HK1190757A

HK1190757A - Methods and compositions for inducing an immune response to egfrviii

Info

Publication number: HK1190757A
Application number: HK14103950.6A
Authority: HK
Inventors: P．M．劳尔; K．巴哈贾特
Original assignee: 艾杜罗生物科技公司; 普罗维登斯医疗与服务俄勒冈州D/B/A普罗维登斯波特兰医疗中心
Priority date: 2010-11-17
Filing date: 2011-11-17
Publication date: 2014-07-11

Abstract

The present invention relates to methods of inducing a T-cell response against a EGFRvIII in a subject. These method comprise administering to a subject a composition which expresses at least one immunogenic polypeptide, the amino acid sequence of which comprise a plurality of EGFRvIII polypeptide sequences, the sequence of which each comprise EEKKGNYV (SEQ ID NO: 3), and/or administering the polypeptide itself.

Description

Methods and compositions for inducing immune responses against EGFRvIII

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention claims priority from U.S. provisional patent application No. 61/414,850 filed on 17.11.2010, the entire contents of which, including all tables, figures and claims, are hereby incorporated by reference.

Background

The following discussion of the background to the invention is provided merely to aid the reader in understanding the invention and is not an admission that it is prior art to describe or constitute the present invention.

The EGFR3 gene (c-erbB-1) is amplified and overexpressed in malignant human tissues. This amplification is often associated with a rearrangement of the gene structure, resulting in the absence of in-frame deletion mutants in the intracellular and transmembrane domains of wild-type c-erb-1. One class of deletion mutants identified in some glioblastomas and non-small cell lung cancers is known as EGFRvIII. EGFRvIII is a mutation wherein amino acids 6-273 (in the extracellular domain, residue 1 isResidue immediately after the signal sequence) was deleted and glycine was inserted between residue 5 and residue 274. The sequence of 10 residues at the N-terminal of the EGFRvIII mutation is LEEKKGNYVV(SEQ ID NO:1)。

Patients with EGFRvIII expressing breast cancer have detectable humoral and cellular immune responses against this peptide, revealing it to act as an immunogenic neo-antigen. A13 amino acid peptide called PEPVIII from this adapter (LEEKKGNYVVTDH; SEQ ID NO:2) has been used for immunization of humans with EGFRvIII expressing tumors. In a recently published study, PEPvIII conjugated to KLH was administered to newly diagnosed glioblastoma multiforme (GBM) patients treated with total resection (>95%), radiation and temozolomide (temozolomide) without radiographic evidence of progression. Humoral immune responses to EGFRvIII were observed in 6 of 14 immunized patients, while 3 of 17 showed positive DTH responses. The median overall survival for patients treated with vaccine and temozolomide was 26.0 months from the time of pathological diagnosis, compared to only 15.0 months for the matching cohort receiving only temozolomide. These encouraging results support the utility of EGFRvIII-expressing vaccines, and reveal that more potent vaccines, i.e., vaccines that elicit robust, durable, and potent antigen-specific T cell responses, can improve the magnitude and duration of anti-EGFRvIII responses.

Listeria monocytogenes (Listeria monocytogenes) are gram-positive intracellular bacteria that have been explored for their use as vaccine vectors. Infection with listeria monocytogenes induces a potent CD8+ T cell response necessary to kill cells infected with listeria monocytogenes and to control infection. The attenuation of Listeria monocytogenes increases the safety of the vector by 100-fold and 1,000-fold while maintaining or enhancing its immunogenicity. The ease with which the vector can be genetically manipulated, and the simple method of production, make listeria monocytogenes an attractive platform for cancer vaccines.

There remains a need in the art for compositions and methods that stimulate an effective immune response against EGFRvIII-expressing malignancies.

Summary of The Invention

The present invention provides compositions and methods for delivering a poly-EGFRvIII antigen vaccine using bacteria that recombinantly encode and express poly-EGFRvIII antigens.

In a first aspect of the invention, the invention relates to a method of inducing a T cell response against EGFRvIII in a subject. These methods comprise administering to the subject a composition comprising a bacterium that expresses one or more immunity polypeptides whose amino acid sequences comprise a plurality (2, 3, 4, 5, or more copies) of EGFRvIII polypeptide sequences, each of which (2, 3, 4, 5, or more copies) comprises EEKKGNYV (SEQ ID NO: 3). In certain embodiments, these EGFRvIII derived sequences may comprise or consist of LEEKKGNYV (SEQ ID NO:4), LEEKKGNYVVTDH (SEQ ID NO:2), or PASRALEEKKGNYVVTDHGSC (SEQ ID NO: 5). As described below, listeria monocytogenes bacteria that express one (more) of the immunogenic polypeptides described herein are most preferred.

As also described herein, such methods can stimulate an immune response in the subject against the recombinantly expressed EGFRvIII polypeptide, including one or more of a humoral response and an antigen-specific T cell (CD 4+ and/or CD8 +) response. The ability of such polypeptides to generate CD4+ and/or CD8+ T cell responses can be demonstrated by a variety of methods described in detail herein and well known in the art. Preferably, when delivered to a subject, the compositions of the invention induce one or more proteins selected from the group consisting of IL-12p70, IFN- γ, IL-6, TNF α and MCP-1, preferably with the serum concentration of each increasing 24 hours after said delivery; and induces CD4+ and/or CD8+ antigen-specific T cell responses against EGFRvIII.

In a related aspect of the invention, the invention relates to compositions useful for inducing a T-cell response against EGFRvIII in a subject. Such compositions comprise bacteria comprising a nucleic acid molecule whose sequence encodes one or more immunogenic polypeptides whose amino acid sequence comprises a plurality (2, 3, 4, 5 or more copies) of an EGFRvIII polypeptide sequence, each of which comprises EEKKGNYV (SEQ ID NO: 3). In certain embodiments, these EGFRvIII derived sequences may comprise or consist of LEEKKGNYV (SEQ ID NO:4), LEEKKGNYVVTDH (SEQ ID NO:2), or PASRALEEKKGNYVVTDHGSC (SEQ ID NO: 5). As described below, listeria monocytogenes bacteria that express one (more) of the immunogenic polypeptides described herein are most preferred. In certain embodiments, the nucleic acid sequence encoding the EGFRvIII-derived sequence is codon optimized for expression in the desired bacterium.

In another related aspect, the invention relates to an isolated nucleic acid molecule whose sequence encodes one or more immunogenic polypeptides whose amino acid sequence comprises a plurality (2, 3, 4, 5 or more copies) of an EGFRvIII polypeptide sequence, each of which comprises EEKKGNYV (SEQ ID NO:3), or its corresponding polypeptide itself. In certain embodiments, these EGFRvIII derived sequences may comprise or consist of LEEKKGNYV (SEQ ID NO:4), LEEKKGNYVVTDH (SEQ ID NO:2), or PASRALEEKKGNYVVTDHGSC (SEQ ID NO: 5).

Methods for obtaining suitable immunogenic polypeptide sequences are described in detail below. In certain embodiments, at least two of the EGFRvIII polypeptide sequences are separated by a polypeptide linker that is configured to be processed by a protease present in the subject. For example, one EGFRvIII polypeptide sequence may be separated from an adjacent EGFR polypeptide sequence by a sequence configured to be cleaved by a proteasome. In certain embodiments, each EGFRvIII polypeptide sequence is flanked by (and thus spaced from) an adjacent EGFR polypeptide sequence(s) by a sequence configured to be cleaved by the proteasome. Suitable polypeptide sequences and nucleic acid sequences encoding them are described in detail below.

In certain embodiments, the immunogenic polypeptide(s) comprise one or more "cleavable" amino acid sequences selected from the group consisting of: ASKVL ↓: ADGSVK, ASKVA ↓: GDGSIK, LSKVL ↓: ADGSVK, LAKSL ↓: ADLAVK, ASVVA ↓: GIGSIA, GVEKI ↓: NAANKG, and DGSKKA ↓: GDGNKK (SEQID NOS: 6-12). In these sequences, the antigenic sequence is located at the position indicated by the arrow, such that the "cleavable" amino acid sequence flanks the antigenic sequence. These sequences may be combined such that any sequence to the left of the arrow may be combined with any sequence to the right of the arrow to create a new side pair.

Strings of such sequences may also be created, e.g.ASKVL↓ADGSVKASKVA↓GDGSIKLSKVL↓ADGSVKASKVA↓GDGSIKLSKVL↓ADGSVK(SEQ ID NO:13), likewise, in which the antigen sequence is located at the position indicated by the arrow. To improve clarity, this list shows the first underlined flanking sequence, the second not underlined flanking sequence, the third underlined flanking sequence, the fourth not underlined flanking sequence, and the fifth underlined flanking sequence. Another example isASKVL↓ADGSVKDGSKKA↓GDGNKKLSKVL↓ADGSVKDGSKKA↓GDGNKKLSKVL↓ADGSVKDGSKKA ↓ GDGNKK (SEQ ID NO: 14). These sequences are merely exemplary in nature. Suitable proteasome cleavage motifs are described in detail in Toes et al, j.exp.med.194:1-12,2001, which is incorporated herein by reference in its entirety. See also Lauer et al, feed. Immun.76:3742-53, 2008; and Sinnathamby et al, (J.Immunother.32: 856-69, 2009.

Many bacterial species have been developed for use as vaccines and may be used as vaccine platforms in the present invention, including, but not limited to, Shigella flexneri (Shigella flexneri), Escherichia coli (Escherichia coli), Listeria monocytogenes, Yersinia enterocolitica (Yersinia enterocolitica), Salmonella typhimurium (Salmonella typhimurium), Salmonella typhi (Salmonella typhi), or Mycobacterium species. This list is not intended to be limiting. The present invention contemplates the use of attenuated, commensal, and/or killed but metabolically active bacterial strains as vaccine platforms. In a preferred embodiment, the bacterium is a listeria monocytogenes comprising a nucleic acid sequence encoding for expression by the bacterium of an EGFRvIII polypeptide sequence of the invention. Such nucleic acids are most preferably integrated into the genome of the bacterium. An attenuated and killed but metabolically active form of listeria monocytogenes is particularly preferred, and listeria monocytogenes containing attenuating mutations in actA and/or inlB are described below in preferred embodiments. Although the invention is described herein with respect to bacterial vectors, suitable agents for delivering the target antigen include additional recombinant vectors, e.g., viruses, and naked DNA.

The vaccine compositions described herein can be administered to a host, alone or in combination with a pharmaceutically acceptable excipient, in an amount sufficient to induce a suitable immune response to prevent or treat a malignancy associated with EGFRvIII expression. Selecting preferred conditions for inducing a T cell response in a subject comprises administering the vaccine platform intravenously to the subject; however, administration can be oral administration, intravenous administration, subcutaneous administration, dermal administration, intradermal administration, intramuscular administration, mucosal administration, parenteral administration, intraorgan administration, intralesional administration, intranasal administration, inhalation administration, intraocular administration, intravascular administration, intranodal administration, administration by scarification, rectal administration, intraperitoneal administration, or any one or combination of a variety of well-known routes of administration.

In certain preferred embodiments, the second vaccine is administered after an effective dose of the vaccine containing the immunogenic polypeptide is administered to the subject to elicit an immune response. This is known in the art as a "prime-boost" regimen. In such embodiments, the compositions and methods of the present invention may be used as a "prime" delivery, as a "boost" delivery, or as both a "prime" and a "boost" delivery. Examples of such schemes are described below.

Preferred listeria monocytogenes for use in the present invention comprise a mutation in the prfA gene that locks the expressed prfA transcription factor into a constitutively active state. For example, PrfA-mutant (G155S) has been demonstrated to enhance functional cellular immunity after prime-boost intravenous or intramuscular immunization regimens.

In certain embodiments, the EGFRvIII polypeptide sequences of the invention are expressed as fusion proteins comprising a box endocrine signal sequence, resulting in their secretion by bacteria as soluble polypeptide(s). A number of exemplary signal sequences are known in the art for use in bacterial expression systems. In the case where the bacterium is listeria monocytogenes, the preferred secretion signal sequence is the listeria monocytogenes signal sequence, most preferably the ActA signal sequence. Additional ActA or other linker amino acids may also be expressed fused to the immunogenic polypeptide(s). In a preferred embodiment, the one or more immunogenic polypeptides are expressed as a fusion protein(s) comprising an in-frame ActA-N100 sequence (e.g., selected from the group consisting of SEQ ID NOS: 37, 38, and 39) or an amino acid sequence having at least 90% sequence identity to the ActA-N100 sequence.

In a preferred embodiment, the vaccine composition comprises listeria monocytogenes expressing a fusion protein comprising:

(a) an ActA-N100 sequence selected from the group consisting of SEQ ID NOs 37, 38 and 39, or an amino acid sequence having at least 90% sequence identity to such ActA-N100 sequence;

(b) an amino acid sequence comprising a plurality (2, 3, 4, 5 or more copies) of EGFRvIII polypeptide sequences, each of the plurality (2, 3, 4, 5 or more copies) of EGFRvIII polypeptide sequences comprising EEKKGNYV (SEQ ID NO: 3); and

(c) a linker amino acid sequence located between at least two EGFRvIII polypeptide sequences, wherein the linker amino acid sequence is configured for proteasomal cleavage,

wherein the fusion protein is expressed from a nucleic acid sequence operably linked to a Listeria monocytogenes ActA promoter.

In another aspect, the invention relates to methods of evaluating an EGFRvIII immune response in a mouse model system. These methods include immunizing mice with an EGFRvIII polypeptide and assessing the EGFRvIII-specific immune response generated thereby by determining reactivity to the sequence, a polypeptide consisting of EEKKGNYV.

