), tetanus toxin [TT] 830-843 (DR2w2a, D
R5w111, DR7, DR8w2, DR8w3, DR9), MBP Y85-
100 (DR2w2b), TT1272-1284 (DR52a), F is DR3
65 kD Y3-13 with Y7 substituted with the sequence YARFQSQTT
Non-natural peptides with LKQKT (DR4w4, DR4w15, DRw53
) (Valli et al., Supra), as well as DR5w12, a naturally processed peptide eluted from cell line C1R, EALIHQLINPYVLS (D
R5w12) and DR6w19 for the 650.22 peptide (TT 83
0-843 A (R) S836 analog).

The radiolabeled peptide was iodinated using the chloramine-T method. Peptide inhibitors were typically tested at concentrations ranging from 1201 μg / ml to 1.2 ng / ml. The data was then plotted and 50% inhibition (IC50
) Was measured. Under appropriate stoichiometric conditions, the IC50 of the unlabeled test peptide against purified DR is a reasonable approximation of the affinity of the interaction (Kd). The peptides were tested in two to four completely independent experiments. The final concentrations of protease inhibitors are as follows: 1 mM PMSF, 1
. 3 nM 1.10 phenanthroline, 73 μM pepstatin A, 8 mM E
DTA, and 200 μM Nα-p-tosyl-L-lysine chloromethylketone (TLCK) [all protease inhibitors from CalBioChem, La Jolla, CA]. The final detergent concentration in the incubation mixture was 0.05% Nonidet P-40. Assays were performed at pH 7.0 except for DR3. DR3 is performed at pH 4.5 and DRw53 is p
H5.0. The pH was described previously (Sette et al., J. Immunol.
148: 844 (1992)).

The class II peptide conjugate was applied to a TSK2000 column (TosoHaas 1
Free peptide was separated by gel filtration on 6215, Montgomeryville, PA). The fraction of bound peptide was then calculated as previously described (Sette et al. (1989) supra). In preliminary experiments, D
R preparations are classified as Class I where 10-20% of the total radioactivity is required for binding.
To determine the concentration of the I molecule, it was titrated in the presence of a fixed amount of radiolabeled peptide. All subsequent inhibition and direct binding assays were performed using these Class II concentrations.

(DRB1 Specificity of DR4w15, DR6w19, DR8w2, DE8w3, and DR9 Assays) Since the antibodies used for purification are α-chain specific, the β1 molecule is β3 (
And / or β4 and β5) molecules. The development and confirmation of assays for DRβ chain specificity is described in detail elsewhere for many of the DR alleles listed in (108) above. Here we describe for the first time the DR4w15, DR6w19, DR8w2, DR8w3, and DR9 assays. Experiments that address the β-chain specificity of these novel assays are described in this section.

The (DR4w15) β4 product, DRw53, is co-expressed with DR4w15, and determining the specificity of the DR4w15 binding assay is complicated in that the same radiolabeled ligand is used in both the DR4w15 and DRw53 binding assays. It is. Typically, the predominant specificity detected in the assay as the β1 chain is expressed at 5-10 fold higher levels than other β chains and all binding assays are performed utilizing a limited amount of DR Is expected to be DR4w15. To confirm that this is indeed the case, 5 in the putative DR4w15-specific assay
The binding pattern of a panel of eight different synthetic peptides was compared to the pattern obtained in a DRw53-specific assay (using DRw53 fibroblasts as a source of class II molecules). Attention was focused on two very different binding patterns.
In some cases, the peptide bound with high affinity to one DR molecule and did not bind to other molecules (data not shown).

