HK1197584A

HK1197584A - Compositions and methods for prevention of escape mutation in the treatment of her2/neu over-expressing tumors

Info

Publication number: HK1197584A
Application number: HK14111317.7A
Authority: HK
Inventors: V．谢哈比
Original assignee: 阿德瓦希斯公司
Priority date: 2011-08-16
Filing date: 2012-08-16
Publication date: 2015-01-30

Description

Compositions and methods for preventing escape mutations during treatment of HER2/NEU overexpressing tumors

Cross Reference to Related Applications

The present application claims priority from U.S. patent application serial No. 13/210,696 filed on 16/8/2011, which is a partial continuation of co-pending U.S. patent application serial No. 12/945,386 filed on 12/11/2010 (which claims benefit of 11/japanese provisional application serial No. 61/260,277 in 2009). These applications are incorporated by reference in their entirety.

Technical Field

The present invention provides compositions and methods for treating and vaccinating non-human animals and inducing an immune response against Her2/neu antigen-expressing tumors.

Background

Her-2/neu (hereinafter "Her-2") is a 185kDa glycoprotein that is a member of the Epidermal Growth Factor Receptor (EGFR) family of tyrosine kinases and consists of an extracellular domain, a transmembrane domain, and an intracellular domain known to be involved in cell signaling (Bargmann CI et al, Nature319:226,1986; King CR et al, Science 229: 974, 1985). In humans, the HER2 antigen is overexpressed in 25% to 40% of all breast cancers, and also in many cancers of the ovary, lung, pancreas, brain, and gastrointestinal tract. Overexpression of Her-2 is associated with uncontrolled cell growth and signaling, both of which contribute to tumor development. Even in the presence of detectable body fluid, CD8, directed against Her-2⁺T cells and CD4⁺T cell response, patients with Her-2 overexpressing cancers exhibit tolerance.

Listeria monocytogenes (Listeria monocytogenes) are intracellular pathogens that primarily infect antigen presenting cells and are adapted to survive in the cytoplasm of these cells. Host cells, such as macrophages, actively phagocytose listeria monocytogenes and most bacteria are degraded in phagolysosomes. Some bacteria escape into the host cytosol by perforating the phagosomal membrane via the action of hemolysin, listeria lysenin o (llo). Once in the cytosol, listeria monocytogenes can polymerize host actin and pass directly from cell to cell, further evading the host immune system and resulting in negligible antibody response to listeria monocytogenes.

The construction and development of a number of listeria monocytogenes (Lm) based vaccines that independently express small fragments of human Her2/neu protein from the extracellular and intracellular domains of the protein have been reported. Her2/neu is too large to fit into Lm, which necessitates the generation of a Her2/neu fragment. All these vaccines were shown to be immunogenic and effective in regressing pre-established tumors in FVB/N mice and delaying the onset of spontaneous breast tumors in Her2/neu expressing transgenic animals. The exciting results from these preliminary experiments suggest that recombinant Listeria (Listeria) -Her2/neu vaccines can be generated that can break tolerance to the Her2/neu autoantigen. However, the listeria-Her 2/neu vaccine developed to date is based on an attenuated listeria platform using an antibiotic marker (cat) for in vitro selection of recombinant bacteria in the presence of chloramphenicol. For clinical use, not only is high attenuation important, but also the lack of resistance to antibiotics is important.

Canine osteosarcoma is a cancer of the long (leg) bone, which is the major killer in dogs over 10 years of age. The standard treatment is amputation immediately after diagnosis, followed by chemotherapy. However, cancer always metastasizes to the lungs. With chemotherapy, dogs survive approximately 12 months, compared to 6 months without treatment. The HER2 antigen is present in up to 50% of osteosarcomas.

Escape mutations (escape mutations) have been reported in tumors to evade host immune responses, which remain a major obstacle to tumor therapy. Therefore, there is a need to develop vaccines that have high therapeutic efficacy and do not generate escape mutations. After discovering the activity of each of the above-described fragments of the vaccine, the present invention integrates all active sites from each of the individual fragments, thereby obtaining a novel immunogenic composition for vaccine production. Furthermore, the need for safe and effective cancer treatments in the animal market is far from being met. The present invention meets this need by providing a recombinant listeria-Her 2/neu vaccine (ADXS31-164) produced using an LmddA vaccine vector with a well defined attenuation mechanism and no antibiotic selection marker. The use of the chimeric antigen did not generate escape mutations, indicating that tumors did not mutate and lost a therapeutically effective response to treatment with the new antigen.

Disclosure of Invention

In one embodiment, the invention relates to an immunogenic composition comprising a fusion polypeptide, wherein said fusion polypeptide comprises a Her2/neu chimeric antigen fused to a further polypeptide, and wherein administration of the fusion protein to a subject having a Her 2/neu-expressing tumor causes evasion of mutations (mutationavoidance). In another embodiment, the mutation avoidance is due to epitope spreading (epitopic prediction). In yet another embodiment, the avoidance of mutations is due to the chimeric nature of the antigen.

In another embodiment, the invention relates to a recombinant listeria vaccine strain comprising a nucleic acid molecule, wherein and in another embodiment, the nucleic acid molecule comprises a first open reading frame encoding a polypeptide, wherein the polypeptide comprises a Her2/neu chimeric antigen, wherein the nucleic acid molecule further comprises a second open reading frame encoding a metabolic enzyme, and wherein the metabolic enzyme complements an endogenous gene that is lacking in the chromosome of the recombinant listeria strain.

In one embodiment, the invention relates to a method of treating Her-2/neu-expressing tumor growth or cancer in a non-human animal, the method comprising the step of administering a recombinant listeria comprising a nucleic acid encoding a fusion polypeptide, wherein the fusion polypeptide comprises a Her2/neu chimeric antigen fused to an additional adjuvant polypeptide.

In another embodiment, the invention relates to a method of preventing Her-2/neu-expressing tumor growth or cancer in a non-human animal, the method comprising the step of administering a recombinant listeria comprising a nucleic acid encoding a fusion polypeptide, wherein the fusion polypeptide comprises a Her2/neu chimeric antigen fused to an additional adjuvant polypeptide.

In one embodiment, the invention relates to a method of eliciting an enhanced immune response against Her-2/neu-expressing tumor growth or cancer in a non-human animal, the method comprising the step of administering a recombinant listeria comprising a nucleic acid encoding a fusion polypeptide, wherein the fusion polypeptide comprises a Her2/neu chimeric antigen fused to an additional adjuvant polypeptide.

Drawings

FIG. 1 shows the construction of ADXS 31-164. (A) plasmid map of pAdv164, which has the Bacillus subtilis dal gene under the control of the constitutive Listeria p60 promoter, was used to complement the chromosomal dal-dat deletion in the LmddA strain. It also contains a truncated LLO (1)_-441) Is fused with a chimeric human Her2/neu gene, which is constructed by directly fusing 3 fragments of Her2/neu, EC1(aa40-170), EC2(aa359-518) and ICI (aa 679-808). (B) The expression and secretion of tLLO-ChHer2 was detected in Lm-LLO-ChHer2(Lm-LLO-138) and LmddA-LLO-ChHer2(ADXS31-164) by Western blot analysis of TCA precipitated cell culture supernatants blotted with anti-LLO antibodies. A differential band of about 104kD corresponds to tLLO-ChHer 2. Endogenous LLO was detected as a 58KD band. The listeria control did not have ChHer2 expression.