As noted above, in certain embodiments, the nucleic acid sequence encoding the antigenic polypeptide(s) is codon optimized for expression by a bacterium (e.g., listeria monocytogenes). As described below, different organisms often exhibit "codon bias", that is, the extent to which a given codon encoding a particular amino acid appears in the genetic code varies significantly between organisms. In general, the more rare codons a gene contains, the less likely it is that a heterologous protein will be expressed at a reasonable level in that particular host system. These levels become even lower if rare codons are present in the cluster or in the N-terminal part of the protein. Replacement of rare codons with other codons that more closely reflect the codon bias of the host system without modification of the amino acid sequence may increase the level of functional protein expression. Methods of codon optimization are described below.

It is to be understood that the invention is not limited in its application to the details of construction and the arrangements of components set forth in the following description or illustrated in the drawings. The invention is capable of embodiments in addition to those described and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the abstract, are for the purpose of description and should not be regarded as limiting.

As such, those skilled in the art will appreciate that the conception upon which this disclosure is based may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.

Brief Description of Drawings

FIG. 1 schematically shows an exemplary expression cassette for use in the present invention.

Fig. 2 shows western blot results demonstrating that recombinant listeria expresses the EGFRvIII antigen.

Figure 3 shows EGFRvIII antigen-specific CD8+ T cells induced by immunization with recombinant listeria.

Figure 4 shows the results obtained from the B3Z T cell activation assay after immunization with recombinant listeria.

Figure 5 shows the results obtained from screening CD8+ T cells for reactivity against specific EGFRvIII peptides following immunization with recombinant listeria.

Figure 6 shows results obtained from a T2 cell assay measuring the induction of class I expression upon EGFRvIII peptide binding.

Figure 7 shows enhanced CD8+ T cell priming following immunization with recombinant listeria expressing multiple copies of EGFRvIII20-40, relative to single copy variants.

Detailed Description

The present invention relates to compositions and methods for delivering immunotherapy using bacteria that encode and express multiple copies of an EGFRvIII-derived antigen.

1. Definition of

Abbreviations used to represent mutations in genes or in bacteria containing genes are shown below. For example, the abbreviation "listeria monocytogenes Δ actA" refers to a partial or complete deletion of the actA gene. The Delta symbol (Δ) refers to the absence. Abbreviations including superscript minus sign (Listeria ActA)^-) Refers to actA gene mutation, such as by way of deletion, point mutation, or frameshift mutation, but is not limited to these types of mutations.

In application to a human, mammal, mammalian subject, animal, veterinary subject, placebo subject, research subject, test subject, cell, tissue, organ, or biological fluid, "administering" refers, without limitation, to contacting an exogenous ligand, agent, placebo, small molecule, pharmaceutical agent, therapeutic agent, diagnostic agent, or composition with the subject, cell, tissue, organ, or biological fluid, and the like. "administration" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, placebo, and test methods. The processing of the cells includes contacting the reagent with the cells, and contacting the reagent with a fluid, wherein the fluid contacts the cells. "administering" also includes in vitro and ex vivo treatment of, for example, a cell with a reagent, a diagnostic, a binding composition, or with another cell.

Where ligands and receptors are involved, an "agonist" comprises a molecule, combination of molecules, complex or combination of agents that stimulates the receptor. For example, an agonist of granulocyte-macrophage colony stimulating factor (GM-CSF) can include GM-CSF, a mutein or derivative of GM-CSF, a peptidomimetic of GM-CSF, a small molecule that mimics the biological function of GM-CSF, or an antibody that stimulates the GM-CSF receptor.

"antagonist" when referring to a ligand and a receptor includes a molecule, combination of molecules, or complex that inhibits, counteracts, downregulates, and/or desensitizes the receptor. An "antagonist" includes any agent that inhibits the constitutive activity of a receptor. Constitutive activity is activity that manifests itself in the absence of ligand/receptor interactions. "antagonist" also includes any agent that inhibits or prevents the stimulated (or modulated) activity of a receptor. For example, antagonists of the GM-CSF receptor include, but are not limited to: an antibody that binds to a ligand (GM-CSF) and prevents it from binding to a receptor, or an antibody that binds to a receptor and prevents a ligand from binding to a receptor, or wherein the antibody locks to the receptor in an inactive conformation.

As used herein, an "analog" or "derivative" with respect to a peptide, polypeptide, or protein refers to another peptide, polypeptide, or protein having a function similar or identical to that of the original peptide, polypeptide, or protein, but which does not necessarily comprise an amino acid sequence or structure similar or identical to that of the original peptide, polypeptide, or protein. The analogue preferably satisfies at least one of the following: (a) a protein agent having an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the original amino acid sequence; (b) a proteinaceous agent encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding the initial amino acid sequence; and (c) a proteinaceous agent encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence encoding the initial amino acid sequence.

An "antigen presenting cell" (APC) is a cell of the immune system that is used to present antigen to T cells. APC includes dendritic cells, monocytes, macrophages, limbusThe regions Kupffer cells, microglia, Langerhans cells, T cells, and B cells. Dendritic cells are produced in at least two lineages. The first lineage included DC1 precursor, bone marrow DC1 and mature DC 1. The second pedigree comprises CD34⁺CD45RA^-Early progenitor pluripotent cells, CD34⁺CD45RA⁺Cell, CD34⁺CD45RA⁺CD4⁺IL-3Rα⁺Pro-DC 2 cells, CD4⁺CD11c^-Plasmacytoid DC2 precursor cells, lymphohuman DC2 plasmacytoid DC2, and mature DC2.

"attenuated" and "attenuated" include bacteria, viruses, parasites, infectious organisms, prions, tumor cells, genes in infectious organisms, and the like, modified to reduce toxicity to the host. The host may be a human or animal host, or an organ, tissue or cell. To give non-limiting examples, bacteria may be attenuated to reduce binding to host cells, to reduce spread from one host cell to another, to reduce extracellular growth, or to reduce intracellular growth in a host cell. Attenuation can be measured, for example, by measuring one or more toxicity indicators, LD₅₀Clearance from organs, or competition index (see, e.g., Auerbuch, et al (2001) feed. Immunity69: 5953-. Generally, attenuation results in LD₅₀Increase and/or increase in clearance of at least 25%; more generally, an increase of at least 50%; most typically, at least 100% (2-fold); at least a 5-fold increase in normal; more normal increases by at least 10-fold; most normally at least 50-fold; often at least 100-fold; more often at least 500-fold; and most often at least 1000-fold; typically at least a 5000-fold increase; more typically at least 10,000-fold; and most typically at least 50,000-fold increase; and most often at least 100,000-fold.

An "attenuated gene" includes a gene that mediates virulence, pathology, or virulence in a host, grows in a host, or survives a host, wherein the gene is mutated in a manner that reduces, or eliminates virulence, pathology, or virulence. Reduction or elimination can be assessed by comparing virulence or toxicity mediated by the mutated gene to virulence or toxicity mediated by the non-mutated (or maternal) gene. "mutant genes" include deletions, point mutations, and frame-shift mutations in the regulatory regions of a gene, the coding region of a gene, the non-coding region of a gene, or any combination thereof.

"conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refers to nucleic acids that encode the same amino acid sequence, or an amino acid sequence having one or more conservative substitutions. An example of a conservative substitution is the replacement of an amino acid in one of the following groups with another amino acid in the same group (U.S. Pat. No. 5,767,063 to Lee, et al, Kyte and Doolittle (1982) J.mol.biol.157: 105-.

(1) Hydrophobicity: norleucine, Ile, Val, Leu, Phe, Cys, Met;

(2) moderate hydrophilicity: cys, Ser, Thr;

(3) acidity: asp and Glu;

(4) alkalinity: asn, Gln, His, Lys, Arg;

(5) residues that influence chain orientation: gly, Pro;

(6) aromatic: trp, Tyr, Phe; and

(7) small amino acids: gly, Ala, Ser.

An "effective amount" includes, but is not limited to, an amount that ameliorates, reverses, alleviates, prevents, or diagnoses symptoms or signs of a medical condition or disorder. Unless otherwise stated explicitly or by context, "effective amount" is not limited to the minimum amount sufficient to ameliorate the condition.

"extracellular fluid" includes, for example, serum, plasma, blood, interstitial fluid, cerebrospinal fluid, secreted fluids, lymph, bile, sweat, fecal matter, and urine. "extracellular fluid" may include colloids or suspensions, such as whole blood or coagulated blood.

The term "fragment" in the context of a polypeptide includes a peptide or polypeptide comprising the amino acid sequence: at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of the amino acid sequence of the larger polypeptide.

"Gene" refers to a nucleic acid sequence encoding an oligopeptide or polypeptide. The oligopeptide or polypeptide can be bioactive, antigenically active, non-bioactive, or non-antigenically active. The term gene includes, for example, the sum of Open Reading Frames (ORFs) encoding a specific oligopeptide or polypeptide; the sum of the ORF plus the nucleic acid encoding the intron; the sum of the ORF and the operably linked promoter(s); the sum of the ORF and the operably linked promoter(s) and any intron; the sum of the ORF and the operably linked promoter(s), intron(s) and promoter(s) and other regulatory elements such as enhancers. In certain embodiments, "gene" includes any sequence required for cis-regulation of gene expression. The term gene may also refer to nucleic acids encoding peptides including peptides, oligopeptides, polypeptides or antigens or antigenically active fragments of proteins. The term gene does not necessarily imply that the encoded peptide or protein has any biological activity, or even that the peptide or protein has antigenic activity. Nucleic acid sequences encoding non-expressible sequences are generally considered pseudogenes. The term gene also includes nucleic acid sequences encoding ribonucleic acids, such as rRNA, tRNA, or ribozymes.

"growth" of bacteria, such as listeria, includes, but is not limited to, bacterial physiology and gene function involving colonization, replication, increased protein content, and/or increased lipid content. Unless otherwise stated explicitly or by context, listeria growth includes growth of bacteria outside of a host cell, and also includes growth of bacteria inside of a host cell. Growth-related genes include, but are not limited to, genes that mediate energy production (e.g., glycolysis, tricarboxylic acid cycle, cytochrome), anabolism and/or catabolism of amino acids, sugars, lipids, minerals, purines, and pyrimidines, nutrient transport, transcription, translation, and/or replication. In some embodiments, "growth" of a listeria bacterium refers to intracellular growth of the listeria bacterium, i.e., growth within a host cell, e.g., a mammalian cell. Although the intracellular growth of listeria bacteria can be measured by light microscopy or Colony Forming Unit (CFU) assays, growth is not limited by any measurement technique. Biochemical parameters such as listeria antigens, listeria nucleic acid sequences, or the amount of listeria bacteria-specific lipids can be used to assess growth. In some embodiments, the gene that mediates growth is a gene that specifically mediates intracellular growth. In some embodiments, genes that specifically mediate intracellular growth include, but are not limited to, genes in which gene inactivation reduces the rate of intracellular growth but does not detectably, substantially, or appreciably reduce the rate of extracellular growth (e.g., growth in broth), or genes in which gene inactivation reduces the rate of intracellular growth to a greater extent than it reduces the rate of extracellular growth. To provide a non-limiting example, in some embodiments, wherein inactivating the gene that reduces the rate of intracellular growth to a greater extent than it reduces extracellular growth includes situations where inactivation reduces intracellular growth to less than 50% of normal or maximum, but only reduces extracellular growth to 1-5%, 5-10%, or 10-15% of maximum. In certain aspects, the invention includes listeria that have reduced intracellular growth but not reduced extracellular growth, listeria that have no reduced intracellular growth and not reduced extracellular growth, and listeria that have no reduced intracellular growth but not reduced extracellular growth.

"hydrophilic assay" refers to the analysis of polypeptide sequences by the Method of Kyte and Doolittle, "A Simple Method for displaying the Hydropathic Character of a Protein", J.mol.biol.157(1982) 105-132. In this method, each amino acid is given a hydrophobicity fraction between 4.6 and-4.6. The score of 4.6 is most hydrophobic, while the score of-4.6 is most hydrophilic. The window size is then set. The window size is the number of amino acids whose hydrophobicity scores are to be averaged and assigned to the first amino acid in the window. The calculation starts with the first window of amino acids and calculates the average of all hydrophobic scores in this window. The window is then shifted down one amino acid and the average of all hydrophobicity scores in the second window is calculated. This pattern continues to the protein end, calculating the average score for each window and assigning it to the first amino acid in the window. The average values are then plotted. The y-axis represents the hydrophobicity fraction and the x-axis represents the window number. The following hydrophobicity fractions were used for the 20 common amino acids.

A "labeled" composition can be detected directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical, isotopic or chemical means. For example, useful markers include³²P、³³P、³⁵S、¹⁴C、³H、¹²⁵I. Stable isotopes, epitope tags, fluorescent dyes, electron-dense reagents, substrates, or enzymes (e.g., enzymes as used in enzyme-linked immunoassays), or fluoroetes (see, e.g., Rozinov and Nolan (1998) chem. biol.5: 713-728).

"ligand" refers to a small molecule, peptide, polypeptide, or membrane associated or membrane bound molecule that is an agonist or antagonist of a receptor. "ligand" also includes binding agents that are not agonists or antagonists and do not have agonist or antagonist properties. Conventionally, if a ligand membrane binds to a first cell, the receptor is typically produced on a second cell. The second cell may have the same characteristics (same name) as the first cell, or it may have different characteristics (different name) from the first cell. The ligand or receptor may be entirely intracellular, i.e., it may be present in the cytosol, nucleus or in some other intracellular compartment. The ligand or receptor may change its position, for example from the intracellular compartment to the outside of the plasma membrane. Complexes of ligands and receptors are referred to as "ligand receptor complexes". If the ligand and receptor are involved in a signaling pathway, the ligand is produced at a position upstream of the signaling pathway and the receptor is produced at a position downstream of the signaling pathway.

"nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-stranded, double-stranded, or multi-stranded form. Non-limiting examples of nucleic acids are e.g. cDNA, mRNA, oligonucleotides and polynucleotides. A particular nucleic acid sequence may also implicitly include "allelic variants" and "splice variants".