(DR6w19) For the DR6w19 assay, DRB30301 (DR52
The EBV-transformed homozygous cell line H0301, co-expressing a), is used as a source of class II molecules. The radiolabeled ligand used in the DR6w19 assay is different from that used in the DR52a assay, but the ligand is associated with a high affinity DR52a binder (ie, is a single substituted analog). As was done for DR4w15, the specificity of the assay was
9 and DR52a were investigated by analyzing the binding capacity of a panel of naturally occurring peptides. The two assays demonstrated completely different binding specificities. For example, with respect to relative binding, TT 1272-1284 provides DR16w
It binds 63 times better in the DR52a assay than in the 19 assay. Conversely, the invariant peptide binds 189 times better than in the DR6w19 assay. In conclusion, these data demonstrated that binding of radiolabeled peptide 650.22 to purified class II MHC from the H0301 cell line was specific for DR6w19.

(DR8w2 and DR8w3) β for DR8w2 and DR8w3 assays
Monospecificity is evident in that β3 (and / or B4 and β5) molecules are not expressed.

(DR9) The specificity of the DR9 assay was determined by a previous study (Ale) showing that the TT 830-843 radiolabeled probe peptide did not bind to the DRw53 molecule.
Xander et al., Immunity 1: 751 (1994)).

(Results) (DR Binding Affinity of Antigenic Peptide Recognized by DR-Restricted T Cells) In order to define a threshold value of DR binding affinity considered to be biologically significant, the present inventors Et al. Compiled the affinities of 32 panel reported cases of DR restriction of a given T cell epitope. In about half the cases, DR binding was associated with an affinity of less than 100 nM, and in half of the other examples, with an IC50% ranging from 100-1000 nM. In only one of the 32 cases (3.1%), DR binding was associated with 50% of the IC with 1000 nM or more. This distribution of affinities is consistent with the previously reported HLA where most of the epitopes bound with an IC50% of less than 50 nM.
It was noted that the affinity distribution of class I epitopes was different (Sette et al., JI, 1994). This relatively low affinity class II restricted epitope interaction may generally explain why activation of class II restricted T cells requires more antigen compared to class I restricted T cells .

In conclusion, this analysis allows 1000 nM to be defined as an affinity threshold associated with immunogenicity in the context of DR molecules, and for this reason a suitable target for our study It was suggested that

(P1 and P6 Anchors Are Required, but Not Sufficient for DRB10401 Binding) Several independent studies have shown that a core region near the N-terminus of the peptide and 9 residues (residues 1-9) A large aromatic or hydrophobic residue at position 1 of DRB1040
An important role in one bond has been pointed out. In addition, an important role has been demonstrated for the residue at position 6 (P6) of this 9 residue core region. Short residues and / or
Alternatively, hydrophobic residues are generally preferred at this position (O'Sullivan et al., JI 147: 2663, 1991; Sette et al., JI 151: 3163.
Hammer et al., Cell 74: 197, 1993, and Ma.
rshall et al., JI 154: 5927, 1995).

In a series of this experiment, a library of 384 peptides was added to DRB104
01 were analyzed for binding ability and screened for the presence of the P1-P6 motif (ie, F, W, Y, L, I, V, or M in P1, and S, T, C, A, P, in P6). P, V, I, L, or M, at least 9 residues away from the C-terminus of the peptide). This set of 384 peptides contains a total of 80 DR4w4 binders (especially 27 good binders [IC less than 100 nM].
50], and 53 medium binders [IC5 in the range 100-1000]
0] was included. 77 of the 80 DR4w4 binders (96%)
Had the P1-P6 motif. However, it should be noted that most non-DR4w4 binding peptides also contained the P1-P6 motif. Of the 384 peptides contained in our database, only 125 contain "
P1-P6 negative ". 77/259 of the "P1-P6 positive" peptide (30
In contrast, only three of them (6%) bound to the extent assessed against purified DR4w4. Therefore, these results indicate that DRB10401
It demonstrates that the presence of appropriate P1 and P6 anchors is required for binding, but not enough.