FIG. 2 shows the immunogenic properties of ADXS 31-164. (A) Cytotoxic T cell responses elicited by the Her2/neu Listeria-based vaccine in splenocytes from immunized mice were tested using NT-2 cells as stimulators and 3T3/neu cells as targets. The Lm-control was based on an LmddA background, which was identical to the vaccine in all respects except that it expressed an unrelated antigen (HPV 16-E7). (B) After stimulating splenocytes from immunized FVB/N mice with mitomycin C treated NT-2 cells in vitro for 24 hours, the secretion of IFN-. gamma.into the cell culture medium by the splenocytes was measured by ELISA. (C) Splenocytes from HLA-A2 transgenic mice immunized with the chimeric vaccine secreted IFN- γ in response to in vitro incubation with peptides from different regions of the protein. As listed in the figure legend, recombinant ChHer 2protein was used as a positive control, and irrelevant or no peptide groups constituted negative controls. Cell culture supernatants harvested after 72 hours co-incubation were used to detect IFN- γ secretion by ELISA assay. Each data point is the mean of the three data +/-standard deviation. P value < 0.001.

FIG. 3 shows a tumor prevention study of the Listeria-ChHer 2/neu vaccine. Six injections of Her2/neu transgenic mice were performed with each recombinant listeria-ChHer 2 or control listeria vaccine. Immunization was started at 6 weeks of age and continued every three weeks until week 21. Appearance of tumors was monitored weekly and expressed as a percentage of tumor-free mice. P <0.05, N =9 per group.

Figure 4 shows the effect of immunization with LmddA-LLO-ChHer2(ADXS31-164) on the percentage of Tregs in the spleen. With 1x10⁶NT-2 cells were inoculated subcutaneously into FVB/N mice and immunized three times with each vaccine at weekly intervals. Spleens were harvested 7 days after the second immunization. After isolation of the immune cells, they were stained for detection of tregs by anti-CD 3, CD4, CD25 and FoxP3 antibodies. Dot plots of tregs from representative experiments show CD25⁺/FoxP3⁺T cell frequency, expressed as total CD3 across different treatment groups⁺Or CD3⁺CD4⁺Percentage of T cells.

Figure 5 shows the effect of immunization with LmddA-LLO-ChHer2(ADXS31-164) on the percentage of Tregs infiltrating tumors in NT-2 tumors. With 1x10⁶NT-2 cells were inoculated subcutaneously into FVB/N mice and immunized three times with each vaccine at weekly intervals. Tumors were harvested 7 days after the second immunization. After isolation of the immune cells, they were stained for Treg detection by anti-CD 3, CD4, CD25 and FoxP3 antibodies. (A) Dot plots of tregs from representative experiments. (B) CD25⁺/FoxP3⁺Frequency of T cells, expressed as Total CD3 across different treatment groups⁺Or CD3⁺CD4⁺Percentage of T cells (left panel) and intratumoral CD8/Treg ratio (right panel). Data are shown as mean ± SEM obtained from 2 independent experiments.

Figure 6 shows that vaccination with LmddA-LLO-ChHer2(ADXS31-164) can delay growth of breast cancer cell lines in the brain. Balb/c mice were immunized three times with ADXS31-164 or a control Listeria vaccine. Intracranial injection of EMT6-Luc cells (5,000) in anesthetized mice. (A) Ex vivo imaging of mice was performed on the indicated day using a Xenogen X-100CCD camera. (B) At a rate of per second per cm²The number of photons of the surface area is plotted against the pixel brightness; this is shown as the average radiation intensity. (C) EMT6-Luc cells, 4T1-Luc and NT-2 cell lines were tested for Her2/neu expression by Western blotting using anti-Her 2/neu antibodies. A2 cells (murine macrophage-like cell line) was used as a negative control.

Detailed Description

In one embodiment, provided herein are compositions and methods for preventing, treating, and vaccinating against Her2-neu antigen-expressing tumors and inducing immune responses against suboptimal epitopes of Her2-neu antigen while causing mutation avoidance. In another embodiment, the mutation is avoided because of epitope spreading. In yet another embodiment, the mutation avoidance is due to the chimeric nature of the antigen.

In another embodiment, provided herein is an immunogenic composition comprising a fusion polypeptide, wherein the fusion polypeptide comprises Her2/neu chimeric antigen fused to another polypeptide, wherein administration of the fusion protein to a subject having a Her 2/neu-expressing tumor prevents escape mutations in the tumor. In another embodiment, provided herein are recombinant listeria vaccine strains comprising an immunogenic composition.

In one embodiment, provided herein is a method of eliciting an enhanced immune response against Her-2/neu-expressing tumor growth or cancer in a non-human animal, the method comprising the step of administering a recombinant listeria comprising a nucleic acid molecule encoding a fusion polypeptide, wherein the fusion polypeptide comprises a Her2/neu chimeric antigen fused to an additional adjuvant polypeptide.

In another embodiment, provided herein is a method of preventing Her-2/neu-expressing tumor growth or cancer in a non-human animal, the method comprising the step of administering a recombinant listeria comprising a nucleic acid encoding a fusion polypeptide, wherein the fusion polypeptide comprises a Her2/neu chimeric antigen fused to an additional adjuvant polypeptide.

In one embodiment, provided herein is a method of treating Her-2/neu-expressing tumor growth or cancer in a non-human animal, the method comprising the step of administering a recombinant listeria comprising a nucleic acid molecule encoding a fusion polypeptide, wherein the fusion polypeptide comprises a Her2/neu chimeric antigen fused to an additional adjuvant polypeptide. In another embodiment, the non-human animal is a canine. In one embodiment, the canine is a dog.

In one embodiment, provided herein is a recombinant listeria vaccine strain comprising a nucleic acid molecule, wherein the nucleic acid molecule comprises a first open reading frame encoding a polypeptide, wherein the polypeptide comprises a Her2/neu chimeric antigen, wherein the nucleic acid molecule further comprises a second open reading frame encoding a metabolic enzyme, wherein the metabolic enzyme complements an endogenous gene that is not present in the chromosome of the recombinant listeria strain. In another embodiment, the recombinant listeria vaccine strain further comprises a nucleic acid molecule comprising a third open reading frame encoding a metabolic enzyme, wherein the metabolic enzyme complements an endogenous gene that is not present in the chromosome of the recombinant listeria strain.

In another embodiment, provided herein is a recombinant listeria vaccine strain comprising a nucleic acid molecule, wherein the nucleic acid molecule comprises a first open reading frame encoding a polypeptide, wherein the polypeptide comprises a Her2/neu chimeric antigen, wherein the nucleic acid molecule further comprises a second and a third open reading frame each encoding a metabolic enzyme, wherein the metabolic enzymes supplement endogenous genes that are not present in the chromosome of said recombinant listeria strain. In one embodiment, the nucleic acid molecule is integrated into the listeria genome. In another embodiment, the nucleic acid molecule is in a plasmid in a recombinant listeria vaccine strain. In one embodiment, the plasmid is stably maintained in the recombinant listeria vaccine strain in the absence of antibiotic selection. In another embodiment, the plasmid does not confer antibiotic resistance upon the recombinant listeria. In another embodiment, the recombinant listeria strain is attenuated. In another embodiment, the recombinant listeria is an attenuated auxotrophic strain. In another embodiment, the high metabolic burden imposed by the expression of the foreign antigen on a bacterium such as one of the present invention is also an important mechanism of attenuation.

In one embodiment, the attenuated strain is LmddA. In another embodiment, the strain exerts a strong adjuvant effect that is inherent to listeria-based vaccines. One manifestation of the adjuvant effect is a 5-fold decrease in the number of intratumoral tregs caused by irrelevant listeria or ADXS-31-164 vaccines (see figure 5 herein). In another embodiment, the LmddA vector expressing an unrelated antigen (HPV16E7) was also associated with a significant decrease in Treg frequency in tumors, likely as a result of an innate immune response.