"operably linked" in the context of a promoter encoding an mRNA and a nucleic acid means that the promoter can be used to initiate transcription of the nucleic acid.

The terms "percent sequence identity" and "% sequence identity" refer to the percentage of sequence similarity found by comparing or aligning two or more amino acid or nucleic acid sequences. Percent identity can be determined as follows: sequence information between two molecules is directly compared by aligning the sequences, counting the number of exact matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. The algorithm used to calculate sequence identity is the Smith-Waterman homology search algorithm (see, e.g., Kann and Goldstein (2002) Proteins48: 367-.

"purified" and "isolated" when referring to a polypeptide means that the polypeptide is present in the substantial absence of other biological macromolecules with which it is associated in nature. The term "purified" as used herein means that the identified polypeptide often constitutes at least 50%, more often at least 60%, typically at least 70%, more typically at least 75%, most typically at least 80%, usually at least 85%, more typically at least 90%, most typically at least 95%, and conventionally at least 98% or more by weight of the polypeptide of the invention. Water, buffers, salts, detergents, reducing agents, protease inhibitors, stabilizers (including added proteins such as albumin), and excipients, and the weight of molecules with molecular weights less than 1000 are generally not used in the determination of polypeptide purity. See, for example, the discussion of purity in U.S. patent No. 6,090,611 to Covacci et al.

"peptide" refers to a short sequence of amino acids, wherein the amino acids are linked to each other by peptide bonds. The peptide may be present in free form or bound to another moiety, such as a macromolecule, lipid, oligosaccharide or polysaccharide, and/or polypeptide. The term "peptide" may still be used to refer specifically to short sequences of amino acids when the peptide is incorporated into a polypeptide chain. The "peptide" may be linked to the other moiety via a peptide bond or some other type of linkage. Peptides are at least two amino acids in length, and generally are less than about 25 amino acids in length, with the maximum length varying with convention or context. The terms "peptide" and "oligopeptide" are used interchangeably.

"protein" generally refers to a sequence of amino acids comprising a polypeptide chain. Protein may also refer to the three-dimensional structure of a polypeptide. "denatured protein" refers to a partially denatured polypeptide having some residual three-dimensional structure, or alternatively, essentially any three-dimensional structure, i.e., all denaturation. The invention includes reagents for and methods of using polypeptide variants, e.g., involving glycosylation, phosphorylation, sulfation, disulfide bond formation, deamidation, isomerization, cleavage points in signal or leader sequence processing, covalently and non-covalently bonded cofactors, oxidized variants, and the like. The formation of disulfide-linked proteins has been described (see, for example, Wycechowsky and Raines (2000) curr. Opin. chem. biol.4: 533-539; Creighton, et al (1995) trends Biotechnol.13: 18-23).

"recombinant" when used with respect to, for example, a nucleic acid, cell, animal, virus, plasmid, vector, or the like, means modified by the introduction of an exogenous non-natural nucleic acid, alteration of a natural nucleic acid, or by being derived in whole or in part from a recombinant nucleic acid, cell, virus, plasmid, or vector. Recombinant protein refers to a protein derived from, for example, recombinant nucleic acids, viruses, plasmids, vectors, and the like. "recombinant bacteria" include bacteria in which the genome is engineered recombinantly, e.g., by mutation, deletion, insertion, and/or rearrangement. "recombinant bacteria" also includes bacteria modified to include recombinant extragenomic nucleic acids, such as plasmids or second chromosomes, or bacteria in which existing extragenomic nucleic acids are altered.

By "sample" is meant a sample from a human, animal, placebo or research sample, such as a cell, tissue, organ, fluid, gas, aerosol, slurry, gel or aggregate material. A "sample" may be tested in vivo, e.g., without removal from a human or animal, or it may be tested in vitro. The sample may be tested after processing, for example by histological methods. "sample" also refers to, for example, cells comprising, or isolated from, a fluid or tissue sample. "sample" may also refer to cells, tissues, organs or fluids freshly extracted from a human or animal, or to processed or stored cells, tissues, organs or fluids.

"selectable marker" includes nucleic acids that allow for selection of or against cells containing the selectable marker. Examples of selectable markers include, but are not limited to, for example: (1) nucleic acids encoding products that provide resistance to other toxic compounds (e.g., antibiotics), or nucleic acids encoding products that are sensitive to other harmful compounds (e.g., sucrose); (2) a nucleic acid encoding a product (e.g., a tRNA gene, an auxotrophic marker) that is deficient in the recipient cell; (3) a nucleic acid encoding a product that inhibits the activity of a gene product; (4) nucleic acids encoding easily identifiable products (e.g., phenotypic markers such as β -galactosidase, Green Fluorescent Protein (GFP), cell surface proteins, epitope tags, FLAG tags); (5) nucleic acids that can be identified by hybridization techniques such as PCR or molecular beacons.

"specifically" or "selectively" binding, when referring to a ligand/receptor, nucleic acid/complementary nucleic acid, antibody/antigen, or other binding pair (e.g., cytokine versus cytokine receptor), refers to a binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other biologies. Thus, under specified conditions, a particular specified ligand binds to a particular receptor and does not bind in significant amounts to other proteins present in the sample. Specific binding may also mean, for example, that the binding compound, nucleic acid ligand, antibody or binding composition derived from the antigen-binding site of an antibody in the contemplated methods binds to its target with an affinity that is often at least 25% greater, more often at least 50% greater, most often at least 100% (2-fold) greater, at least 10-fold greater than normal, at least 20-fold greater than normal, and at least 100-fold greater than the affinity for any other binding compound.

In typical embodiments, the affinity of the antibody is greater than about 10, for example, as determined by Scatchard analysis (Munsen, et al (1980) Analyt. biochem.107:220-⁹Liter/mol. The skilled artisan will recognize that some binding compounds may specifically bind to more than one target, e.g., an antibody specifically binds to its antigen, binds to a lectin via the oligosaccharide of the antibody, and/or binds to an Fc receptor via the Fc region of the antibody.

"spreading" of bacteria includes "cell-to-cell spreading" as e.g. mediated by vesicles, i.e. transfer of bacteria from a first host cell to a second host cell. Functions involving diffusion include, but are not limited to, for example, formation of actin tails, formation of pseudopodoid-like extensions (pseudopodia-likeextensions), and formation of double-membrane vacuoles.

The term "subject" as used herein refers to a human or non-human organism. Thus, the methods and compositions described herein may be applicable to both human and veterinary disease. In certain embodiments, the subject is a "patient," i.e., a living human receiving medical care for a disease or condition. This includes persons who do not have a defined disease and who have been investigated for signs of pathology. Preferably a subject suffering from a malignancy that expresses EGFRvIII. In certain embodiments, the subject suffers from glioma (e.g., GBM), head and neck squamous cell carcinoma, large bowel cancer, or breast cancer.

The "target" of the recombinase is a Nucleic acid sequence or region for recognition, binding and/or action by the recombinase (see, e.g., U.S. Pat. No. 6,379,943 to Graham et al; Smith and Thorpe (2002) mol. Microbiol.44: 299-307; Groth and calcium (2004) J. mol. biol.335: 667-678; Nunes-Duby, et al (1998) Nucleic Acids Res.26: 391-406).

A "therapeutically effective amount" is defined as the amount of an agent or pharmaceutical composition sufficient to exhibit patient benefit, i.e., to alleviate, inhibit or ameliorate symptoms of the condition being treated. When the agent or pharmaceutical composition comprises a diagnostic agent, a "diagnostically effective amount" is defined as an amount sufficient to produce a signal, image, or other diagnostic parameter. The effective amount of the pharmaceutical agent will vary depending on a variety of factors, such as the sensitivity of the individual, the age, sex, and weight of the individual, and the specific constitutional response of the individual (see, e.g., U.S. Pat. No. 5,888,530 to Netti et al).

"Treatment" or "treating" (in terms of a condition or disease) is a means for obtaining a beneficial or desired result, including and preferably a clinical result. For purposes of the present invention, beneficial or desired results with respect to disease include, but are not limited to, one or more of the following: ameliorating a condition associated with a disease, curing a disease, slowing the severity of a disease, delaying the progression of a disease, alleviating one or more symptoms associated with a disease, improving the quality of life of a patient suffering from a disease, and/or prolonging survival. Also, for purposes of the present invention, beneficial or desired results with respect to a condition include, but are not limited to, one or more of the following: ameliorating a condition, curing a condition, slowing the severity of a condition, delaying the progression of a condition, alleviating one or more symptoms associated with a condition, increasing the quality of life of a patient having a condition, and/or prolonging survival.

"vaccine" includes prophylactic vaccines. Vaccines also include therapeutic vaccines, such as vaccines that are administered to mammals having a condition or disorder associated with the antigen or epitope provided by the vaccine.

2. Exemplary EGFRvIII antigens

The EGFRvIII antigen may comprise a sequence encoding at least one MHC class I epitope and/or at least one MHC class II epitope. The prediction algorithm "BIMAS" ranks potential HLA-binding epitopes according to the predicted half-life dissociation of the peptide/HLA complexes. The "SYFPEITHI" algorithm ranks peptides according to the fraction of primary and secondary HLA-binding anchor residues specified. Both computerized algorithms score candidate epitopes based on amino acid sequences within a given protein, which have similar binding motifs to previously published HLA binding epitopes. Other algorithms may also be used to identify candidates for further biological testing.

As indicated above, the EGFRvIII antigenic peptide of the present invention comprises multiple (2, 3, 4, 5, or multiple copies) EGFRvIII polypeptide sequences, each of which comprises EEKKGNYV (SEQ ID NO: 3). This sequence represents the H2-Kk-restricted epitope. Preferred EGFRvIII sequences comprise multiple copies of what are referred to herein as EGFRvIII_20-40Sequence PASRALEEKKGNYVVTDHGSC (SEQ ID NO: 4). This 21-AA sequence spans a new adapter created by the 267-AA deletion relative to native EGFR. This also increases the chance of finding a class I-binding epitope that includes a novel glycine at the EGFRvIII splice site (45 unique at EGFRvIII)_20-40The 7 to 11-AA novel peptides within PEPvIII) without including excessive amounts of native EGFR sequences. In addition, this expanded EGFRvIII peptide increased potential class II-binding epitopes (30 in EGFRvIII)_20-40In (1)>Number of 9-AA New peptides and 11 peptides in PEPVIII)However, vaccine titers do not necessarily depend on the presence of EGFRvIII-specific class II restriction epitopes.

As described below, the antigen dose is a critical determinant of vaccine potency. To maximize the number of EGFRvIII peptide-MHC complexes after immunization, preferably at least 5 copies of the EGFRvIII polypeptide sequence are included in a single protein construct, wherein each copy is separated by a sequence predicted to promote proteasomal cleavage. As an example, such an amino acid sequence may be ASKVLPASRALEEKKGNYVVTDHGSCADGSVKTSASKVAPASRA LEEKKGNYVVTDHGSCGDGSIKLSKVLPASRALEEKKGNYVVTD HGSCADGSVKASKVAPASRALEEKKGNYVVTDHGSCGDGSIKLSKVLPASRALEEKKGNYVVTDHGSCADGSVKTS (SEQ ID NO: 15). For clarity, in this sequence, the exemplary EGFRvIII-derived polypeptide sequence is underlined, while the exemplary "cleavable" sequence is not underlined.

The term "immunogenic" as used herein means that the antigen is capable of inducing an antigen-specific humoral or T-cell response (CD 4+ and/or CD8 +). The selection of one or more antigens or derivatives thereof for use in the vaccine compositions of the invention may be performed in a variety of ways, including assessing the ability of the selected bacteria to successfully express and secrete recombinant antigen(s); and/or the ability of the recombinant antigen(s) to elicit an antigen-specific CD4+ and/or CD8+ T cell response. As discussed below, to accomplish the final selection of antigen(s) for use with a particular bacterial delivery vehicle, it is preferred to combine these attributes of the recombinant antigen(s) with the ability of the complete vaccine platform (meaning the selected bacterial expression system for the selected antigen (s)) to elicit both an innate immune response and an antigen-specific T cell response against the recombinantly expressed antigen(s). Initial assays for suitable antigens can be performed by selecting an antigen(s) or antigen fragment(s) that is successfully recombinantly expressed by the selected bacterial host (e.g., listeria) and is immunogenic.

Direct detection of recombinant antigen expression via western blotting can be performed using antibodies that detect recombinantly produced antigen sequences, or using antibodies that detect introduced sequences ("tags") that are expressed as fusion proteins with EGFRvIII-derived antigens. For example, the antigen(s) can be expressed as a fusion with the N-terminal portion of the Listeria ActA protein, and anti-ActA antibodies raised against a synthetic peptide corresponding to the mature N-terminal 18 amino acids of ActA (ATDSEDSSLNTDEWEEEK (SEQ ID NO:27)) can be used to detect the expressed protein product.

Assays for testing the immunogenicity of antigens are described herein and are well known in the art. For example, antigens recombinantly produced by selected bacteria can optionally be constructed to contain a peptide encoding the eight amino SIINFEKL (SEQ ID NO:28) (also known as SL8 and ovalbumin)_257-264) A nucleotide sequence in frame at the carboxy terminus of the antigen. Compositions such as the C-terminal SL8 epitope act as surrogates to: (i) demonstrates that the recombinant antigen is expressed in its entirety from N-terminus to C-terminus; and (ii) demonstrating the ability of antigen presenting cells to present recombinant antigen via the MHC class I pathway using an in vitro antigen presentation assay. Such presentation assays can be performed using the cloned C57 BL/6-derived dendritic cell line DC2.4 and B3Z T cell hybridoma cell line, as described below.