Detailed Map of DRB10401 Peptide Interactions Next, for each P1-P6 aligned core region, similar to the strategy previously used to probe peptide class I interactions, The average binding affinity of the peptide with a particular residue was calculated for each position compared to the rest of the group. According to this method, a table of average relative binding (ARB) values was compiled.
The table also presents a map of the positive or negative effects of each of the 20 naturally occurring amino acids on DRB10401 binding ability when occupying a particular position compared to the primary P1-P6 anchor (FIG. 1). .

A change in ARB value of more than 4-fold (ARB ≧ 4 or ≦ 0.25) is arbitrarily determined to be significant and indicative of a secondary effect of a given residue on DR peptide interaction. Thought. Most secondary effects were associated with positions 4, 7, and 9.
These positions correspond to secondary anchors that engage a shallow pocket on the DR molecule. Furthermore, a significant secondary effect was the third M (ARB = 12.8), the third T (A
RB = 4.34), and I at position 5 (ARB = 4.4).

(Development of DRB10401-Specific Algorithm) Next, a DRB10401-specific algorithm was developed using an ARB table. To infer the 0401 binding propensity, each aligned P1-P6 sequence was
Each position was scored by multiplying by the ARB value of the appropriate amino acid. Following this procedure, a numerical "algorithm score" was derived. Multiple P1-P
If 6 alignments were possible, the binding score was calculated for each and the best score was selected. The efficiency of this method in estimating the binding capacity of 0401 is shown in Table IIa.

Considering only those peptides with an algorithm score greater than -17.00 reduced the set of putative peptides to 156. This set still contained 72 (90%) of all 80 high or medium DR binders. Increasing the cutoff to an algorithm score of -16.44 or higher allowed the identification of yet another 60 (75%) of the 80 DR4w4 binding peptides. Of the entire set of 107 peptides, 25 of them were either good or moderate binders. In other words, as expected, increasing the stringency of the algorithm score predicted a smaller portion of the total binder present in the set, but at the same time, identified fewer false positive peptides.

Blind Test of the Estimation Ability of the DRB10401-Specific Algorithm It can be seen that the estimation ability of our algorithm is not merely a reflection of utilizing the same set of data to test and define the algorithm itself. To confirm, we further tested its efficiency in a blind estimation test. To this end, we utilized data from an independent set of 50 peptides whose binding affinity was known but not used in the derivation of the algorithm. As shown in Table IIb, the algorithm was effective in estimating the DR4w4 binding capacity of this independent set of peptides. An algorithm score of -17.00 identified a total of 18 peptides. This set includes 3 of all 3 good binders (100%) of the total of the 50 peptide sets tested and 8 of all 11 medium binders (70%). Cutoff value is -16
. By increasing to 44, a set of nine peptides was identified. Seven of them (78%) were either good or medium binders. This set contained 7 (50%) of the 14 binders included in the blind putative peptide set. Taken together, these data support the validity of the DR4w4-specific algorithm described above.

Detailed Map of DRB10401, DRB10101, and DRB10701 Peptide Binding Specificities Next, we utilized the 38 to define the DR4w4 algorithm.
The same set of four peptides was analyzed for binding to purified DR1 and DR7 molecules. This set was found to contain 120 and 59 binders for the DR1 and DR7 alleles, respectively. A total of 158 peptides could bind to either DR1, DR4w4, or DR7. These high proportions (73/158; 46%) were also degenerate binders, binding to more than one of the three alleles considered so far. In addition, we found that more than 90% of the good and moderate binders of DR1 or DR7 had a P1-P6 motif. Most importantly, 7
Seventy-two (99%) of the three degenerate DR binders had this motif (data not shown). Taken together, this analysis suggests that an algorithm based on P1-P6 can be used to efficiently predict degenerate DR binders.

As above for the DR4w4 molecule, specific algorithms were designed for the DR1 and DR7 alleles. 2A and 2B detail allele-specific maps defined according to this method.