In one embodiment, the invention provides nucleic acid sequences for cloning her-2-chimeras (see Table 1 of the examples herein).

Direct fusion by SOEing PCR method and using each isolated hHer-2/neu segment as template generates a Her-2/neu chimeric construct. The primers are shown in table 2 (see examples herein).

In one embodiment, LmddA-LLO-ChHer2(ADXS31-164) is generated according to the examples provided herein. In one embodiment, the chimeric Her2 gene (ChHer2) gene was excised from pAdv138 using XhoI and SpeI restriction enzymes and cloned in frame with a truncated, non-hemolytic LLO fragment in the Lmdd shuttle vector pAdv 134. In another embodiment, the sequences of the insert, LLO and hly promoter are confirmed by DNA sequencing analysis. In another embodiment, the plasmid is then electroporated into an electrically competent actA, dal, dat mutant listeria monocytogenes strain and LmddA and positive clones are selected on Brain Heart Infusion (BHI) agar plates containing streptomycin (250 μ g/ml). In another embodiment, a similar Listeria strain expressing a hHer2/neu (Lm-hHer2) fragment is used for comparative purposes. In another embodiment, an unrelated listeria construct (Lm-control) is included to account for the antigen-independent effects of listeria on the immune system. In another embodiment, the Lm-control is based on the same listeria platform as LmddA-LLO-ChHer2(ADXS31-164), but expresses a different antigen, such as HPV16-E7 or NY-ESO-1. In another embodiment, expression and secretion of the fusion protein from listeria is detected and confirmed using methods known in the art, including, but not limited to, immunoblotting, northern blotting, immunofluorescence, and the like. In another embodiment, each construct is passaged twice in vivo (see examples herein).

In one embodiment, the attenuated auxotrophic listeria vaccine strain LmddA-LLO-ChHer2(ADXS31-164) strain is based on a listeria vaccine vector that is attenuated by deletion of the causative gene actA and retains a plasmid for expression of Her2/neu in vivo and in vitro by supplementation with the dal gene. In one embodiment, ADXS31-164 expresses and secretes a chimeric Her2/neu protein fused to the first 441 amino acids of listeria lysate o (llo). In another embodiment, the construct utilizes an appropriate promoter and signal sequence or signal peptide known in the art to allow expression and secretion of the fusion protein. Some of these promoters and signal sequences include, but are not limited to, the listeria actA promoter and signal sequence, the hly promoter and signal sequence, the p60 promoter, and other promoters and signal sequences known in the art, some of which are described in U.S. patent No. 5,830,702, which is incorporated by reference herein in its entirety.

In one embodiment, ADXS31-164 exerts a strong and antigen-specific anti-tumor response in the transgenic animal with the ability to break HER2/neu tolerance in the transgenic animal (see example 2). In another embodiment, ADXS31-164 is capable of eliciting an anti-Her 2/neu specific immune response to human epitopes located in different domains of the target antigen (see example 2). In another embodiment, the ADXS31-164 strain is highly attenuated and has a better safety profile than the listeria vaccine of the previous generation, as it is cleared more rapidly from the spleen of immunized mice. In another embodiment, although ADXS31-164 is more attenuated, it is more effective than Lm-LLO-ChHer2 in preventing the appearance of spontaneous breast tumors in Her2/neu transgenic animals (see example 3).

In another embodiment, ADXS31-164 delays tumor onset in transgenic animals longer than Lm-LLO-ChHer2, Lm-LLO-ChHer2 being an antibiotic resistant and more pathogenic variant of the vaccine (see FIG. 3). In another embodiment, the ADXS31-164 strain is highly immunogenic, is capable of breaking tolerance to Her2/neu autoantigen in Her2/neu transgenic animals and prevents tumor formation in transgenic animals. In another embodiment, ADXS31-164 results in a significant decrease in intratumoral T regulatory cells (tregs) (see example 5 herein). In another embodiment, a lower frequency of tregs in tumors treated with the LmddA vaccine resulted in an increased intratumoral CD8/Treg ratio, suggesting that a more favorable tumor microenvironment could be obtained after immunization with the LmddA vaccine (see example 5 herein). In another embodiment, the use of the chimeric antigen does not produce escape mutations, indicating that the tumor is free of mutations and loses a therapeutically effective response to treatment with the neoantigen (see example 6 herein). In another embodiment, peripheral immunization with ADXS31-164 delays the growth of a metastatic breast cancer cell line in the brain (see example 7 herein). In another embodiment, peripheral administration of ADXS31-164 to the central nervous system demonstrates that the LmddA based vaccine provides a potential treatment of CNS tumors (see example 7 herein).

Canine osteosarcoma is a cancer of the long (leg) bone, which is the major killer in dogs over 10 years of age. The standard treatment is amputation immediately after diagnosis, followed by chemotherapy. However, cancer always metastasizes to the lungs. With chemotherapy, dogs survive approximately 18 months, compared to 6-12 months without treatment. The HER2 antigen is thought to be present in up to 50% of osteosarcomas. In one embodiment, the vaccine ADXS31-164 provided herein generates an immune challenge to cells expressing this antigen and treats human breast cancer.

Dogs histologically diagnosed with osteosarcoma showed evidence of HER2/neu expression by malignant cells. Thus, in one embodiment, provided herein is a method of treating canine osteosarcoma, the method comprising the steps of: a vaccine composition comprising ADXS31-164 and an adjuvant is administered to a canine having an osteosarcoma, thereby treating the canine osteosarcoma (see example 8 herein). In another embodiment, provided herein is a method of treating canine osteosarcoma, the method comprising the steps of: a vaccine composition consisting of ADXS31-164 and an adjuvant is administered to a canine having an osteosarcoma, thereby treating the canine osteosarcoma (see example 8 herein). In another embodiment, provided herein is a method of treating canine osteosarcoma, the method comprising the steps of: a vaccine composition consisting of ADXS31-164 is administered to canines with osteosarcoma, thereby treating the canine osteosarcoma (see example 8).

In another embodiment, the vaccines of the present invention are administered to canines using various methods known in the art, including intravenous, intraperitoneal, intramuscular, oral, intranasal, and the like, as understood by those skilled in the art. In another embodiment, the vaccine is administered in a manner that delivers the vaccine at or near the basal cells of the tongue of the canine subject.

In another embodiment, administration may be a single event, or multiple booster doses may be administered at different time intervals to increase the immune response. In the case of administration to puppies, a typical administration regimen is an initial administration at, for example, 2-3 weeks of age, followed by booster doses at 4 and 6 weeks. In another embodiment, administration is prophylactic, i.e., before tumor growth occurs or is suspected of occurring, but may also follow that fact, i.e., therapeutically, e.g., after the occurrence of disease symptoms associated with tumor or cancer growth.

In one embodiment, the Lm-LLO-ChHer2 strain is Lm-LLO-138.

In one embodiment, recombinant attenuated, antibiotic-free listeria expressing a chimeric antigen can be used for the prevention and treatment of cancer or solid tumors, as exemplified herein. In another embodiment, the tumor is a Her2/neu positive tumor. In another embodiment, the cancer is a Her 2/neu-expressing cancer. In another embodiment, the cancer is breast cancer, Central Nervous System (CNS) cancer, head and neck cancer, osteosarcoma, canine osteosarcoma, or any cancer known in the art. In another embodiment, the tumor is a bone tumor, a breast tumor, a head and neck tumor, or any other antigen expressing tumor known in the art. In another embodiment, recombinant listeria expressing chimeric Her2/neu can be used as a therapeutic vaccine for the treatment of Her2/neu overexpressing solid tumors. In another embodiment, the Her2/neu chimeric antigens provided herein can be used to treat Her 2/neu-expressing tumors and prevent escape mutations thereof. In another embodiment, the term "escape mutation" refers to a tumor mutation that loses a therapeutically effective response to treatment.