Alternatively or additionally, immunogenicity may be tested using the ELISPOT assay as described below. The ELISPOT assay was originally developed for counting B-cells secreting antigen-specific antibodies, but has subsequently been modified for a variety of tasks, particularly the identification and counting of cytokine-producing cells at the single cell level. Spleens can be harvested from animals vaccinated with a suitable bacterial vaccine and the isolated spleens incubated overnight in the presence or absence of peptides derived from one or more EGFRvIII antigens expressed by the bacterial vaccine. The immobilized antibody captures any secreted IFN- γ, thus allowing subsequent measurement of secreted IFN- γ and assessment of the immune response to the vaccine.

3. Recombinant expression system, vaccine platform "

The choice of vaccine platform for antigen delivery is a key component of an effective vaccine. Recombinant vectors are prepared using standard techniques known in the art and contain suitable control elements operably linked to a nucleic acid sequence encoding a target antigen. See, e.g., Plotkin, et al (eds) (2003) Vaccines, 4 th edition, w.b. saunders, co., philia., PA.; sikora, et al, (1996) Tumor Immunology Cambridge University Press, Cambridge, UK; hackett and Harn (eds.) Vaccine Adjuvants, Humana Press, Totowa, NJ; isaacson (eds.) (1992) Recombinant DNA Vaccines, MarcelDekker, NY, NY; morse, et al, (eds.) (2004) Handbook of Cancer Vaccines, Humana Press, Totowa, NJ), Liao, et al (2005) Cancer Res.65: 9089-; dean (2005) Expert opin. drug Deliv.2: 227-; arlen, et al (2003) Expert Rev. vaccine 2: 483-493; dela Cruz, et al (2003) Vaccine21: 1317-1326; johansen, et al (2000) Eur.J.Pharm.Biopharm.50: 413-417; excler (1998) Vaccine16: 1439-1443; disis, et al (1996) J.Immunol.156: 3151-3158). Peptide vaccines have been described (see, e.g., McCabe, et al (1995) Cancer Res.55: 1741-.

Suitable viral-derived antigen delivery vectors include viruses, modified viruses, and virions. The virus-derived vector can be administered directly to a mammalian subject, or it can be introduced into Antigen Presenting Cells (APCs) ex vivo, and then the APCs are administered to the subject. Viral vectors can be based on, for example, togaviruses including alphaviruses and flaviviruses; alphaviruses, such as Sindbis virus (Sindbis virus), Sindbis strain SAAR86, Semliki Forest Virus (SFV), Venezuelan Equine Encephalitis (VEE), Eastern Equine Encephalitis (EEE), western equine encephalitis, ross river virus, aigren virus (Sagiyami virus), aniron nian virus (O' Nyong-Nyong virus), plateau J virus, flaviviruses, such as yellow fever virus, yellow fever strain 17D, japanese encephalitis virus, st louisis encephalitis (st. louisiencephalitis), Tick-borne encephalitis (Tick-borne encephatis), dengue virus (Denguevirus), West Nile virus (wenile virus), kuru (subtype of West Nile virus); arteriviruses, such as equine arteritis virus; and rubella viruses (rubivirus), such as rubella virus (rubella virus), herpes viruses, Modified Vaccinia Ankara (MVA); an avipox virus vector; a avipox carrier; vaccinia virus vectors; an influenza virus vector; adenovirus vectors, human papilloma virus vectors; bovine papilloma virus vectors, and the like. Viral vectors can be based on the genus orthopoxvirus, such as variola (smallpox), vaccinia (smallpox vaccine), Ankara (Ankara, MVA) or Copenhagen strain (Copenhagen strain), camelpox, monkeypox or vaccinia. The viral vector may be based on an avipoxvirus-like virus, such as avipoxvirus or canarypox virus.

Adenoviral vectors and adeno-associated viral vectors (AAV) can be used, wherein adenoviral vectors include adenovirus serotype 5 (adenovirus 5; Ad 5), gland 6, gland 11, and gland 35. There are at least 51 human adenovirus serotypes available, which are divided into 6 subgroups (subgroups A, B, C, D, E and F). Adenovirus proteins that can be used, for example, to assess an immune response against an "empty" adenovirus vector include hexon proteins, such as hexon 3 protein, fiber protein, and penton-like protein, and human immune responses against adenovirus proteins have been described (see, e.g., Wu, et al (2002) J.Virol.76: 12775-. The following table describes exemplary viral-derived vaccine vectors for use in the present invention:

antigen Presenting Cell (APC) vectors, such as Dendritic Cell (DC) vectors, including cells loaded with antigen, cells loaded with tumor lysate, or cells transfected with a composition comprising a nucleic acid, wherein the nucleic acid can be, for example, a plasmid, mRNA, or virus. DC/tumor fusion vaccines can also be used. See, e.g., Di Nicola, et al (2004) Clin. cancer Res.10: 5381-5390; cerundolo, et al (2004) Nature Immunol.5: 7-10; parmiani, et al (2002) J.Natl.cancer Inst.94: 805-818; kao, et al (2005) Immunol.Lett.101: 154-; geiger, et al (2005) j.trans.med.3: 29; osada, et al (2005) Cancer immunol.immunother.nov.5,1-10[ pre-press electronic publication ]; malowany, et al (2005) mol.Ther.13: 766-775; morse and Lyerly (2002) World J.Surg.26: 819. 825; gabrilovich (2002) Curr. Opin. mol. Ther.4: 454-458; morse, et al (2003) Clin. Breast cancer3suppl.4: S164-S172; morse, et al (2002) Cancer Chemother.biol.response Modif.20: 385-390; arlen, et al (2003) Expert Rev. vaccine 2: 483-493; morse and Lyerly (1998) Expert Opin. investig. drugs7: 1617-; hirschowitz, et al (2004) J.Clin.Oncol.22: 2808-2815; vasir, et al (2005) Br.J.Haematol.129: 687-700; koido, et al (2005) Gynecol.Oncol.99: 462-471.

Tumor cells, e.g., autologous and allogeneic tumor cells, can be used as vaccines (Arlen, et al (2005) Semin. Oncol.32: 549-. The vaccine may also comprise modified tumor cells, e.g., tumor cell lysates, or irradiated tumor cells. Tumor cells can also be modified by the incorporation of nucleic acids encoding molecules such as: cytokines (GM-CSF, IL-12, IL-15, etc.), NKG2D ligand, CD40L, CD80, CD86, etc. (cf., for example, Dranoff (2002) Immunol. Rev.188: 147-154; Jain, et al (2003) Ann. Surg. Oncol.10: 810-820; Borrello and Pardoll (2002) Cytokine GrowthFactor Rev.13: 185-193; Chen, et al (2005) Cancer Immunol. Immunol.27: 1-11; Kjaergaard, et al (2005) J. Neurocur. 103: 156-164; Tai, et al (2004) J. biomed. Sci.11: waab 228; Schwaab, et al (2004) J. Urol. 1042.1042: 643; Bri. Cleis.70: 35; Bri. 134: 97; Bright. Clonline;. 134: 35; Bright. 134: 97; Bright. Cloney. 134: 97; Bright. Ito. 19823; Bright. 134; Bright. 19832; Bright. 3195: 96; Bright. 134: 97; Bright. 134; Bright.

Naked DNA vectors and naked RNA vectors may also be used to provide an antigen expression platform. These nucleic acid-containing vaccines can be administered by gene gun, electroporation, bacterial membrane contrast (bacterial ghost), microspheres, microparticles, liposomes, polycationic nanoparticles, and the like (see, e.g., Donnelly, et al (1997) Ann. Rev. Immunol.15:617-648; Mincheff, et al (2001) Crit. Rev. Oncol. Hematol.39:125-132; Song, et al (2005) J. Virol.79:9854-9861; Estcourt, et al (2004) Immunol. Rev.199: 144-155). Reagents and methods for administering naked nucleic acids by, for example, gene gun, intradermal, intramuscular, and electroporation are available. The Nucleic acid Vaccine may comprise Locked Nucleic Acid (LNA), wherein the LNA enables the attachment of a functional moiety to plasmid DNA, and wherein the functional moiety may be an adjuvant (see, e.g., Fensterle, et al (1999) J. Immunol.163: 4510-4518; Strugnell, et al (1997) Immunol.cell biol.75: 364-369; Hertoughs, et al (2003) Nucleic Acids Res.31: 5817-5830; Trimble, et al (2003) Vaccine21: 4036-4042; Nishitani, et al (2000) mol. Urol.4: 47-50; Tuting (1999) curr. Opin. mol. Ther.1: 216-225). Nucleic acid vaccines can be used in combination with agents that promote migration of immature dendritic cells to the vaccine, and agents that promote migration of mature DCs to the primordial draining lymph nodes, where these agents include MIP-1 α and Flt3L (see, e.g., Kutzler and Weiner (2004) J.Clin.invest.114: 1241-.

Many bacterial species have been developed for use as vaccines and can be used in the present invention, including, but not limited to, Shigella flexneri, Escherichia coli, Listeria monocytogenes, Yersinia enterocolitica, Salmonella typhimurium, Salmonella typhi, or Mycobacterium species. This list is not intended to be limiting. See, for example, WO04/006837, WO07/103225, and WO07/117371, the entire contents of which, including all tables, figures, and claims, are hereby incorporated by reference. The bacterial vector used in the vaccine composition may be a facultative intracellular bacterial vector. Bacteria can be used to deliver the polypeptides described herein to antigen presenting cells in a host organism. As described herein, listeria monocytogenes provides a preferred vaccine platform for expression of the antigens of the present invention.

Both attenuated and commensal microorganisms have been successfully used as vectors for vaccine antigens, but bacterial vectors for such antigens are optionally attenuated or killed but have metabolic activity (KBMA). The genetic background of the vector strain used in the formulation, the type of mutation selected to achieve attenuation, and the inherent nature of the immunogen to optimize the extent and quality of the induced immune response. General factors to be considered for optimizing the immune response stimulated by the bacterial vector include: selecting a carrier; the specific background of the strain; attenuating mutations and attenuation levels; stability of the attenuated phenotype and determination of optimal dosage. Other antigen-related factors to consider include: the inherent nature of the antigen; an expression system; stability of the antigen-displayed form and the recombinant phenotype; co-expression of regulatory molecules and immunization protocols.

The vaccine platform is preferably characterized by the ability to elicit both an innate immune response as well as an antigen-specific T cell response against the recombinantly expressed antigen(s). For example, listeria monocytogenes expressing one (more) of the antigens described herein can induce the downstream cascade of type 1 interferon (IFN- α/β) and chemokines and cytokines in the liver. In response to this intrahepatic immune stimulation, NK cells and Antigen Presenting Cells (APCs) are recruited to the liver. In certain embodiments, the vaccine platform of the invention induces an increase in serum concentration of one or more (preferably all) of the cytokines and chemokines selected from the group consisting of IL-12p70, IFN- γ, IL-6, TN, and IL-12p70, preferably at 24 hours after delivery of the vaccine platform to a subjectF α, and MCP-1; and inducing a CD4+ and/or CD8+ antigen-specific T cell response against one or more EGFRvIII-derived antigens expressed by the vaccine platform. In other embodiments, the vaccine platform of the invention also induces maturation of resident immature liver NK cells as evidenced by upregulation of activation markers, e.g., DX5, CD11b, and CD43, in a mouse model system, or by using as target cells⁵¹The measured NK cell-mediated cytolytic activity of Cr-labeled YAC-1 cells.

In various embodiments, the vaccines and immunogenic compositions of the invention can comprise listeria monocytogenes configured to express one (more) desired EGFRvIII-derived antigens. The ability of Listeria monocytogenes to be used as vaccine vectors is reviewed in Wesikirch et al, Immunol.Rev.158:159-169 (1997). Many of the desirable features of the natural biological community of listeria monocytogenes make it an attractive platform for application in therapeutic vaccines. The main reason is that the intracellular life cycle of listeria monocytogenes is able to efficiently stimulate CD4+ and CD8+ T cell immunity. A variety of pathogen-associated assay mode (PAMP) receptors, including TLRs (TLR2, TLR5, TLR9) and nucleotide-binding oligomerization domains (NODs), are triggered following infection in response to interaction with listeria monocytogenes macromolecules, leading to pan-activation of innate immune effectors and release of Th-1 polarized cytokines, and profound effects on the development of CD4+ and CD8+ T cell responses to expressed antigens.

Strains of listeria monocytogenes have recently been developed as efficient intracellular delivery vehicles for heterologous proteins that deliver antigens to the immune system to induce immune responses to clinical conditions that do not allow for the injection of pathogens, such as cancer and HIV. See, for example, U.S. patent No. 6,051,237; gunn et al, J.Immunol.,167:6471-6479 (2001); liau, et al, Cancer Research,62: 2287-; U.S. patent No. 6,099,848; WO 99/25376; WO 96/14087; and U.S. patent No. 5,830,702) which includes all tables, figures and claims, are hereby incorporated by reference in their entirety. Recombinant Listeria monocytogenes vaccines expressing lymphocytic choriomeningitis virus (LCMV) antigens have also been shown to induce protective cell-mediated immunity against the antigens (Shen et al, Proc. Natl. Acad. Sci. USA,92: 3987-.

Attenuated and killed but metabolically active forms of listeria monocytogenes have been produced that can be used in immunogenic compositions. WO07/103225 and WO07/117371, which include all tables, figures and claims, are hereby incorporated by reference in their entirety. The ActA protein of listeria monocytogenes is sufficient to promote actin recruitment and aggregation events responsible for intracellular motility. Human safety studies have reported that oral administration of an actA/plcB-deleted attenuated form of listeria monocytogenes does not cause severe sequelae in adults (Angelakopoulos et al, Infection and Immunity,70:3592-3601 (2002)). Other types of attenuated forms of listeria monocytogenes have also been described (see, e.g., WO99/25376 and U.S. patent No. 6,099,848, which describe auxotrophic, attenuated strains of listeria that express heterologous antigens).