As in the case of DRB10401, most of the secondary effects were concentrated at positions 4, 7, and 9. The fourth position was particularly remarkable in the case of DR1. On the other hand, position 7 was the most prominent secondary anchor of DR7. Specific algorithms were developed based on these maps, and the cutoff values required to predict 75% or 90% of the binder were -19.32 and -20 for DR1, respectively.
. 28 and was found to be 20.91 and -21.63 for DR7. Depending on the particular allele or cutoff value chosen, 40
〜60% of the putative peptides were in fact good or moderate binders (data not shown).

(Development of DR1-4-7 Combination Algorithm) Finally, the present inventors examined whether the combination algorithm can estimate degenerate binders. To this end, the sequences of 384 peptides in our database were screened simultaneously using three (DR1, 4w4, and 7) specific algorithms. As many as 100 peptides
It was found that binding to either two or three of the alleles considered was assumed (using a 75% cutoff). This set includes 59 of 73 peptides that can actually bind degenerate 1-4-7 (defined as the ability to bind to more than one of the DR1, 4w4, or 7 alleles). (81%) (Table III).

Defining a Target Set of DR Specificity Representative of a Worldwide Population The data presented in the section above shows that peptides that can bind to multiple DR alleles are combined as “1-4-7” 4 illustrates a method that can be identified by use of an algorithm. Next, we wanted to test if peptides exhibiting degenerate 1-4-7 binding behavior would also bind to other common DR types as well. As a first step in our experimental strategy, we define a representative set of target DR types for a high proportion (≧ 80%) of the world's population, regardless of the ethnic group from which they originated. Explored that. For this purpose, seven additional DR antigens were considered. For each of the DR antigens considered in this study (including DR1, 4, and 7), the estimated frequencies in various ethnic groups according to the most recent HLA workshop (11th, 1991) were identified previously. The various subtypes are shown in Table IVa.

For the purpose of measuring the binding affinity of a peptide for various DR molecules, each D
One representative subtype was selected for the R antigen (Table I). For most antigens, whether one subtype is much more abundant or whether there appears to be a significant degree of similarity in the binding patterns presented by the other most abundant subtype of each DR antigen It should be noted that either is the case (see the note column in Table IVb). One exception to this general trend is represented by the DR4 antigen, for which significant differences in peptide specificity between 0401 and 0405 have been reported. Since both alleles are very frequent (in Caucasians and Orientals, respectively), we have identified a representative D
The set of R-binding assays included both DR 0401 and 0405.

Our representative set of assays mostly focuses on the allelic products of genes. Because these molecules are the most abundantly expressed, act as dominant constraining elements of most human class III responses analyzed to date, and are most readily available for serology and DNA typing. Because it seems to be an accurate method. However, we also considered representative species of DRB3 / 4/5 molecules in our analytical assays (Table IVc). These molecules can act as functional constraints elements, and their peptide binding specificity, some shown in have a certain similarity previously with respect to the specificity of the common DR beta ₁ allele products Have been.

General Strategies for Estimating DR Degenerate Binders The 1-4-7 combinatorial algorithm also describes how to estimate whether degenerate binding to other common DR types is estimated. We have found that purified HL
The ability of three different groups of synthetic peptides to bind to a panel of ADR molecules was measured. The set of three different peptides was as follows: A) 36 peptides not scored positive in combination 1-4-7 algorithm (not estimated), B) 1-4-7 algorithm , Scored positive at a cut-off level of 75%, but were found not to be degenerate 1-4-7 binders during actual testing ("wrong" estimate) And C) 1-4
-7 algorithm scored positive and, in experimental testing,
29 peptides proved to be 4-7 degenerate binders (correct presumption)
. The results of this analysis are shown in Table V.