In one embodiment, provided herein are nucleic acid molecules comprising a first open reading frame encoding an immunogenic composition, wherein the nucleic acid molecule is located in a recombinant listeria vaccine strain. In another embodiment, the nucleic acid molecules provided herein are used to transform listeria to obtain a recombinant listeria. In another embodiment, the nucleic acids provided herein are free of a disease causing gene. In another embodiment, the nucleic acid molecule integrated into the listeria genome carries a non-functional virulence gene. In another embodiment, the virulence gene is mutated in a recombinant listeria. In yet another embodiment, the nucleic acid molecule is used to inactivate an endogenous gene present in the listeria genome. In yet another embodiment, the disease-causing gene is an ActA gene. In another embodiment, the disease-causing gene is a PrfA gene. As understood by those skilled in the art, the virulence gene can be any gene known in the art that is associated with the pathogenicity of recombinant listeria.

In one embodiment, the listeria strain is free of metabolic genes, virulence genes, etc., in its chromosome. In another embodiment, the listeria strain is free of metabolic genes, virulence genes, etc., in its chromosome and any episomal genetic elements. In another embodiment, the pathogenic strain has no metabolic genes, pathogenic genes, etc. in its genome. In one embodiment, the disease-causing gene is mutated in the chromosome. In another embodiment, the disease causing gene is deleted from the chromosome. Each possibility represents a separate embodiment of the invention.

In another embodiment, the nucleic acids and plasmids provided herein do not confer antibiotic resistance upon the recombinant listeria.

In another embodiment, a "nucleic acid molecule" refers to a plasmid. In another embodiment, the term refers to an integrating vector. In another embodiment, the term refers to a plasmid comprising an integrating vector. In another embodiment, the integration vector is a site-specific integration vector. In another embodiment, the nucleic acid molecules of the methods and compositions of the present invention are comprised of any type of nucleotide known in the art. Each possibility represents a separate embodiment of the invention.

In another embodiment, "metabolic enzyme" refers to an enzyme involved in the synthesis of nutrients required by the host bacterium. In another embodiment, the term refers to an enzyme required for the synthesis of a nutrient required by the host bacterium. In another embodiment, the term refers to an enzyme involved in the synthesis of nutrients utilized by the host bacterium. In another embodiment, the term refers to an enzyme involved in the synthesis of nutrients required for continued growth of the host bacterium. In another embodiment, the enzyme is required for nutrient synthesis. Each possibility represents a separate embodiment of the invention.

In another embodiment, "stably maintained" refers to the nucleic acid molecule or plasmid remaining without selection (e.g., antibiotic selection) for 10 passages without detectable loss. In another embodiment, the period is 15 generations. In another embodiment, the period is 20 generations. In another embodiment, the cycle is 25 generations. In another embodiment, the period is 30 generations. In another embodiment, the period is 40 generations. In another embodiment, the period is 50 generations. In another embodiment, the cycle is 60 generations. In another embodiment, the cycle is 80 generations. In another embodiment, the cycle is 100 generations. In another embodiment, the period is 150 generations. In another embodiment, the cycle is 200 generations. In another embodiment, the cycle is 300 generations. In another embodiment, the period is 500 generations. In another embodiment, the period is greater than generation. In another embodiment, the nucleic acid molecule or plasmid is stably maintained in vitro (e.g., in culture). In another embodiment, the nucleic acid molecule or plasmid is stably maintained in vivo. In another embodiment, the nucleic acid molecule or plasmid is stably maintained both in vitro and in vitro. Each possibility represents a separate embodiment of the invention.

In one embodiment, the invention provides recombinant listeria strains that express an antigen. The invention also provides recombinant peptides comprising a Listeria Lysenin (LLO) protein fragment or a PEST peptide or an ActA N-terminal fragment fused to Her-2 chimeric protein or a fragment thereof, vaccines and immunogenic compositions comprising the recombinant peptides, and methods of inducing an immune response against Her-2 and treating Her-2 expressing tumors and vaccinating against them comprising the recombinant peptides.

In another embodiment, the recombinant listeria strains of the present invention have been passaged through an animal host. In another embodiment, the passaging maximizes the efficacy of the strain as a vaccine vector. In another embodiment, the listeria strain is passage-stabilized for immunogenicity. In another embodiment, passaging stabilizes the pathogenicity of the listeria strain. In another embodiment, passaging increases the immunogenicity of the listeria strain. In another embodiment, passaging increases the pathogenicity of the listeria strain. In another embodiment, passaging removes unstable sublines of listeria strain. In another embodiment, passaging reduces the prevalence of unstable sublines of listeria strains. In another embodiment, the listeria strain contains a genomic insertion of a gene encoding a recombinant peptide comprising an antigen. In another embodiment, the listeria strain harbors a plasmid comprising a gene encoding a recombinant peptide comprising an antigen. In another embodiment, passaging is performed by any other method known in the art.

In one embodiment, the polypeptide provided herein is a fusion protein comprising an additional polypeptide selected from the group consisting of: a) a non-hemolytic LLO protein or an N-terminal fragment, b) a PEST sequence, or c) an ActA N-terminal fragment, and further wherein the further polypeptide is fused to an Her2/neu chimeric antigen. In another embodiment, the additional polypeptide is functional. In another embodiment, a fragment of the additional polypeptide is immunogenic. In another embodiment, the additional polypeptide is immunogenic.

In another embodiment, the polypeptide provided herein is a fusion protein comprising a non-hemolytic LLO protein or an N-terminal fragment fused to Her2/neu chimeric antigen. In another embodiment, the fusion protein of the methods and compositions of the invention comprises an ActA sequence from a listeria organism. The ActA protein and fragments thereof increase antigen presentation and immunity in a similar manner to LLO.

In another embodiment of the methods and compositions of the present invention, the fusion protein comprises Her2/neu antigen and an additional adjuvant polypeptide. In one embodiment, the additional polypeptide is a nonhemolytic LLO protein or fragment thereof (see examples herein). In another embodiment, the additional polypeptide is a PEST sequence. In another embodiment, the additional polypeptide is an ActA protein or fragment thereof. The ActA protein and fragments thereof increase antigen presentation and immunity in a similar manner to LLO.

In another embodiment, the additional polypeptide of the methods and compositions of the invention is a Listerial Lysin (LLO) peptide. In another embodiment, the additional polypeptide is an ActA peptide. In another embodiment, the additional polypeptide is a peptide of PEST-like sequence. In another embodiment, the additional polypeptide is any other peptide capable of increasing the immunogenicity of the antigenic peptide. Each possibility represents a separate embodiment of the invention.

Fusion proteins comprising Her2/neu chimeric antigen can be prepared by any suitable method, including, for example, cloning and restriction enzyme digestion (restriction) of the appropriate sequence or by direct chemical synthesis by the methods described below. Alternatively, the subsequence may be cloned and the appropriate subsequence cleaved using an appropriate restriction enzyme. The fragments can then be ligated to produce the desired DNA sequence. In one embodiment, DNA encoding the antigen is generated using a DNA amplification method, such as Polymerase Chain Reaction (PCR). First, segments of native DNA on either side of the new end are amplified separately. The 5 'end of one amplified sequence encodes a peptide linker, while the 3' end of the other amplified sequence also encodes a peptide linker. Since the 5 'end of the first fragment is complementary to the 3' end of the second fragment, both fragments (after partial purification, e.g., on LMP agarose) can be used as overlapping templates in the third PCR reaction. The amplification sequence contains a codon, a stretch on the carboxy side of the open site (now forming an amino sequence), a linker and a sequence on the amino side of the open site (now forming a carboxy sequence). The antigen is ligated to a plasmid. Each representing a separate embodiment of the invention.