In certain embodiments, the listeria monocytogenes used in the vaccine compositions of the present invention are live attenuated strains comprising an attenuating mutation in actA and/or inlB, preferably a deletion of all or a portion of actA and inlB (referred to herein as "Lm Δ actA/Δ inlB"), and containing recombinant DNA encoding for expression of one (more) EGFRvIII-derived antigen(s) of interest. The EGFRvIII-derived antigen(s) is preferably under the control of bacterial expression sequences and is stably integrated into the listeria monocytogenes genome. Thus, such listeria monocytogenes vaccine strains do not use eukaryotic transcription or translation elements.

The invention also contemplates listeria with at least one regulatory factor, such as a promoter or transcription factor, attenuated. The following relates to promoters. ActA expression is regulated by two different promoters (Vazwuez-Boland, et al (1992) infection. Immun.60: 219-230). inlA together with inlB expression is regulated by 5 promoters (Lingnau, et al (1995) feed. Immun.63: 3896-3903). The transcription factor prfA is required for transcription of many listeria monocytogenes genes, such as hly, plcA, ActA, mpl, prfA, and iap. The regulatory properties of PrfA are mediated, for example, by PrfA-dependent promoters (PinlC) and PrfA-cassettes. In certain embodiments, the invention provides an inactivated, mutated or deleted nucleic acid encoding at least one of the ActA promoter, the inlB promoter, PrfA, PinlC, PrfA cassettes and the like (see, e.g., Lalic Mullthaler, et al (2001) mol. Microbiol.42: 111-120; Shetron-Rama, et al (2003) mol. Microbiol.48: 1537-1551; Luo, et al (2004) mol. Microbiol.52: 39-52). PrfA can be constitutively active by a Gly145Ser mutation, a Gly155Ser mutation or a Glu77Lys mutation (see, e.g., Mueller and Freitag (2005) infection. Immun.73: 1917-1926; Wong and Freitag (2004) J.Bacteriol.186: 6265-6276; Ripio, et al (1997) J.Bacteriol.179: 1533-1540).

Attenuation can be achieved by, for example, heat treatment or chemical modification. Attenuation can also be achieved by genetically modifying nucleic acids that modulate, for example, metabolism, extracellular growth, or intracellular growth, genetically modifying nucleic acids encoding virulence factors (e.g., listeria prfA, actA, Listeria Lysin (LLO)), adhesion-mediating factors (e.g., internalizing proteins such as inlA or inlB), mpl, phosphatidylcholine phospholipase C (PC-PLC), phosphatidylinositol-specific phospholipase C (PI PLC; plcA gene), any combination of the above, and the like. Attenuation can be assessed by comparing the biological function of the attenuated listeria with the corresponding biological function exhibited by the appropriate parent listeria.

In other embodiments, the invention provides listeria attenuated by treatment with nucleic acid targeting agents, such as cross-linking agents, psoralens, nitrogen mustards, cisplatin, bulky adducts, ultraviolet light, gamma radiation, combinations thereof, and the like. Typically, the damage produced by one crosslinker molecule involves the crosslinking of both strands of a double helix. The listeria of the present invention can also be attenuated by attenuating at least one nucleic acid repair gene, such as uvrA, uvrB, uvrAB, uvrC, uvrD, uvrAB, phrA, and/or a gene that mediates recombinant repair, such as a recA mutation. Furthermore, the present invention provides listeria attenuated by both a nucleic acid targeting agent and by mutating a nucleic acid repair gene. In addition, the invention includes treatment with a photosensitive nucleic acid targeting agent, such as psoralen, and/or a photosensitive nucleic acid cross-linking agent, such as psoralen, followed by exposure to ultraviolet light.

Attenuated listeria useful in the present invention are described, for example, in U.S. patent publication nos. 2004/0228877 and 2004/0197343, which are incorporated herein by reference in their entirety. Various assays for assessing whether a particular strain of listeria has a desired attenuation are provided, for example, in U.S. patent publication nos. 2004/0228877, 2004/0197343, and 2005/0249748, which are incorporated herein by reference in their entirety.

In other embodiments, the listeria monocytogenes used in the vaccine compositions of the present invention is a Killed But Metabolically Active (KBMA) platform derived from Lm Δ actA/Δ inlB, and further lacks uvrA and uvrB, i.e., the genes encoding the DNA repair enzymes of the Nucleotide Excision Repair (NER) pathway, and contains recombinant DNA encoding for the expression of one (more) EGFRvIII-derived antigen(s) of interest. The antigen(s) of interest are preferably under the control of bacterial expression sequences and are stably integrated into the listeria monocytogenes genome. The KBMA platform is extremely sensitive to photochemical inactivation by the combined treatment of synthetic psoralens, S-59 and long wavelength UV light. Despite being killed, kbmal vaccines can transiently express their gene products, allowing them to escape phagolysosomes and induce functional cellular immunity and protection against wild-type WT Lm and vaccine virus challenge.

In certain embodiments, an attenuated or KBMA listeria monocytogenes vaccine strain comprises a PrfA gene (referred to herein as a PrfA x mutant) that has constitutive activity. PrfA is an intracellular activated transcription factor that induces expression of virulence genes and encoded heterologous antigens (Ags) in suitable engineered vaccine strains. As noted above, expression of the actA gene is responsive to PrfA, and the actA promoter is a PrfA responsive regulatory element. Introduction of the prfA G155s allele can confer a significantly enhanced vaccine potency on live attenuated or KBMA vaccines. Preferred PrfA MUTANTs are described in U.S. provisional patent application 61/054,454 entitled composition synthesizing PrfA multiple LISTERIA OF USE theof filed on 19.5.2008, which includes all tables, figures and claims hereby incorporated by reference in their entirety.

The sequence of Listeria monocytogenes PrfA comprising a glycine at residue 155 is as follows (SEQ ID NO: 16):

MNAQAEEFKK YLETNGIKPK QFHKKELIFN QWDPQEYCIF LYDGITKLTS 50ISENGTIMNL QYYKGAFVIM SGFIDTETSV GYYNLEVISE QATAYVIKIN 100ELKELLSKNL THFFYVFQTL QKQVSYSLAK FNDFSINGKL GSICGQLLIL 150TYVYGKETPD GIKITLDNLT MQELGYSSGI AHSSAVSRII SKLKQEKVIV 200YKNSCFYVQN LDYLKRYAPK LDEWFYLACP ATWGKLN 237

the sequence of Listeria monocytogenes PrfA including serine at residue 155 is as follows (SEQ ID NO: 17):

MNAQAEEFKK YLETNGIKPK QFHKKELIFN QWDPQEYCIF LYDGITKLTS 50ISENGTIMNL QYYKGAFVIM SGFIDTETSV GYYNLEVISE QATAYVIKIN 100ELKELLSKNL THFFYVFQTL QKQVSYSLAK FNDFSINGKL GSICGQLLIL 150TYVYSKETPD GIKITLDNLT MQELGYSSGI AHSSAVSRII SKLKQEKVIV 200YKNSCFYVQN LDYLKRYAPK LDEWFYLACP ATWGKLN 237

4. antigenic constructs

The antigenic constructs expressed by the vaccine strains of the invention comprise at least a nucleic acid encoding a secretory sequence operable within the vaccine platform to support secretion, fused to the EGFRvIII-derived antigen(s) to be expressed. In the case of a bacterial platform, the resulting fusion protein is operably linked to regulatory sequences (e.g., promoters) required for expression of the fusion protein by the bacterial vaccine platform. The invention is not limited to secreted polypeptides and peptide antigens, but also includes polypeptides and peptides that are not secreted or secreted from listeria or other bacteria. Preferably, however, the EGFRvIII-derived antigen(s) is expressed by the bacterial vaccine strain in a soluble secreted form when the bacterial vaccine strain is inoculated into a recipient.

Table 1 discloses a number of non-limiting examples of signal peptides for fusion with fusion protein partner sequences, such as heterologous antigens. Signal peptides often contain three domains: positively charged N-termini (1-5 residues long); an intermediate hydrophobic domain (7-15 residues long); and a neutral but polar C-terminal domain.

TABLE 1 bacterial signalling pathways Signal peptides are identified by Signal peptidase sites

In certain exemplary embodiments described below, the EGFRvIII-derived sequence(s) can be expressed as a single polypeptide fused to the amino-terminal portion of a listeria monocytogenes ActA protein that allows the bacterium to express and secrete the fusion protein in an immunized host. In these embodiments, the antigenic construct may be a polynucleotide comprising a promoter operably linked to a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises (a) a modified ActA and (b) one or more EGFRvIII-derived epitopes to be expressed as the fusion protein following the modified ActA sequence.

"modified ActA" refers to a contiguous portion of a listeria monocytogenes ActA protein that comprises at least the ActA signal sequence, but does not contain the entire ActA sequence, or that has at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, or at least about 98% sequence identity to such an ActA sequence. The ActA signal sequence is MGLNRFMRAMMVVFITANCITINPDIIFA (SEQ ID NO: 27). In some embodiments, the promoter is the ActA promoter from WO07/103225 and WO07/117371, the entire contents of which are incorporated herein by reference.

For example, the modified ActA may comprise at least the first 59 amino acids of the ActA, or a sequence that is at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, or at least about 98% sequence identity to at least the first 59 amino acids of the ActA. In some embodiments, the modified ActA comprises at least the first 100 amino acids of the ActA, or a sequence that is at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, or at least about 98% sequence identity to at least the first 100 amino acids of the ActA. In other words, in some embodiments, the modified ActA sequence corresponds to an N-terminal fragment of ActA truncated at residues 100 or 100 later (including the ActA signal sequence).

ActA-N100 has the following sequence (SEQ ID NO: 28):

VGLNRFMRAM MVVFITANCI TINPDIIFAA TDSEDSSLNT DEWEEEKTEE 50QPSEVNTGPR YETAREVSSR DIEELEKSNK VKNTNKADLI AMLKAKAEKG 100

in this sequence, the first residue is depicted as valine; the polypeptide is synthesized by listeria using the methionine at this position. Thus, ActA-N100 can also have the following sequence (SEQ ID NO: 29):

MGLNRFMRAM MVVFITANCI TINPDIIFAA TDSEDSSLNT DEWEEEKTEE 50QPSEVNTGPR YETAREVSSR DIEELEKSNK VKNTNKADLI AMLKAKAEKG 100

ActA-N100 may also comprise one or more additional residues located between the C-terminal residue of the modified ActA and the EGFRvIII derived antigen sequence. In the following sequence, ActA-N100 was extended by adding two residues by introducing the BamH1 site:

VGLNRFMRAM MVVFITANCI TINPDIIFAA TDSEDSSLNT DEWEEEKTEE 50QPSEVNTGPR YETAREVSSR DIEELEKSNK VKNTNKADLI AMLKAKAEKG 100GS(SEQ ID NO：30)

when synthesized with the first residue methionine, the ActA-N100 has the sequence:

MGLNRFMRAM MVVFITANCI TINPDIIFAA TDSEDSSLNT DEWEEEKTEE 50QPSEVNTGPR YETAREVSSR DIEELEKSNK VKNTNKADLI AMLKAKAEKG 100GS(SEQ ID NO：31)。

thus, exemplary constructs are as follows:

vglnrfmram mvvfitanci tinpdiifaa tdsedsslnt deweeektee 50qpsevntgpr yetarevssr dieeleksnk vkntnkadli amlkakaekg 100gsASKVLPAS RALEEKKGNY VVTDHGSCAD GSVKTSASKV APASRALEEK 150KGNYVVTDHG SCGDGSIKLS KVLPASRALE EKKGNYVVTD HGSCADGSVK 200ASKVAPASRA LEEKKGNYVV TDHGSCGDGS IKLSKVLPAS RALEEKKGNY 250

VVTDHGSCAD GSVKTS (SEQ ID NO: 32). In this sequence, the ActA-N100 sequence is lower case, the EGFRvIII derived antigen sequence is underlined, and the linker "cleavable" sequence is not underlined. The corresponding DNA sequences are as follows: gtgggattaaatagatttatgcgtgcgatgatggtagttttcattactgccaactgcattacgattaaccccgacataatatttgcagcgacagatagcgaagattccagtctaaacacagatgaatgggaagaagaaaaaacagaagagcagccaagcgaggtaaatacgggaccaagatacgaaactgcacgtgaagtaagttcacgtgatattgaggaactagaaaaatcgaataaagtgaaaaatacgaacaaagcagacctaatagcaatgttgaaagcaaaagcagagaaaggtggatCCGCAAGCAAAGTATTGCCAGCTAGTCGTGCATTAGAGGAGAAAAAGGGGAATTACGTGGTGACGGATCATGGATCGTGTGCCGATGGCTCAGTAAAGACTAGTGCGAGCAAAGTGGCCCCTGCATCACGAGCACTTGAAGAGAAAAAAGGAAACTATGTTGTGACCGATCATGGTAGCTGCGGAGATGGTTCAATTAAATTATCAAAAGTCTTACCAGCATCTAGAGCTTTAGAGGAAAAGAAGGGTAACTATGTCGTAACAGATCATGGAAGTTGTGCTGACGGAAGTGTTAAAGCGTCGAAAGTAGCTCCAGCTTCTCGCGCATTAGAAGAAAAGAAAGGCAATTATGTTGTAACAGACCATGGTAGTTGTGGTGATGGCTCGATCAAATTGTCAAAAGTTCTACCGGCTTCTCGTGCGCTAGAAGAGAAGAAAGGAAATTACGTAGTTACAGACCACGGCTCTTGCGCGGAT

GGTTCCGTTAAAACTAGT (SEQ ID NO: 33). In this sequence, the ActA-N100 sequence is lower case, the BamHI cloning site is underlined, while the EGFRvIII derived antigen and linker "cleavable" sequences are not underlined. This sequence may be preceded by an Acta promoter sequence, e.g.

gggaagcagttggggttaactgattaacaaatgttagagaaaaattaattctccaagtgatattcttaaaataattcatgaatattttttcttatattagctaattaagaagataattaactgctaatccaatttttaacggaataaattagtgaaaatgaaggccgaattttccttgttctaaaaaggttgtattagcgtatcacgaggagggagtataa(SEQ ID NO：34)。

Exemplary constructs are described below and in WO07/103225, which is incorporated herein by reference. ANZ-100 (originally referred to as CRS-100; BB-IND 12884 and clinicalrials. gov identifier NCT00327652) consists of the Listeria monocytogenes Δ actA/Δ inlB platform without any foreign antigen expression sequences. In the exemplary construct described in WO07/103225, this platform was engineered to express a fusion of human mesothelin and ActA-N100. Mesothelin expressing vaccines were evaluated in subjects with advanced cancer with liver metastases using CRS-207(BB-IND13389 and clinicaltrials. gov identifier NCT 00585845). The present invention contemplates the modification of such vaccines by replacing the mesothelin sequence with an EGFRvIII derived antigen sequence.