From the set of “not putative” peptides (Table Va), 3 out of 34 (
9%) bound at least two of the DR1, DR4w4, or DR7 molecules. Interestingly, two of three of these peptides (1136
. 04 and 1136.29) are also relatively cross-reactive and even more of the DR form (in the case of 1136.04, DR2w2 β2, DR4w15, DR5w1
1, and DR8w2, and for 1136.29, DR2w2 β2, D
R4w15, 9 and DR5w12). Peptides from the "misplaced" set of peptides (Table V5), as the name suggests, have at most one D
Binds to the R1, DR4w4, or DR7 molecule and is also poorly degenerate;
Or other DR forms with only two peptides (1136.22 and 1188.35) binding to all three DR molecules. In this set of peptides, none of the peptides bound more than three of the tested DR molecules (data not shown).

These results were obtained with a set of peptides corresponding to the peptides initially estimated by using the combinatorial 1,4,7 algorithm and then degenerate DR1-
In contrast to data found experimentally to be 4-7 bonds. Tested 2
Fourteen (48%) of the nine peptides bound to a total of five or more alleles. Four of these (1188.16, 1188.32, 1188.34)
, And F107.09) are significantly degenerate, and the 11 D
Bound to a total of 9 of the R molecules. Taken together, these results demonstrate that a strategy based on the combined use of a combined DR1,4,7 algorithm and a quantitative DR1,4,7 binding assay can be used to identify widely cross-reactive DR binding peptides. Suggest that it can be done.

Definition of the HLA-DR1-4-7 supertype The data presented above also suggested that some common DR types were characterized by a largely overlapping peptide binding repertoire. This problem,
32 from Tables Va and Vb, which were the actual DR1-7 degenerate binders
A more detailed analysis was performed by analyzing the binding patterns of the individual peptides. Thirty-one (97%) of them bind DR1 and 22 (69%) DR4w
4 and 21 (66%) bound to DR7. These files contrast with the low binding rate observed among those that remain non-degenerate binding peptides (17/67 for DR1, DR4w4, and DR7, respectively).
(25%), 8/67 (12%), and 7/67 (10%)) (Table VII).

Interestingly, a large fraction of the 1-4-7 degenerate binder also bound to certain other common DR forms. 16 (50%) bind to DR2w2a and 18 (
56%) bind to DR6w19, 18 (56%) bind to DR2w2b,
Twenty (62%) bound to DR9. In all cases, non-
The binding frequency of the set of 7 degenerate peptides was much lower (Table VIII).

[0108] The frequency of low but significant cross-reactions was also higher for DR4w15, DR5w11, and D
R8w2 was noted (range 28-37%). Finally, negligible levels of cross-reactivity were observed in the case of DR3 and DR5w12, and DR53. Further studies address whether any of these two groups of molecules (DR4w15, DR5w11 and DR8w2 for one and DR3, DR53 and DR5w12 for the other) may belong to different DR supertypes. I do.

To summarize, these data are DR1, DR4w4, DR2w2a, DR2
It demonstrates that a large set of DR molecules, including w2b, DR7, DR9, and DR6w19, are characterized by a largely overlapping peptide binding repertoire.

DISCUSSION In the report of the present invention, we analyzed the peptide binding specificity of a set of 13 different DR molecules, representative of a common DR type within a broad population.
Detailed maps of secondary anchors and secondary interactions were derived for three of them (DR4w4, DR1, and DR7). In addition, we have demonstrated that at least seven different sets of DR types share overlapping peptide binding repertoires; and as a result, a wide range of degenerate HLA DR binding peptides appear relatively common Prove that. The present study also describes computerized procedures that should greatly assist in the work of identifying such degenerate peptides.

We consider data in the context of our current understanding of peptide-class II interactions, and in the context of the recently described class I supermotif. Finally, the potential relationship of a wide range of degenerate class II epitopes for epitope-based vaccine design should also be considered.