The results of the present invention demonstrate that administration of the compositions of the present invention is effective in inducing the formation of antigen-specific T cells (e.g., cytotoxic T cells) that recognize and kill tumor cells (see examples herein).

In one embodiment, the invention provides a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to a Her-2 chimeric protein or to a fragment thereof. In one embodiment, the invention provides a recombinant polypeptide consisting of an N-terminal fragment of an LLO protein fused to a Her-2 chimeric protein or to a fragment thereof.

In another embodiment, the Her-2 chimeric protein of the methods and compositions of the invention is a human Her-2 chimeric protein. In another embodiment, the Her-2 protein is a mouse Her-2 chimeric protein. In another embodiment, the Her-2 protein is a rat Her-2 chimeric protein. In another embodiment, the Her-2 protein is a primate Her-2 chimeric protein. In another embodiment, the Her-2 protein is a Her-2 chimeric protein of a human or any other animal species or a combination thereof known in the art. Each possibility represents a separate embodiment of the invention.

In another embodiment, the Her-2 protein is a protein designated "HER-2/neu", "Erbb 2", "v-erb-b 2", "c-erb-b 2", "neu", or "cNeu". Each possibility represents a separate embodiment of the invention.

In one embodiment, the Her2-neu chimeric protein has two extracellular and one intracellular fragments of Her2/neu antigen displaying MHC-class I epitope clusters of oncogenes, while in another embodiment, the chimeric protein has 3H 2Dq and at least 17 localized human MHC-class I epitopes of Her2/neu antigen (fragments EC1, EC2 and IC1) (see figure 1 and example 1). In another embodiment, the chimeric protein has at least 13 human MHC-class I epitopes (fragments EC2 and IC1) located. In another embodiment, the chimeric protein has at least 14 human MHC-class I epitopes (fragments EC1 and IC1) mapped. In another embodiment, the chimeric protein has at least 9 localized human MHC-class I epitopes (fragments EC1 and IC 2). In another embodiment, the Her2-neu chimeric protein is fused to a non-hemolytic listeria lysate o (llo). In another embodiment, the Her2-neu chimeric protein is fused to the first amino acid 441 of the listeria monocytogenes listeriolysin o (llo) protein and is expressed and secreted by the listeria monocytogenes attenuated auxotrophic strain LmddA. In another embodiment, the expression and secretion of the fusion protein tLLO-ChHer2 from an attenuated auxotrophic strain expressing chimeric Her2/neu antigen/LLO fusion protein provided herein is comparable to the expression and secretion of Lm-LLO-ChHer2 in cell culture supernatants of TCA pellets after 8 hours of in vitro growth (see fig. 1B).

In one embodiment, the animal is a non-immunized animal injected with an unrelated Listeria vaccineOr no CTL activity was detected in the mice (see fig. 2A). In yet another embodiment, the attenuated auxotrophic strain (ADXS31-164) provided herein is capable of stimulating secretion of IFN- γ from splenocytes from a wild-type FVB/N mouse (FIG. 2B).

In another embodiment, the metabolic enzyme of the methods and compositions provided herein is an amino acid metabolizing enzyme, and in another embodiment, the metabolic enzyme is an alanine racemase. In another embodiment, the metabolic enzyme is a D-amino acid transferase enzyme. In another embodiment, the metabolic enzyme catalyzes the formation of an amino acid for cell wall synthesis in a recombinant listeria strain, while in another embodiment, the metabolic enzyme is an alanine racemase enzyme.

In another embodiment, the gene encoding the metabolic enzyme is expressed under the control of the listeria p60 promoter. In another embodiment, an inlA (encoding an internalization protein) promoter is used. In another embodiment, the hly promoter is used. In another embodiment, an ActA promoter is used. In another embodiment, the integrase gene is expressed under the control of any other gram-positive promoter. In another embodiment, the gene encoding the metabolic enzyme is expressed under the control of any other promoter functional in listeria. One skilled in the art will appreciate that other promoters or polycistronic expression cassettes may be used to drive expression of a gene. Each possibility represents a separate embodiment of the invention.

In another embodiment, the Her-2 chimeric protein is encoded by the following nucleic acid sequence as set forth in SEQ ID NO 1

gagacccacctggacatgctccgccacctctaccagggctgccaggtggtgcagggaaacctggaactcacctacctgcccaccaatgccagcctgtccttcctgcaggatatccaggaggtgcagggctacgtgctcatcgctcacaaccaagtgaggcaggtcccactgcagaggctgcggattgtgcgaggcacccagctctttgaggacaactatgccctggccgtgctagacaatggagacccgctgaacaataccacccctgtcacaggggcctccccaggaggcctgcgggagctgcagcttcgaagcctcacagagatcttgaaaggaggggtcttgatccagcggaacccccagctctgctaccaggacacgattttgtggaagaatatccaggagtttgctggctgcaagaagatctttgggagcctggcatttctgccggagagctttgatggggacccagcctccaacactgccccgctccagccagagcagctccaagtgtttgagactctggaagagatcacaggttacctatacatctcagcatggccggacagcctgcctgacctcagcgtcttccagaacctgcaagtaatccggggacgaattctgcacaatggcgcctactcgctgaccctgcaagggctgggcatcagctggctggggctgcgctcactgagggaactgggcagtggactggccctcatccaccataacacccacctctgcttcgtgcacacggtgccctgggaccagctctttcggaacccgcaccaagctctgctccacactgccaaccggccagaggacgagtgtgtgggcgagggcctggcctgccaccagctgtgcgcccgagggcagcagaagatccggaagtacacgatgcggagactgctgcaggaaacggagctggtggagccgctgacacctagcggagcgatgcccaaccaggcgcagatgcggatcctgaaagagacggagctgaggaaggtgaaggtgcttggatctggcgcttttggcacagtctacaagggcatctggatccctgatggggagaatgtgaaaattccagtggccatcaaagtgttgagggaaaacacatcccccaaagccaacaaagaaatcttagacgaagcatacgtgatggctggtgtgggctccccatatgtctcccgccttctgggcatctgcctgacatccacggtgcagctggtgacacagcttatgccctatggctgcctcttagactaa(SEQ ID NO:1)。

In another embodiment, the Her-2 chimeric protein has the following sequence:

ETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKNIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLD(SEQ ID NO:2)。

in one embodiment, Her2 chimeric protein or fragment thereof of the methods and compositions provided herein does not include a signal sequence thereof. In another embodiment, due to the high hydrophobicity of the signal sequence, omission of the signal sequence enables successful expression of the Her2 fragment in listeria. Each possibility represents a separate embodiment of the invention.

In another embodiment, fragments of Her2 chimeric proteins of the methods and compositions of the invention do not include their transmembrane domain (TM). In one embodiment, omission of the TM enables successful expression of the Her-2 fragment in listeria due to its high hydrophobicity. Each possibility represents a separate embodiment of the invention.