Because the sequence encoded by an organism is not necessarily subject to codon optimization for optimal expression in a selected vaccine platform bacterial strain, the invention also provides nucleic acids altered by codon optimization for expression of the nucleic acid by bacteria such as listeria monocytogenes.

In various embodiments, at least 1% of any non-optimal codons are altered to provide optimal codons, more typically at least 5% are altered, most typically at least 10% are altered, often at least 20% are altered, more typically at least 30% are altered, most typically at least 40% are altered, typically at least 50% are altered, more typically at least 60% are altered, most typically at least 70% are altered, ideally at least 80% are altered, more ideally at least 90% are altered, most ideally at least 95% are altered, and conventionally 100% of any non-optimal codons are subjected to codon optimization for expression by listeria (table 2).

TABLE 2 optimal codons for expression in Listeria

The present invention provides a number of listeria species and strains useful for preparing or engineering the vaccine platforms of the present invention. The listeria of the present invention is not limited by the species and strains disclosed in table 3.

TABLE 3 Listeria strains suitable for use in the present invention, e.g., as a vaccine or nucleic acid source

Targeting antigens against endocytic receptors on professional Antigen Presenting Cells (APCs) is also an attractive strategy to enhance vaccine efficacy. Such APC-targeted vaccines have the specific ability to direct foreign protein antigens into vesicles that efficiently process antigens for major histocompatibility complex class I and class II presentation. Efficient targeting requires not only high specificity for receptors abundantly expressed on the surface of APCs, but also the ability to be rapidly internalized and loaded into compartments containing elements of the antigen processing machinery. In these embodiments, the antigens of the invention are provided as fusion constructs comprising an immunogenic EGFRvIII-derived antigenic polypeptide and a desired endocytic receptor-targeting moiety. Suitable APC endocytic receptors include DEC-205, mannose receptor, CLEC9, Fc receptor. This list is not intended to be limiting. The receptor targeting moiety may be coupled to the immunogenic EGFRvIII-derived antigenic polypeptide by recombinants or using chemical cross-linking.

4. Therapeutic compositions

The vaccine compositions described herein can be administered to a host alone or in combination with a pharmaceutically acceptable excipient in an amount sufficient to induce a suitable immune response. Immune responses may include, but are not limited to, specific immune responses, non-specific immune responses, specific and non-specific responses, innate responses, primary immune responses, adaptive immunity, secondary immune responses, memory immune responses, immune cell activation, immune cell proliferation, immune cell differentiation, and cytokine expression. The vaccines of the present invention may be stored as a suspension, as a cell paste, or as a complex with a solid or colloidal matrix, e.g., frozen, lyophilized.

In certain embodiments, the second vaccine is administered after administering to the subject an effective dose of a vaccine containing an immunogenic EGFRvIII-derived antigenic polypeptide to elicit an immune response. This is known in the art as a "prime-boost" regimen. In such a scenario, the compositions and methods of the present invention may be used as a "prime" delivery, as a "boost" delivery, or as both a "prime" and a "boost" delivery.

For example, a first vaccine comprising killed but metabolically active listeria encoding and expressing antigenic polypeptide(s) can be delivered as a "prime vaccine", while a second vaccine comprising attenuated (live or killed but metabolically active) listeria encoding antigenic polypeptide(s) can be delivered as a "boost vaccine". However, it is to be understood that neither a prime vaccine nor a boost vaccine need utilize the methods and compositions of the present invention. Rather, the present invention contemplates the use of other vaccine forms with the bacterial vaccine methods and compositions of the present invention. The following are examples of suitable hybrid prime-boost schemes: DNA (e.g., plasmid) vaccine prime/bacterial vaccine boost; viral vaccine prime/bacterial vaccine boost; protein vaccine prime/bacterial vaccine boost; DNA prime/bacterial vaccine boost plus protein vaccine boost; bacterial vaccine prime/DNA vaccine boost; bacterial vaccine prime/viral vaccine boost; bacterial vaccine prime/protein vaccine boost; bacterial vaccine prime/bacterial vaccine boost plus protein vaccine boost, etc. This list is not intended to be limiting.

The prime and boost vaccines may be administered by the same route or by different routes. The term "different routes" includes, but is not limited to, different sites on the body, such as oral, parenteral, enteral, parenteral, rectal, intranodal (lymph node), intravenous, arterial, subcutaneous, intramuscular, intratumoral, peritumoral, infusion, mucosal, nasal, sites in the cerebrospinal space or cerebrospinal fluid, and the like, as well as different modes, such as oral, intravenous and intramuscular.

An effective amount of a prime or boost vaccine may be administered in a single dose, but is not limited to a single dose. Thus, administration can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more vaccine administrations. If there is more than one administration of the vaccine or vaccines in the methods of the invention, administration may be separated at intervals of 1 minute, 2 minutes, 3, 4, 5, 6, 7, 8, 9, 10 or more minutes, at intervals of about 1 hour, 2 hours, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, etc. In the context of hours, the term "about" means any time interval within plus or minus 30 minutes. The administration can be spaced at intervals of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, and combinations thereof. The present invention is not limited to dosing intervals that are equally spaced in time, but includes dosing at unequal intervals, e.g., a priming regimen consisting of administration at 1 day, 4 days, 7 days, and 25 days, merely to provide non-limiting examples.

In certain embodiments, administration of the booster vaccination may be initiated about 5 days after initiation of the priming immunization, about 10 days after initiation of the priming immunization, about 15 days, about 20 days, about 25 days, about 30 days, about 35 days, about 40 days, about 45 days, about 50 days, about 55 days, about 60 days, about 65 days, about 70 days, about 75 days, about 80 days, about 6 months and about 1 year after initiation of administration of the priming immunization. Preferably, one or both of the prime and boost immunizations comprises delivery of a composition of the invention.

"pharmaceutically acceptable excipient" or "diagnostically acceptable excipient" includes, but is not limited to, sterile distilled water, saline, phosphate buffer, amino acid based buffer, or bicarbonate buffer. The excipients selected and the amounts of excipients used will depend on the mode of administration. Administration can be oral administration, intravenous administration, subcutaneous administration, dermal administration, intradermal administration, intramuscular administration, mucosal administration, parenteral administration, intraorgan administration, intralesional administration, intranasal administration, inhalation administration, intraocular administration, intramuscular administration, intravascular administration, intranodal administration, administration by scarification, rectal administration, intraperitoneal administration, or any one or combination of a variety of well-known routes of administration. Administration may include injection, infusion, or a combination thereof.

Tolerance can be avoided by administering the vaccine of the invention by a non-oral route. Methods of intravenous administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, oral administration, mucosal administration, administration through the urinary tract, administration through the reproductive tract, administration through the gastrointestinal tract, or administration by inhalation are known in the art.

The effective amount for a particular patient may vary with factors such as the condition being treated, the general health of the patient, the route and dosage of administration, and the severity of the side effects. Guidelines for therapeutic and diagnostic methods are available (see, e.g., Maynard, et al (1996) A handbook SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, FL; Dent (2001) Good Laboratory and Good Clinical Practice, Urch publication, London, UK).

The vaccines of the present invention may be administered in doses or amounts wherein each dose comprises at least 100 bacterial cells/kg body weight or more, in certain embodiments 1000 bacterial cells/kg body weight or more, normally at least 10,000 cells/kg body weight, more normally at least 100,000 cells/kg body weight, most normally at least 100 million cells/kg body weight, often at least 1000 million cells/kg body weight, more often at least 10000 million cells/kg body weight, typically at least 10 million cells/kg body weight, usually at least 100 million cells/kg body weight, conventionally at least 1000 cells/kg body weight, and sometimes at least 1 trillion cells/kg body weight. The present invention provides the above dose wherein the unit of bacterial administration is Colony Forming Unit (CFU), equivalent of CFU before psoralen treatment, or wherein the unit is bacterial cell number.

The vaccines of the present invention may be administered in doses or amounts wherein each dose comprises 10⁷To 10⁸Individual bacteria/70 kg body weight (or `)1.7 square meters surface area, or/1.5 kg liver weight), 2 × 10⁷To 2x10⁸Bacteria/70 kg body weight (or/1.7 square meter surface area, or/1.5 kg liver weight), 5 × 10⁷To 5x10⁸Bacteria/70 kg body weight (or/1.7 square meter surface area, or/1.5 kg liver weight), 10⁸To 10⁹Bacteria/70 kg body weight (or/1.7 square meter surface area, or/1.5 kg liver weight), 2.0x10⁸To 2.0x10⁹Each bacterium 70kg (or/1.7 square meter surface area, or/1.5 kg liver weight), 5.0x10⁸To 5.0x10⁹Bacteria/70 kg (or/1.7 square meter surface area, or/1.5 kg liver weight), 10⁹To 10¹⁰Each bacterium 70kg (or/1.7 square meter surface area, or/1.5 kg liver weight), 2 × 10⁹To 2x10¹⁰Each bacterium 70kg (or/1.7 square meter surface area, or/1.5 kg liver weight), 5 × 10⁹To 5x10¹⁰Bacteria/70 kg (or/1.7 square meter surface area, or/1.5 kg liver weight), 10¹¹To 10¹²Each bacterium 70kg (or/1.7 square meter surface area, or/1.5 kg liver weight), 2 × 10¹¹To 2x10¹²Each bacterium 70kg (or/1.7 square meter surface area, or/1.5 kg liver weight), 5 × 10¹¹To 5x10¹²Bacteria/70 kg (or/1.7 square meter surface area, or/1.5 kg liver weight), 10¹²To 10¹³Bacteria/70 kg (or/1.7 square meter surface area), 2X10¹²To 2x10¹³Each bacterium 70kg (or/1.7 square meter surface area, or/1.5 kg liver weight), 5 × 10¹²To 5x10¹³Bacteria/70 kg (or/1.7 square meter surface area, or/1.5 kg liver weight), 10¹³To 10¹⁴Each bacterium 70kg (or/1.7 square meter surface area, or/1.5 kg liver weight), 2 × 10¹³To 2x10¹⁴Each bacterium 70kg (or/1.7 square meter surface area, or/1.5 kg liver weight), 5 × 10¹³To 5x10¹⁴Bacteria/70 kg (or/1.7 square meter surface area, or/1.5 kg liver weight), 10¹⁴To 10¹⁵Each bacterium 70kg (or/1.7 square meter surface area, or/1.5 kg liver weight), 2 × 10¹⁴To 2x10¹⁵Each bacterium 70kg (or 1.7 square meter)Surface area, or/1.5 kg liver weight) and the like.

Also provided are one or more of the above doses, wherein the dose is administered by injection once daily, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, or once every 7 days, wherein the injection regimen is maintained, for example, for only 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or longer. The invention also includes combinations of the above dosages and regimens, e.g., using a relatively large initial bacterial dose followed by a relatively small subsequent dose, or using a relatively small initial dose followed by a large dose.

The invention may employ a dosing regimen of, for example, 1 time/week, 2 times/week, 3 times/week, 4 times/week, 5 times/week, 6 times/week, 7 times/week, 1 time every 2 weeks, 1 time every 3 weeks, 1 time every 4 weeks, 1 time every 5 weeks, etc. The dosing regimen includes a total dosing period of, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, and 12 months.

Cycles of the above dosing regimen are provided. The cycle may repeat, for example, approximately every 7 days, every 14 days, every 21 days, every 28 days, every 35 days, 42 days, every 49 days, every 56 days, every 63 days, every 70 days, etc. There may be a non-dosing interval between cycles, wherein the interval may be about, e.g., 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, 56 days, 63 days, 70 days, etc. In this context, the term "about" means plus or minus 1 day, plus or minus 2 days, plus or minus 3 days, plus or minus 4 days, plus or minus 5 days, plus or minus 6 days, or plus or minus 7 days.

The invention includes methods of orally administering listeria. Also provided are methods of administering listeria intravenously. Also, methods of administering listeria orally, intramuscularly, intravenously, intradermally, and/or subcutaneously are provided. The present invention provides listeria bacteria, or a culture or suspension of listeria bacteria, prepared by growing in a meat-based medium or a medium containing a polypeptide derived from a meat or animal product. The invention also provides a listeria bacterium, or a culture or suspension of a listeria bacterium, prepared by growth in a medium that does not contain meat or animal products, prepared by growth in a medium that contains plant polypeptides, prepared by growth in a medium that is not based on yeast products, or prepared by growth in a medium that contains yeast polypeptides.

Methods of administration in combination with additional therapeutic agents are well known in The art (Hardman, et al (eds.) (2001) Goodman and Gilman's The Pharmacological Basis of therapeutics, 10 th edition, McGraw-Hill, New York, NY; Poole and Peterson (eds.) (2001) Pharmacological for Advanced Practice: A practical approach, Lippincott, Williams & Wilkins, Phila., PA; Chamner and Long (eds.) (2001) Cancer therapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., PA).