First, our study analyzed the DR4 analyzed in this study (data not shown).
Most of the peptides that bind with good affinity to w4, DR1, DR7, and most of the other DR types were all reduced by the P1-P6 motif, which is consistent with the motif originally proposed by O'Sullivan et al. A method for characterizing is described. Crystallographic analysis of the DR-1 peptide complex shows that residues occupying these positions engage two complementary pockets on the DR1 molecule, with position P1 being the most important anchor residue and the deepest hydrophobicity. Clarified that it corresponds to the pocket. Our analysis also describes how other "secondary anchor" positions can dramatically affect peptide binding capacity in an allele-specific manner. 4th place is DR1
Position 9 was found to be particularly important for DR4w4 and position 7 for DR7. These data are consistent with previous results that first described such allele-specific anchors, and with crystallographic data describing how these residues engage a shallow pocket on the DR molecule. .

Second, our work is based on the alignment and calculation of average relative binding values of large peptide libraries, which allows the definition of quantitative algorithms to predict binding capacity. The method will be described. The present study extends these observations to two other common HLA-DR types, and also
7 illustrates how the combined use of seven algorithms can help identify widely degenerate DR binding peptides.

The data presented herein represent a group of common DR alleles (at least DR
1, DR2w2a, DR2w2b, DR4w4, DR6w19, DR7 and D
R9) share a largely overlapping peptide repertoire. A degenerate peptide that binds to multiple DR alleles and recognizes the same epitope in the context of multiple DR types was originally described in Lanzavechia, Sinig.
Allia and Rothbard. This research is
DR1, DR2w2a, DR2w2b, DR4w4, DR7, DR9, DR6w
Provides a classification of alleles belonging to the major HLA-DR supertype (DR1-4-7-like), including 19; Based on the data presented herein, there are at least two additional groups of alleles. The first group, despite being significant, has a 1-4-7-like supertype (DR4w15, DR8w2, DR8w2,
5w11) and encode molecules with greatly reduced overlap. The second group of alleles (DR5w12, DR3w17, and DRw53) are clearly 1-4-
Has little repertoire associated with the 7 supertype. In this context, Hammer et al. Show that a good DR5w11 binding peptide is often characterized by a positively charged, poorly compatible P6 anchor with the 1-4-7 supermotif proposed herein. It is interesting to pay attention to what you pay attention to. It is also interesting that Sidney et al. Considered that DR3w17 binds to a set of peptides that are largely distinct from those bound by other common DR types. Future studies indicate that any of the molecules listed above may have additional DR
You must decide whether you can be grouped into supertypes. Our group is currently investigating whether analysis of the polymorphic residues that line the peptide binding pocket of DR can be used to aid in the classification and prediction of HLA DR supertypes.

We comment on the similarities and differences between the HLA DR supertype described herein and the currently described HLA class I supermotif. Class I supermotifs are clear and generally non-overlapping. Four of these are described almost equally frequently in the broad population. In contrast, the repertoire defining the HLA DR supertype described herein is not clear and at least partially overlaps the repertoire of other alleles. Also, based on the data presented in Tables I and IV, DR 1-4-7 appears to be by far the most abundantly shown worldwide, even when other DR supertypes are present. It is.

Finally, we point to a possible relationship of these data with respect to the development of epitope-based vaccines. Class II restricted HTL has been implicated in prevention from many important diseases and cessation of the disease. The inclusion of a well-defined class II epitope in a prophylactic or therapeutic vaccine allows the immune response to be focused on conserved or semi-dominant epitopes and avoids determinants of suppressiveness. Based on the data presented herein (Table IV), the DR1-4-7 supertype has a 50% dependence on the ethnicity considered.
To 80%. Thus, it is possible that broad and ethnically unbiased population coverage can be achieved by considering very limited peptide binding specificities.

Based on the above results, the sequences of various antigens of interest were scanned for the presence of the DR1-4-7 motif. Peptides identified using this approach
Broadly cross-reactive, class II restricted T cell epitope. Table VIII
Shown is a list of such peptides from various antigens, including representative epitopes that bind one or more DR alleles with an IC ₅₀ of 100 nM or less. Table VI
The information in II includes the antigen from which the peptide was derived and, as indicated,
Contains binding data expressed as IC ₅₀ values for the specified DR allele.