In one embodiment, the nucleic acid sequence of the rat-Her 2/neu gene is CCGGAATCGCGGGCACCCAAGTGTGTACCGGCACAGACATGAAGTTGCGGCTCCCTGCCAGTCCTGAGACCCACCTGGACATGCTCCGCCACCTGTACCAGGGCTGTCAGGTAGTGCAGGGCAACTTGGAGCTTACCTACGTGCCTGCCAATGCCAGCCTCTCATTCCTGCAGGACATCCAGGAAGTTCAGGGTTACATGCTCATCGCTCACAACCAGGTGAAGCGCGTCCCACTGCAAAGGCTGCGCATCGTGAGAGGGACCCAGCTCTTTGAGGACAAGTATGCCCTGGCTGTGCTAGACAACCGAGATCCTCAGGACAATGTCGCCGCCTCCACCCCAGGCAGAACCCCAGAGGGGCTGCGGGAGCTGCAGCTTCGAAGTCTCACAGAGATCCTGAAGGGAGGAGTTTTGATCCGTGGGAACCCTCAGCTCTGCTACCAGGACATGGTTTTGTGGAAGGACGTCTTCCGCAAGAATAACCAACTGGCTCCTGTCGATATAGACACCAATCGTTCCCGGGCCTGTCCACCTTGTGCCCCCGCCTGCAAAGACAATCACTGTTGGGGTGAGAGTCCGGAAGACTGTCAGATCTTGACTGGCACCATCTGTACCAGTGGTTGTGCCCGGTGCAAGGGCCGGCTGCCCACTGACTGCTGCCATGAGCAGTGTGCCGCAGGCTGCACGGGCCCCAAGCATTCTGACTGCCTGGCCTGCCTCCACTTCAATCATAGTGGTATCTGTGAGCTGCACTGCCCAGCCCTCGTCACCTACAACACAGACACCTTTGAGTCCATGCACAACCCTGAGGGTCGCTACACCTTTGGTGCCAGCTGCGTGACCACCTGCCCCTACAACTACCTGTCTACGGAAGTGGGATCCTGCACTCTGGTGTGTCCCCCGAATAACCAAGAGGTCACAGCTGAGGACGGAACACAGCGTTGTGAGAAATGCAGCAAGCCCTGTGCTCGAGTGTGCTATGGTCTGGGCATGGAGCACCTTCGAGGGGCGAGGGCCATCACCAGTGACAATGTCCAGGAGTTTGATGGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTTTGCCGGAGAGCTTTGATGGGGACCCCTCCTCCGGCATTGCTCCGCTGAGGCCTGAGCAGCTCCAAGTGTTCGAAACCCTGGAGGAGATCACAGGTTACCTGTACATCTCAGCATGGCCAGACAGTCTCCGTGACCTCAGTGTCTTCCAGAACCTTCGAATCATTCGGGGACGGATTCTCCACGATGGCGCGTACTCATTGACACTGCAAGGCCTGGGGATCCACTCGCTGGGGCTGCGCTCACTGCGGGAGCTGGGCAGTGGATTGGCTCTGATTCACCGCAACGCCCATCTCTGCTTTGTACACACTGTACCTTGGGACCAGCTCTTCCGGAACCCACATCAGGCCCTGCTCCACAGTGGGAACCGGCCGGAAGAGGATTGTGGTCTCGAGGGCTTGGTCTGTAACTCACTGTGTGCCCACGGGCACTGCTGGGGGCCAGGGCCCACCCAGTGTGTCAACTGCAGTCATTTCCTTCGGGGCCAGGAGTGTGTGGAGGAGTGCCGAGTATGGAAGGGGCTCCCCCGGGAGTATGTGAGTGACAAGCGCTGTCTGCCGTGTCACCCCGAGTGTCAGCCTCAAAACAGCTCAGAGACCTGCTTTGGATCGGAGGCTGATCAGTGTGCAGCCTGCGCCCACTACAAGGACTCGTCCTCCTGTGTGGCTCGCTGCCCCAGTGGTGTGAAACCGGACCTCTCCTACATGCCCATCTGGAAGTACCCGGATGAGGAGGGCATATGCCAGCCGTGCCCCATCAACTGCACCCACTCCTGTGTGGATCTGGATGAACGAGGCTGCCCAGCAGAGCAGAGAGCCAGCCCGGTGACATTCATCATTGCAACTGTAGTGGGCGTCCTGCTGTTCCTGATCTTAGTGGTGGTCGTTGGAATCCTAATCAAACGAAGGAGACAGAAGATCCGGAAGTATACGATGCGTAGGCTGCTGCAGGAAACTGAGTTAGTGGAGCCGCTGACGCCCAGCGGAGCAATGCCCAACCAGGCTCAGATGCGGATCCTAAAAGAGACGGAGCTAAGGAAGGTGAAGGTGCTTGGATCAGGAGCTTTTGGCACTGTCTACAAGGGCATCTGGATCCCAGATGGGGAGAATGTGAAAATCCCCGTGGCTATCAAGGTGTTGAGAGAAAACACATCTCCTAAAGCCAACAAAGAAATTCTAGATGAAGCGTATGTGATGGCTGGTGTGGGTTCTCCGTATGTGTCCCGCCTCCTGGGCATCTGCCTGACATCCACAGTACAGCTGGTGACACAGCTTATGCCCTACGGCTGCCTTCTGGACCATGTCCGAGAACACCGAGGTCGCCTAGGCTCCCAGGACCTGCTCAACTGGTGTGTTCAGATTGCCAAGGGGATGAGCTACCTGGAGGACGTGCGGCTTGTACACAGGGACCTGGCTGCCCGGAATGTGCTAGTCAAGAGTCCCAACCACGTCAAGATTACAGATTTCGGGCTGGCTCGGCTGCTGGACATTGATGAGACAGAGTACCATGCAGATGGGGGCAAGGTGCCCATCAAATGGATGGCATTGGAATCTATTCTCAGACGCCGGTTCACCCATCAGAGTGATGTGTGGAGCTATGGAGTGACTGTGTGGGAGCTGATGACTTTTGGGGCCAAACCTTACGATGGAATCCCAGCCCGGGAGATCCCTGATTTGCTGGAGAAGGGAGAACGCCTACCTCAGCCTCCAATCTGCACCATTGATGTCTACATGATTATGGTCAAATGTTGGATGATTGACTCTGAATGTCGCCCGAGATTCCGGGAGTTGGTGTCAGAATTTTCACGTATGGCGAGGGACCCCCAGCGTTTTGTGGTCATCCAGAACGAGGACTTGGGCCCATCCAGCCCCATGGACAGTACCTTCTACCGTTCACTGCTGGAAGATGATGACATGGGTGACCTGGTAGACGCTGAAGAGTATCTGGTGCCCCAGCAGGGATTCTTCTCCCCGGACCCTACCCCAGGCACTGGGAGCACAGCCCATAGAAGGCACCGCAGCTCGTCCACCAGGAGTGGAGGTGGTGAGCTGACACTGGGCCTGGAGCCCTCGGAAGAAGGGCCCCCCAGATCTCCACTGGCTCCCTCGGAAGGGGCTGGCTCCGATGTGTTTGATGGTGACCTGGCAATGGGGGTAACCAAAGGGCTGCAGAGCCTCTCTCCACATGACCTCAGCCCTCTACAGCGGTACAGCGAGGACCCCACATTACCTCTGCCCCCCGAGACTGATGGCTATGTTGCTCCCCTGGCCTGCAGCCCCCAGCCCGAGTATGTGAACCAATCAGAGGTTCAGCCTCAGCCTCCTTTAACCCCAGAGGGTCCTCTGCCTCCTGTCCGGCCTGCTGGTGCTACTCTAGAAAGACCCAAGACTCTCTCTCCTGGGAAGAATGGGGTTGTCAAAGACGTTTTTGCCTTCGGGGGTGCTGTGGAGAACCCTGAATACTTAGTACCGAGAGAAGGCACTGCCTCTCCGCCCCACCCTTCTCCTGCCTTCAGCCCAGCCTTTGACAACCTCTATTACTGGGACCAGAACTCATCGGAGCAGGGGCCTCCACCAAGTAACTTTGAAGGGACCCCCACTGCAGAGAACCCTGAGTACCTAGGCCTGGATGTACCTGTA (SEQ ID NO: 45).