Additional agents useful for enhancing cytolytic T cell responses may also be used. Such agents are referred to herein as carriers. These include, but are not limited to, B7 co-stimulatory molecules, interleukin-2, interferon- γ, GM-CSF, CTLA-4 antagonists, OX-40/OX-40 ligands, CD40/CD40 ligands, sargramostim (sargramostim), levamisole (levamisol), vaccinia virus, Bacillus Calmette-Guerin (BCG), liposomes, alum, Freund's complete or incomplete adjuvant, detoxified endotoxin, mineral oil, surface active substances such as lipolecithins (lipofectin), polyols (pluronic polyols), polyanions, peptides, and oil or hydrocarbon emulsions. Vectors that induce a T cell immune response that preferentially stimulate a cytolytic T cell response relative to an antibody response are preferred, but those that stimulate both types of responses may also be used. Where the agent is a polypeptide, the polypeptide itself or a polynucleotide encoding the polypeptide may be administered. The carrier may be a cell, such as an Antigen Presenting Cell (APC) or a dendritic cell. Antigen presenting cells include cell types such as macrophages, dendritic cells and B cells. Other professional antigen-presenting cells include monocytes, marginal zone Kupffer cells, microglia, langerhans cells, dendritic cells (dendritic cells), follicular dendritic cells and T cells. Facultative antigen-presenting cells may also be used. Examples of facultative antigen-presenting cells include astrocytes, follicular cells, endothelium and fibroblasts. The vector may be a bacterial cell transformed to express the polypeptide or to deliver the polynucleotide which is subsequently expressed in the cells of the immunized individual. Adjuvants such as aluminum hydroxide or aluminum phosphate may be added to increase the ability of the vaccine to trigger, enhance, or prolong the immune response. Additional materials such as the following are also potential adjuvants: cytokines, chemokines, and CpG-like bacterial nucleic acid sequences, toll-like receptor (TLR)9 agonists, and additional agonists for TLR2, TLR4, TLR5, TLR7, TLR8, TLR9, including lipoproteins, LPS, monophosphoryl lipid a, lipoteichoic acid, imiquimod (imiquimod), resiquimod (resiquimod), and other similar immune modulators used alone or in combination with the composition. Other representative examples of adjuvants include the synthetic adjuvant QS-21, which comprises a homogeneous saponin purified from the bark of the Quillaja saponaria tree (Quillaja saponaria) and Corynebacterium parvum (McCune et al, Cancer,1979;43: 1619). It is understood that the adjuvant is subject to optimization. In other words, the skilled person may perform routine experiments to determine the best adjuvant to use.

An effective amount of a therapeutic agent is an amount that reduces or ameliorates the symptoms by at least 10% normal, by at least 20% more normal, by at least 30% most normal, typically by at least 40%, more typically by at least 50%, most typically by at least 60%, often by at least 70%, more often by at least 80%, and most often by at least 90%, conventionally by at least 95%, more conventionally by at least 99%, and most conventionally by at least 99.9%.

The reagents and methods of the invention provide a vaccine comprising only one immunization; or a vaccine comprising a first immunization; or a vaccine comprising at least one booster immunization, at least two booster immunizations, or at least three booster immunizations. Parameter guidelines for booster immunizations are available. See, e.g., Marth (1997) Biologicals25: 199-203; ramsay, et al (1997) Immunol.cell biol.75: 382-388; gherardi, et al (2001) Histol. Histopathiol.16: 655-667; Leroux-Roels, et al (2001) ActA Clin. Belg.56: 209-219; greiner, et al (2002) cancer Res.62: 6944-6951; smith, et al (2003) J.Med.Virol.70: Suppl.1: S38-S41; Sepulveda-Amor, et al (2002) Vaccine20: 2790-.

Therapeutic agent formulations may be prepared, for example, as lyophilized powders, slurries, aqueous solutions or suspensions by mixing with physiologically acceptable carriers, excipients or stabilizers (see, e.g., Hardman, et al (2001) Goodman and Gilman's The pharmacological basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al (eds. (1993) Pharmaceutical Dosage Forms: commercial medicines, Marcel Dekker, NY; Lierman, et al (eds. (1990) Pharmaceutical dosages: Tablets, NY Dekkebker, et al (1990) Pharmaceutical Dosage Forms: Lipofenker, N.S., Lipoferman, et al (1990) incorporated, Inc.: New York, Inc.).

Examples

The following examples serve to illustrate the invention. These examples are in no way intended to limit the scope of the invention.

Example 1 bacterial strains and antigen selection

Lm vaccine strains were constructed in two strain backgrounds, live attenuated (Lm11, aka Lm Δ actA/Δ inlB) and KBMAPrfA (Lm677, aka Lm Δ actA/Δ inlB/Δ uvrAB/prfA G155S). The expression cassette was designed to contain 1 and 5 copies of sequence PASRALEEKKGNYVVTDHGSC (SEQ ID NO:4) (denoted PvIII in FIG. 1), each copy flanking an eggProteasome cleavage sequence (black box in fig. 1). The expression cassette was codon optimized for expression in listeria monocytogenes, cloned downstream of the actA promoter as a BamHI-SpeI fragment and in-frame with the 100 amino-terminal acid of actA ("actA-N100"), and labeled SIINFEKL (SL8) on the carboxy-terminus of the expression cassette, i.e., an alternative T-cell epitope that facilitates evaluation of expression and secretion of the encoded heterologous antigen. The construct was cloned into a derivative of pPL2 integration vector and stably integrated into the bacterial chromosomal tRNA^ArgAt the locus of the gene.

The strains tested are summarized in the following table:

bacterial strains	Construct	Background
			BH137	ActAN100-AH1A5-OVA	Lm11(ΔactAΔinlB)
Lm11	Negative control
			PL712	ActAN100-PepVIIIx5-SL8	Lm11
PL714	ActAN100-PepVIIIx5-SL8	Lm677(prfA*)
			PL716	ActAN100-PepVIIIx1-SL8	Lm11
PL717	ActAN100-PepVIIIx1-SL8	Lm677(prfA*)

Example 2 in vitro cell culture

J774 cells were cultured in T cell culture medium (RPMI medium (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (Hyclone, Logan, UT), penicillin/streptomycin (Mediatech, Manassas, VA), 1x non-essential amino acids (Mediatech, Manassas, VA), 2mM L-glutamine (Mediatech, Manassas, VA), HEPES buffer (Invitrogen, Carlsbad, CA), 1mM sodium pyruvate (Sigma, st.louis, MO), and 50 μ M β -mercaptoethanol (Sigma, st.louis, MO)). B3Z hybridoma cells were cultured in T cell medium without penicillin/streptomycin.

Example 4 immunization

Female C3H/HeJ mice 6-12 weeks old were obtained from Charles River Laboratories (Wilmington, MA). The study was conducted according to the Animal protocol approved by the Institutional Animal Care and Use Committee. Live attenuated bacteria for immunization were prepared from overnight grown cultures in yeast extract medium. The bacteria were diluted in Hank's balanced salt solution for injection (HBSS). Live attenuated bacteria were administered intravenously into the tail vein in a volume of 200 μ L. Injection stocks of live attenuated bacteria were plated to determine Colony Forming Units (CFU). At intervals of 30 days, with 1X10⁷CFU LmΔactAΔinlBprfA*-5xEGFRvIII_20-40For female animalsPrime and boost, and the frequency of EGFRvIII-specific CD8+ T cells determined by intracellular cytokine staining. (FIG. 3).

Example 5 evaluation of antigen expression and immune response

a. Western blot

For growth to OD in Yeast extract Medium₆₀₀Western blotting of the broth culture was performed for an equal amount of TCA pellet supernatant of 0.7 (late log phase) of the bacterial culture. For western blot of Lm infected DC2.4 cells, the cells were seeded at multiplicity of infection (MOI) of 10 for 1 hour and washed 3 times with PBS and DMEM medium supplemented with 50 μ g/mL gentamicin. Cells were harvested 7 hours post infection. Cells were lysed with SDS sample buffer, collected and electrophoresed on a 4-12% polyacrylamide gel, and transferred to nitrocellulose membranes for western blot analysis. All western blots utilized polyclonal antibodies raised against the mature N-terminus of the ActA protein and normalized to p60 expression using an anti-p 60 monoclonal antibody (unrelated Lm protein). Antigen detection was either visualized by Enhanced Chemiluminescence (ECL) or visualized and quantified using the Licor Odyssey IR detection system (fig. 2).

B3Z assay

DC2.4 cells were infected with the selected strain and the cells were incubated with OVA_257-264Specific T cell hybridomas, B3Z were incubated together. Assessment of SIINFEKL epitopes at H-2K by measuring beta-galactosidase expression using chromogenic substrates^bPresentation on class I molecules (fig. 4).

c. Reagents for flow cytometry

CD4FITC (clone GK 1.5) and MHC class II FITC (clone M5/114.15.2) were purchased from eBioscience (San Diego, Calif.). The CD8a antibody PerCP (clone 53-6.7) was purchased from BD Biosciences (San Jose, Calif.). The Kk-EEKKGNYV (SEQ ID NO:3) Tetramer was folded using NIH Tetramer Core and conjugated to APC.

Tetramer staining of EGFRvIII-specific T cells

Splenocytes (lymphocytes from the spleen) were incubated with anti-CD 4, anti-CD 8, anti-MHC class II and Kk-EGFRvIII tetramer for 15 minutes at room temperature. Cells were washed three times with HBSS. Samples were taken using a LSRII flow cytometer (BD Biosciences). The data was gated to specifically include CD8+, CD 4-class II events, and the percentage of these cells that bound the Kk-EEKKGNYV (SEQ ID NO:3) tetramer was then determined. Data were analyzed using FlowJo software (Treestar, Ashland, OR).

Example 6 results

As can be seen from FIG. 2, all EGFRvIII detected within J774 macrophages_20-40Constructs. Furthermore, we found that increasing EGFRvIII_20-40The copy number of (a) appears to enhance intracellular secretion (fig. 2). Secreted 5xEGFRvIII_20-40The amount of the egg albumin exceeds the amount of the egg albumin of the 'gold standard'. After receiving 5xEGFRvIII_20-40In the animals of the construct, greater than 30% of CD8+ T cells in the spleen were EGFRvIII-specific 5 days after booster immunization (fig. 3). B3Z T cells recognized all EGFRvIII tested_20-40Constructs indicating that listeria monocytogenes strains successfully expressed and presented T cell epitopes at levels approximately equivalent to model ovalbumin antigens (fig. 4).

These data reveal that EGFRvIII_20-40Expression constructs, in particular poly-EGFRvIII_20-40The expression constructs are well suited for use in this listeria monocytogenes vaccine platform.

Example 7 evaluation of immune response Using EEKKGNYV (SEQ ID NO:3)

The precise identification of class I restriction epitopes provides a means for assessing immunogenicity in vivo, thereby simplifying the comparison of our vaccine candidates. To identify mouse class I-restricted epitopes, and to perform a rough assessment of immunogenicity, 1x10 was used⁷ColoniesEach EGFRvIII form a Unit (CFU)_20-40Expression strain immunization C57BL/6 (H-2)^b)、BALB/c(H-2^d)、C3H/HeJ(H-2^k) And SJL (H-2)^s) Mice, and the frequency and specificity of EGFRvIII-specific T cells was determined by IFN- γ Intracellular Cytokine Staining (ICS). Probably due to EGFRvIII in our vaccine_20-40Short sequences of peptides, and desirably due to known N-terminal processing problems associated with these libraries, this library uses 15-AA peptides that span 14-AA. C3H mouse strain (H-2)^k) The most intense reaction in (1), among which in the spleen>1% of total CD8+ T cells responded to one of the 15-AA peptides (data not shown).

To confirm the results of this study and to refine the exact sequence of class I binding peptides, C3H mice were primed and boosted with Lm-egfrviii x5 strain and then rescreened with a peptide library. In addition, we introduced peptides that were completed according to the 15-mer identified in the original immunogenicity assay. CD8+ T cells from these primed and boosted mice recognize several 15-AA peptides and the reactivity depends on the two N-terminal glutamic acid residues. Assuming that these two glutamate residues are required for MHC binding, we used 7-, 8-and 9-mers of this sequence and screened for reactivity (fig. 5).

Identification of class I-restricted EGFRvIII epitopes in C3H mice.Female C3H mice were primed and boosted with Lm-EGFRvIII x5, then the spleens were harvested five days later and tested for reactivity using the EGFRvIII peptide library and 7-, 8-and 9-mers derived from the peptides with the strongest reactivity in the preliminary screen. Data are expressed as% IFN- γ + events within the CD8+ T cell gate. These experiments showed that CD8+ T cells from immunized C3H mice recognized the 8-AA peptide EEKKGNYV (SEQ ID NO: 3).

TAP-deficient T2 cells were not loaded with class I molecules and peptides, resulting in instability and recycling from the cell surface. When exogenous peptides are added that bind to the expressed MHC molecule, the molecule is stabilized on the cell surface along with Lm-EGFRvIII. To confirm the interaction of the EEKKGNYV (SEQ ID NO:3) peptide withk^kUsing T2 cells measuring such induction of class I expression after peptide binding, essentially as described in the following documents: hansen and Myers (2003) Peptide indication of surface expression of class I MHC, p.18.11.1-18.11.8, see J.E.Colgan, A.M.Kruisbeer, D.H.Marguiles, and W.Strober (eds.), Current Protocols in Immunology, vol.4.John Wiley&Sons, New York, n.y. Will express K^kWas incubated overnight with each EGFRvIII peptide at the peptide concentrations indicated in fig. 6. Cells were washed and directed against surface K using class I specific antibodies^kThe expression was stained. The data indicate that EEKKGNYV binds K^kAnd are optimal relative to larger peptides containing such sequences. These data support that the defined EEKKGNYV peptide binds K based on it^kAnd provides an MHC-peptide complex that is recognized by CD8+ T cells.