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

[0119]

[Table 1]

[0120]

[Table 2]

[0121]

[0122]

[Table 3]

[0123]

[Table 4]

[0124]

[Table 5]

[0125]

[0126]

[Table 6]

[0127]

[Table 7]

[0128]

[Table 8]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[Table 9]

[Brief description of the drawings]

FIG. 1 shows the effect of positive or negative effects on the DR4w4 binding ability of each of the 20 naturally occurring amino acids when occupying a particular site compared to the primary P1-P6 anchor. Show the map.

FIG. 2A shows the effect of each positive or negative effect on the DR1 binding ability of each of the 20 naturally occurring amino acids when occupying a particular site compared to the primary P1-P6 anchor. Show the map.

FIG. 2B shows the positive or negative effect of each of the 20 naturally occurring amino acids on DR7 binding ability when occupying a particular site compared to the primary P1-P6 anchor. Show the map.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) A61P 1/16 A61P 1/16 13/12 13/12 21/04 21/04 25/00 25/00 29 / 00 101 29/00 101 31/12 31/12 35/00 35/00 37/06 37/06 37/08 37/08 C07K 7/08 C07K 7/08 G01N 33/53 G01N 33/53 D 33 / 574 33/574 A // C12N 15/09 C12N 15/00 A (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, A , BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Sydney, John USA 92130, San Diego, Mistral Place 4395 F-term (reference) 4B024 AA01 AA11 BA31 BA33 BA35 BA36 BA80 HA17 4C085 AA05 BA32 BA69 BA89 BA 92 BB01 CC32 DD08 DD24 DD33 DD63 EE01 GG01 4H045 AA30 BA10 BA71 CA02 CA05 CA20 CA40 DA86 EA22 EA28 EA29 EA51 EA52 EA53

Claims (13)

[Claims] 1. A pharmaceutical composition comprising a unit dosage form of a peptide comprising an epitope according to Table VIII or an analog thereof, which binds to an HLA class II molecule with an IC ₅₀ of less than 1,000 nM. 2. The method according to claim 1, wherein the peptide is carcinoembryonic antigen (CEA), p53, MAGE.
2. The composition of claim 1, wherein the composition is derived from a tumor antigen selected from the group consisting of -2, MAGE-3, or Her2 / neu. 3. The method of claim 1, wherein said immunogenic peptide is derived from a viral antigen.
A composition according to claim 1. 4. The composition of claim 3, wherein said viral antigen is derived from HIV, HBV, or HCV. 5. The method according to claim 5, wherein the antigen is Plasmodium falciparum.
The composition of claim 1, wherein 6. The method according to claim 1, wherein the epitope is Y at position 1 from the N-terminus of the epitope.
Including an amino acid that is F, W, L, I, V, or M, and an amino acid that is S, T, C, A, P, V, I, L, or M at position 6 from the N-terminus of the epitope ,
The composition according to claim 1. 7. The composition of claim 1, wherein said composition is a nucleic acid encoding said peptide. 8. A method for inducing a helper T cell response in a patient, comprising:
A method comprising contacting a helper T cell with the composition of claim 1. 9. The method of claim 8, wherein said composition is a nucleic acid encoding said peptide. 10. A composition comprising an epitope according to Table VIII or an analogue thereof, wherein said epitope binds to an HLA class II molecule with an IC ₅₀ of 1,000 nM or less, wherein said epitope is present on antigen presenting cells. A composition conjugated to a Class II molecule. 11. A composition comprising at least two peptides according to claim 1. 12. A composition comprising at least three peptides according to claim 1. 13. A unit dosage form of the peptide comprising 500 μg and 50,000
The composition of claim 1, wherein the composition is in a range between 0 μg.