Using this defined class I restricted peptide, we compared the strength of the initial EGFRvIII-specific CD8+ T cell response after immunization with each strain. With 1x10⁵Female C3H mice were immunized with 1x or 5x strains of CFU, and the spleens were harvested 7 days later, and EGFRvIII was determined by ICS_26-33Frequency of specific CD8+ T cells. Consistent with our hypothesis, multiple copies of EGFRvIII_20-40The introduction of (a) resulted in enhanced priming of CD8+ T cells relative to single copy variants (fig. 7).

These data demonstrate the ability to express constructs that encode repeated epitope sequences but alter codon usage to maximize genetic stability and antigen expression/secretion. This approach allows for increased potency (i.e., by administering a large dose of vaccine) without increasing the potential risk to the patient.

Those skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

It will be apparent to those skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the spirit and scope of the invention.

All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations, which is/are not specifically disclosed herein. Thus, for example, in each example herein, any of the terms "comprising," consisting essentially of, "and" consisting of may be substituted with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

Other embodiments are set forth in the following claims.

Claims

1. A method of inducing a T cell response to EGFRvIII in a subject, the method comprising:

recombinantly expressing in the subject at least one immunogenic polypeptide under conditions selected to induce the T cell response in the subject, the amino acid sequence of the immunogenic polypeptide comprising a plurality of EGFRvIII polypeptide sequences, the sequences of the plurality of EGFRvIII polypeptide sequences each comprising EEKKGNYV (SEQ ID NO: 3).

2. The method of claim 1, wherein the plurality of EGFRvIII polypeptide sequences comprise one or more amino acid sequences selected from the group consisting of: LEEKKGNYV (SEQ ID NO:4), LEEKKGNYVVTDH (SEQ ID NO:2), and PASRALEEKKGNYVVTDHGSC (SEQ ID NO: 5).

3. The method of claim 2, wherein the plurality of EGFRvIII polypeptide sequences comprises at least 3 copies of PASRALEEKKGNYVVTDHGSC (SEQ ID NO: 5).

4. The method of claim 2, wherein the plurality of EGFRvIII polypeptide sequences comprises at least 5 copies of PASRALEEKKGNYVVTDHGSC (SEQ ID NO: 5).

5. The method of any one of claims 1 to 4, wherein each EGFRvIII polypeptide sequence is flanked by sequences configured to be cleaved by the proteasome.

6. The method of claim 5, wherein each EGFRvIII polypeptide sequence is flanked by sequences selected from the group consisting of: ASKVL ↓: ADGSVK, ASKVA ↓: GDGSIK, LSKVL ↓: ADGSVK, LAKSL ↓: ADLAVK, ASVVA ↓: GIGSIA, GVEKI ↓: NAANKG, and DGSKKA ↓: GDGNKK (SEQID NOS:6-12), wherein each arrow mark represents EGFRvIII polypeptide sequence.

7. The method of any one of claims 1-6, wherein said immunogenic polypeptide is expressed in said subject by administering a Listeria monocytogenes bacterium comprising a nucleic acid sequence encoding said immunogenic polypeptide integrated into the genome of said bacterium operably linked to control sequences that cause expression of said immunogenic polypeptide in said subject.

8. The method of claim 7, wherein the bacterium is an actA deletion mutant or an actA insertion mutant, an inlB deletion mutant or an inlB insertion mutant or a Δ actA/Δ inlB mutant comprising both an actA deletion or an actA insertion and an inlB deletion or an inlB insertion.

9. The method of claim 8, wherein the nucleic acid sequence has been integrated into a virulence gene of the bacterium, and integration of the nucleic acid sequence disrupts expression of the virulence gene or disrupts the coding sequence of the virulence gene.

10. The method of claim 9, wherein the virulence gene is actA or inlB.

11. The method of any one of claims 1-10, wherein the bacterium is an attenuated listeria monocytogenes.

12. The method of claim 11, wherein the bacteria is Lm Δ actA/Δ inlB.

13. The method of claim 12, wherein the bacterium further comprises a genetic mutation that attenuates the ability of the bacterium to repair nucleic acids.

14. The method of claim 13, wherein the genetic mutation is a mutation in one or more genes selected from phrB, uvrA, uvrB, uvrC, uvrD and recA.

15. The method of claim 11, wherein said bacterium is a listeria monocytogenes prfA mutant, the genome of which encodes a prfA protein that is constitutively active.

16. The method of claim 11, wherein the bacteria are killed but metabolically active listeria monocytogenes.

17. The method of claim 16, wherein said bacterium is a listeria monocytogenes prfA mutant, the genome of which encodes a prfA protein that is constitutively active.

18. The method of claim 7, wherein the nucleic acid sequence is codon optimized for expression by Listeria monocytogenes.

19. The method of any one of claims 7-18, wherein selecting said conditions for inducing said T cell response in said subject comprises administering said listeria monocytogenes to said subject by one or more routes of administration selected from the group consisting of: oral administration, intramuscular administration, intravenous administration, intradermal administration, and subcutaneous administration.

20. The method of any one of claims 1 to 19, wherein the immunogenic polypeptide is expressed as a fusion protein comprising a secretion signal sequence.

21. The method of claim 20, wherein the secretion signal sequence is a listeria monocytogenes ActA signal sequence.

22. The method according to claim 20, wherein the immunogenic polypeptide is expressed as a fusion protein comprising an in-frame ActA-N100 sequence selected from the group consisting of SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, and SEQ ID No. 31, or an amino acid sequence having at least 90% sequence identity to the ActA-N100 sequence.

23. The method of claim 7, wherein the method comprises administering Listeria monocytogenes expressing a fusion protein comprising:

an ActA-N100 sequence selected from the group consisting of SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, and SEQ ID No. 31, or an amino acid sequence having at least 90% sequence identity to said ActA-N100 sequence; and

a plurality of amino acid sequences having sequence PASRALEEKKGNYVVTDHGSC (SEQ ID NO:5), each of the plurality of amino acid sequences flanked by a sequence configured to be cleaved by the proteasome,

24. The method of any one of claims 1-23, wherein the subject has an EGFRvIII-expressing tumor.

25. The method of claim 24, wherein the subject has a glioma.

26. The method of any one of claims 1-25, wherein the composition, when delivered to the subject, induces an increase in serum concentration of one or more proteins selected from the group consisting of IL-12p70, IFN- γ, IL-6, TNF α, and MCP-1 at 24 hours after the delivery; and inducing a CD4+ and/or CD8+ antigen-specific T cell response against EGFRvIII.

27. A composition, comprising:

a bacterium or virus comprising a nucleic acid encoding at least one immunogenic polypeptide having an amino acid sequence comprising a plurality of EGFRvIII polypeptide sequences, each of which comprises EEKKGNYV (SEQ ID NO: 3).

28. The composition of claim 27 wherein the plurality of EGFRvIII polypeptide sequences comprise one or more amino acid sequences selected from the group consisting of: LEEKKGNYV (SEQ ID NO:4), LEEKKGNYVVTDH (SEQ ID NO:2), and PASRALEEKKGNYVVTDHGSC (SEQ ID NO: 5).

29. The composition of claim 28, wherein said plurality of EGFRvIII polypeptide sequences comprises at least 3 copies of PASRALEEKKGNYVVTDHGSC (SEQ ID NO: 5).

30. The composition of claim 28, wherein said plurality of EGFRvIII polypeptide sequences comprises at least 5 copies of PASRALEEKKGNYVVTDHGSC (SEQ ID NO: 5).

31. The method of any one of claims 27-30, wherein each EGFRvIII polypeptide sequence is flanked by sequences configured to be cleaved by the proteasome.

32. The composition of any one of claims 27-31, wherein said composition comprises listeria monocytogenes comprising said nucleic acid integrated into the genome of said bacterium.

33. The composition of claim 32, wherein the bacterium is an actA deletion mutant or an actA insertion mutant, an inlB deletion mutant or an inlB insertion mutant or a Δ actA/Δ inlB mutant comprising both an actA deletion or an actA insertion and an inlB deletion or an inlB insertion.

34. The composition of claim 32, wherein the nucleic acid has been integrated into a virulence gene of the bacterium, and integration of the polynucleotide disrupts expression of the virulence gene or disrupts the coding sequence of the virulence gene.

35. The composition of claim 34, wherein the virulence gene is actA or inlB.

36. The composition of claim 32, wherein the bacterium is an attenuated listeria monocytogenes.

37. The composition of claim 36, wherein the bacterium is Lm Δ actA/Δ inlB.

38. The composition of claim 34, wherein the bacterium further comprises a genetic mutation that attenuates the ability of the bacterium to repair nucleic acids.

39. The composition of claim 38, wherein the genetic mutation is a mutation in one or more genes selected from phrB, uvrA, uvrB, uvrC, uvrD and recA.

40. The composition of claim 36, wherein said bacterium is a listeria monocytogenes prfA mutant, the genome of which encodes a prfA protein that is constitutively active.

41. The composition of claim 37, wherein the bacteria is killed but metabolically active listeria monocytogenes.

42. The composition of claim 32, wherein said bacterium is a listeria monocytogenes prfA mutant, the genome of which encodes a prfA protein that is constitutively active.

43. The composition of claim 32, wherein said nucleic acid is codon optimized for expression by listeria monocytogenes.

44. The composition of any one of claims 27-43, wherein the composition further comprises a pharmaceutically acceptable excipient.

45. The composition of any one of claims 27-44, wherein the nucleic acid encodes the immunogenic polypeptide as a fusion protein comprising a secretion signal sequence.

46. The composition of claim 45, wherein the secretion signal sequence is a Listeria monocytogenes ActA signal sequence.

47. The composition of claim 46, wherein the nucleic acid molecule encodes the immunogenic polypeptide as a fusion protein comprising an in-frame ActA-N100 sequence selected from the group consisting of SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, and SEQ ID NO 31, or an amino acid sequence having at least 90% sequence identity to the ActA-N100 sequence.

48. The composition of any one of claims 32-47, wherein the composition comprises Listeria monocytogenes comprising a nucleic acid whose sequence encodes a fusion protein comprising:

49. A method of treating an EGFRvIII-expressing malignancy in a subject, the method comprising:

administering to the subject a composition according to any one of claims 27-48 under conditions selected to induce a T cell response in the subject.

50. A pharmaceutical composition comprising:

the composition of any one of claims 27-49; and

a pharmaceutically acceptable excipient.

51. An isolated nucleic acid molecule encoding at least one immunogenic polypeptide having an amino acid sequence comprising a plurality of EGFRvIII polypeptide sequences, each of the plurality of EGFRvIII polypeptide sequences comprising EEKKGNYV (SEQ ID NO: 3).

52. The isolated nucleic acid molecule of claim 51, wherein said plurality of EGFRvIII polypeptide sequences comprises one or more amino acid sequences selected from the group consisting of: LEEKKGNYV (SEQ ID NO:4), LEEKKGNYVVTDH (SEQ ID NO:2), and PASRALEEKKGNYVVTDHGSC (SEQ ID NO: 5).

53. The isolated nucleic acid molecule of claim 51, wherein said plurality of EGFRvIII polypeptide sequences comprises at least 3 copies of PASRALEEKKGNYVVTDHGSC (SEQ ID NO: 5).

54. The isolated nucleic acid molecule of claim 51, wherein said plurality of EGFRvIII polypeptide sequences comprises at least 5 copies of PASRALEEKKGNYVVTDHGSC (SEQ ID NO: 5).

55. The isolated nucleic acid molecule of any one of claims 51-54, wherein each EGFRvIII polypeptide sequence is flanked by sequences configured to be cleaved by the proteasome.

56. The isolated nucleic acid molecule of any one of claims 51-55, wherein said nucleic acid molecule is codon-optimized for expression by Listeria monocytogenes.

57. The isolated nucleic acid molecule of any one of claims 51-56, wherein the nucleic acid molecule encodes the immunogenic polypeptide as a fusion protein comprising a secretion signal sequence.

58. The isolated nucleic acid molecule of claim 57, wherein the secretion signal sequence is a Listeria monocytogenes ActA signal sequence.

59. The isolated nucleic acid molecule according to claim 58, wherein said nucleic acid molecule encodes said immunogenic polypeptide as a fusion protein comprising an in-frame ActA-N100 sequence selected from the group consisting of SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, and SEQ ID NO 31, or an amino acid sequence having at least 90% sequence identity to said ActA-N100 sequence.

60. The isolated nucleic acid molecule of claim 59, wherein the nucleic acid molecule encodes the immunogenic polypeptide as a fusion protein comprising:

wherein the nucleic acid molecule is operably linked to a Listeria monocytogenes ActA promoter.

61. A recombinantly produced or chemically synthesized polypeptide having an amino acid sequence comprising a plurality of EGFRvIII polypeptide sequences, each of the sequences of the plurality of EGFRvIII polypeptide sequences comprising EEKKGNYV (SEQ ID NO: 3).

62. The polypeptide of claim 61, wherein the plurality of EGFRvIII polypeptide sequences comprise one or more amino acid sequences selected from the group consisting of: LEEKKGNYV (SEQ ID NO:4), LEEKKGNYVVTDH (SEQ ID NO:2), and PASRALEEKKGNYVVTDHGSC (SEQ ID NO: 5).

63. The polypeptide of claim 61, wherein the plurality of EGFRvIII polypeptide sequences comprises at least 3 copies of PASRALEEKKGNYVVTDHGSC (SEQ ID NO: 5).

64. The polypeptide of claim 61, wherein the plurality of EGFRvIII polypeptide sequences comprises at least 5 copies of PASRALEEKKGNYVVTDHGSC (SEQ ID NO: 5).

65. The polypeptide of any one of claims 61-64, wherein each EGFRvIII polypeptide sequence is flanked by sequences configured to be cleaved by the proteasome.

66. The polypeptide of any one of claims 61-64, further comprising a moiety configured to target the polypeptide to a cell surface receptor of an antigen presenting cell.