In one embodiment, the nucleic acid sequence encoding the rat/her 2/neu EC1 fragment is: CCCAGGCAGAACCCCAGAGGGGCTGCGGGAGCTGCAGCTTCGAAGTCTCACAGAGATCCTGAAGGGAGGAGTTTTGATCCGTGGGAACCCTCAGCTCTGCTACCAGGACATGGTTTTGTGGAAGGACGTCTTCCGCAAGAATAACCAACTGGCTCCTGTCGATATAGACACCAATCGTTCCCGGGCCTGTCCACCTTGTGCCCCCGCCTGCAAAGACAATCACTGTTGGGGTGAGAGTCCGGAAGACTGTCAGATCTTGACTGGCACCATCTGTACCAGTGGTTGTGCCCGGTGCAAGGGCCGGCTGCCCACTGACTGCTGCCATGAGCAGTGTGCCGCAGGCTGCACGGGCCCCAAGCA (SEQ ID NO: 46).

In another embodiment, the nucleic acid sequence encoding the rat her2/neu EC2 fragment is: GGTCACAGCTGAGGACGGAACACAGCGTTGTGAGAAATGCAGCAAGCCCTGTGCTCGAGTGTGCTATGGTCTGGGCATGGAGCACCTTCGAGGGGCGAGGGCCATCACCAGTGACAATGTCCAGGAGTTTGATGGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTTTGCCGGAGAGCTTTGATGGGGACCCCTCCTCCGGCATTGCTCCGCTGAGGCCTGAGCAGCTCCAAGTGTTCGAAACCCTGGAGGAGATCACAGGTTACCTGTACATCTCAGCATGGCCAGACAGTCTCCGTGACCTCAGTGTCTTCCAGAACCTTCGAATCATTCGGGGACGGATTCTCCACGATGGCGCGTACTCATTGACACTGCAAGGCCTGGGGATCCACTCGCTGGGGCTGCGCTCACTGCGGGAGCTGGGCAGTGGATTGGCTCTGATTCACCGCAACGCCCATCTCTGCTTTGTACACACTGTACCTTGGGACCAGCTCTTCCGGAACCCACATCAGGCCCTGCTCCACAGTGGGAACCGGCCGGAAGAGGATTGTGGTCTCGAGGGCTTGGTCTGTAACTCACTGTGTGCCCACGGGCACTGCTGGGGGCCAGGGCCCACCCA (SEQ ID NO: 47).

In another embodiment, the nucleic acid sequence encoding rat her2/neu IC1 fragment is:

CGCCCAGCGGAGCAATGCCCAACCAGGCTCAGATGCGGATCCTAAAAGAGACGGAGCTAAGGAAGGTGAAGGTGCTTGGATCAGGAGCTTTTGGCACTGTCTACAAGGGCATCTGGATCCCAGATGGGGAGAATGTGAAAATCCCCGTGGCTATCAAGGTGTTGAGAGAAAACACATCTCCTAAAGCCAACAAAGAAATTCTAGATGAAGCGTATGTGATGGCTGGTGTGGGTTCTCCGTATGTGTCCCGCCTCCTGGGCATCTGCCTGACATCCACAGTACAGCTGGTGACACAGCTTATGCCCTACGGCTGCCTTCTGGACCATGTCCGAGAACACCGAGGTCGCCTAGGCTCCCAGGACCTGCTCAACTGGTGTGTTCAGATTGCCAAGGGGATGAGCTACCTGGAGGACGTGCGGCTTGTACACAGGGACCTGGCTGCCCGGAATGTGCTAGTCAAGAGTCCCAACCACGTCAAGATTACAGATTTCGGGCTGGCTCGGCTGCTGGACATTGATGAGACAGAGTACCATGCAGATGGGGGCAAGGTGCCCATCAAATGGATGGCATTGGAATCTATTCTCAGACGCCGGTTCACCCATCAGAGTGATGTGTGGAGCTATGGAGTGACTGTGTGGGAGCTGATGACTTTTGGGGCCAAACCTTACGATGGAATCCCAGCCCGGGAGATCCCTGATTTGCTGGAGAAGGGAGAACGCCTACCTCAGCCTCCAATCTGCACCATTGATGTCTACATGATTATGGTCAAATGTTGGATGATTGACTCTGAATGTCGCCCGAGATTCCGGGAGTTGGTGTCAGAATTTTCACGTATGGCGAGGGACCCCCAGCGTTTTGTGGTCATCCAGAACGAGGACTTGGGCCCATCCAGCCCCATGGACAGTACCTTCTACCGTTCACTGCTGGAA(SEQ ID NO:48)。

in one embodiment, the nucleic acid sequence of the human Her2/neu gene is:

ATGGAGCTGGCGGCCTTGTGCCGCTGGGGGCTCCTCCTCGCCCTCTTGCCCCCCGGAGCCGCGAGCACCCAAGTGTGCACCGGCACAGACATGAAGCTGCGGCTCCCTGCCAGTCCCGAGACCCACCTGGACATGCTCCGCCACCTCTACCAGGGCTGCCAGGTGGTGCAGGGAAACCTGGAACTCACCTACCTGCCCACCAATGCCAGCCTGTCCTTCCTGCAGGATATCCAGGAGGTGCAGGGCTACGTGCTCATCGCTCACAACCAAGTGAGGCAGGTCCCACTGCAGAGGCTGCGGATTGTGCGAGGCACCCAGCTCTTTGAGGACAACTATGCCCTGGCCGTGCTAGACAATGGAGACCCGCTGAACAATACCACCCCTGTCACAGGGGCCTCCCCAGGAGGCCTGCGGGAGCTGCAGCTTCGAAGCCTCACAGAGATCTTGAAAGGAGGGGTCTTGATCCAGCGGAACCCCCAGCTCTGCTACCAGGACACGATTTTGTGGAAGGACATCTTCCACAAGAACAACCAGCTGGCTCTCACACTGATAGACACCAACCGCTCTCGGGCCTGCCACCCCTGTTCTCCGATGTGTAAGGGCTCCCGCTGCTGGGGAGAGAGTTCTGAGGATTGTCAGAGCCTGACGCGCACTGTCTGTGCCGGTGGCTGTGCCCGCTGCAAGGGGCCACTGCCCACTGACTGCTGCCATGAGCAGTGTGCTGCCGGCTGCACGGGCCCCAAGCACTCTGACTGCCTGGCCTGCCTCCACTTCAACCACAGTGGCATCTGTGAGCTGCACTGCCCAGCCCTGGTCACCTACAACACAGACACGTTTGAGTCCATGCCCAATCCCGAGGGCCGGTATACATTCGGCGCCAGCTGTGTGACTGCCTGTCCCTACAACTACCTTTCTACGGACGTGGGATCCTGCACCCTCGTCTGCCCCCTGCACAACCAAGAGGTGACAGCAGAGGATGGAACACAGCGGTGTGAGAAGTGCAGCAAGCCCTGTGCCCGAGTGTGCTATGGTCTGGGCATGGAGCACTTGCGAGAGGTGAGGGCAGTTACCAGTGCCAATATCCAGGAGTTTGCTGGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTCTGCCGGAGAGCTTTGATGGGGACCCAGCCTCCAACACTGCCCCGCTCCAGCCAGAGCAGCTCCAAGTGTTTGAGACTCTGGAAGAGATCACAGGTTACCTATACATCTCAGCATGGCCGGACAGCCTGCCTGACCTCAGCGTCTTCCAGAACCTGCAAGTAATCCGGGGACGAATTCTGCACAATGGCGCCTACTCGCTGACCCTGCAAGGGCTGGGCATCAGCTGGCTGGGGCTGCGCTCACTGAGGGAACTGGGCAGTGGACTGGCCCTCATCCACCATAACACCCACCTCTGCTTCGTGCACACGGTGCCCTGGGACCAGCTCTTTCGGAACCCGCACCAAGCTCTGCTCCACACTGCCAACCGGCCAGAGGACGAGTGTGTGGGCGAGGGCCTGGCCTGCCACCAGCTGTGCGCCCGAGGGCACTGCTGGGGTCCAGGGCCCACCCAGTGTGTCAACTGCAGCCAGTTCCTTCGGGGCCAGGAGTGCGTGGAGGAATGCCGAGTACTGCAGGGGCTCCCCAGGGAGTATGTGAATGCCAGGCACTGTTTGCCGTGCCACCCTGAGTGTCAGCCCCAGAATGGCTCAGTGACCTGTTTTGGACCGGAGGCTGACCAGTGTGTGGCCTGTGCCCACTATAAGGACCCTCCCTTCTGCGTGGCCCGCTGCCCCAGCGGTGTGAAACCTGACCTCTCCTACATGCCCATCTGGAAGTTTCCAGATGAGGAGGGCGCATGCCAGCCTTGCCCCATCAACTGCACCCACTCCTGTGTGGACCTGGATGACAAGGGCTGCCCCGCCGAGCAGAGAGCCAGCCCTCTGACGTCCATCGTCTCTGCGGTGGTTGGCATTCTGCTGGTCGTGGTCTTGGGGGTGGTCTTTGGGATCCTCATCAAGCGACGGCAGCAGAAGATCCGGAAGTACACGATGCGGAGACTGCTGCAGGAAACGGAGCTGGTGGAGCCGCTGACACCTAGCGGAGCGATGCCCAACCAGGCGCAGATGCGGATCCTGAAAGAGACGGAGCTGAGGAAGGTGAAGGTGCTTGGATCTGGCGCTTTTGGCACAGTCTACAAGGGCATCTGGATCCCTGATGGGGAGAATGTGAAAATTCCAGTGGCCATCAAAGTGTTGAGGGAAAACACATCCCCCAAAGCCAACAAAGAAATCTTAGACGAAGCATACGTGATGGCTGGTGTGGGCTCCCCATATGTCTCCCGCCTTCTGGGCATCTGCCTGACATCCACGGTGCAGCTGGTGACACAGCTTATGCCCTATGGCTGCCTCTTAGACCATGTCCGGGAAAACCGCGGACGCCTGGGCTCCCAGGACCTGCTGAACTGGTGTATGCAGATTGCCAAGGGGATGAGCTACCTGGAGGATGTGCGGCTCGTACACAGGGACTTGGCCGCTCGGAACGTGCTGGTCAAGAGTCCCAACCATGTCAAAATTACAGACTTCGGGCTGGCTCGGCTGCTGGACATTGACGAGACAGAGTACCATGCAGATGGGGGCAAGGTGCCCATCAAGTGGATGGCGCTGGAGTCCATTCTCCGCCGGCGGTTCACCCACCAGAGTGATGTGTGGAGTTATGGTGTGACTGTGTGGGAGCTGATGACTTTTGGGGCCAAACCTTACGATGGGATCCCAGCCCGGGAGATCCCTGACCTGCTGGAAAAGGGGGAGCGGCTGCCCCAGCCCCCCATCTGCACCATTGATGTCTACATGATCATGGTCAAATGTTGGATGATTGACTCTGAATGTCGGCCAAGATTCCGGGAGTTGGTGTCTGAATTCTCCCGCATGGCCAGGGACCCCCAGCGCTTTGTGGTCATCCAGAATGAGGACTTGGGCCCAGCCAGTCCCTTGGACAGCACCTTCTACCGCTCACTGCTGGAGGACGATGACATGGGGGACCTGGTGGATGCTGAGGAGTATCTGGTACCCCAGCAGGGCTTCTTCTGTCCAGACCCTGCCCCGGGCGCTGGGGGCATGGTCCACCACAGGCACCGCAGCTCATCTACCAGGAGTGGCGGTGGGGACCTGACACTAGGGCTGGAGCCCTCTGAAGAGGAGGCCCCCAGGTCTCCACTGGCACCCTCCGAAGGGGCTGGCTCCGATGTATTTGATGGTGACCTGGGAATGGGGGCAGCCAAGGGGCTGCAAAGCCTCCCCACACATGACCCCAGCCCTCTACAGCGGTACAGTGAGGACCCCACAGTACCCCTGCCCTCTGAGACTGATGGCTACGTTGCCCCCCTGACCTGCAGCCCCCAGCCTGAATATGTGAACCAGCCAGATGTTCGGCCCCAGCCCCCTTCGCCCCGAGAGGGCCCTCTGCCTGCTGCCCGACCTGCTGGTGCCACTCTGGAAAGGGCCAAGACTCTCTCCCCAGGGAAGAATGGGGTCGTCAAAGACGTTTTTGCCTTTGGGGGTGCCGTGGAGAACCCCGAGTACTTGACACCCCAGGGAGGAGCTGCCCCTCAGCCCCACCCTCCTCCTGCCTTCAGCCCAGCCTTCGACAACCTCTATTACTGGGACCAGGACCCACCAGAGCGGGGGGCTCCACCCAGCACCTTCAAAGGGACACCTACGGCAGAGAACCCAGAGTACCTGGGTCTGGACGTGCCAGTGTGAACCAGAAGGCCAAGTCCGCAGAAGCCCTGA(SEQ ID NO:49)。

in another embodiment, the nucleic acid sequence encoding the human her2/neu EC1 fragment used in the chimera spans 120-510bp of the human EC1 region and is shown (SEQ ID NO: 50).

GAGACCCACCTGGACATGCTCCGCCACCTCTACCAGGGCTGCCAGGTGGTGCAGGGAAACCTGGAACTCACCTACCTGCCCACCAATGCCAGCCTGTCCTTCCTGCAGGATATCCAGGAGGTGCAGGGCTACGTGCTCATCGCTCACAACCAAGTGAGGCAGGTCCCACTGCAGAGGCTGCGGATTGTGCGAGGCACCCAGCTCTTTGAGGACAACTATGCCCTGGCCGTGCTAGACAATGGAGACCCGCTGAACAATACCACCCCTGTCACAGGGGCCTCCCCAGGAGGCCTGCGGGAGCTGCAGCTTCGAAGCCTCACAGAGATCTTGAAAGGAGGGGTCTTGATCCAGCGGAACCCCCAGCTCTGCTACCAGGACACGATTTTGTGGAAG(SEQ ID NO:50)。

In one embodiment, the entire EC1 human her2/neu fragment spans 58-979bp of the human her2/neu gene and is as shown (SEQ ID NO: 54).

GCCGCGAGCACCCAAGTGTGCACCGGCACAGACATGAAGCTGCGGCTCCCTGCCAGTCCCGAGACCCACCTGGACATGCTCCGCCACCTCTACCAGGGCTGCCAGGTGGTGCAGGGAAACCTGGAACTCACCTACCTGCCCACCAATGCCAGCCTGTCCTTCCTGCAGGATATCCAGGAGGTGCAGGGCTACGTGCTCATCGCTCACAACCAAGTGAGGCAGGTCCCACTGCAGAGGCTGCGGATTGTGCGAGGCACCCAGCTCTTTGAGGACAACTATGCCCTGGCCGTGCTAGACAATGGAGACCCGCTGAACAATACCACCCCTGTCACAGGGGCCTCCCCAGGAGGCCTGCGGGAGCTGCAGCTTCGAAGCCTCACAGAGATCTTGAAAGGAGGGGTCTTGATCCAGCGGAACCCCCAGCTCTGCTACCAGGACACGATTTTGTGGAAGGACATCTTCCACAAGAACAACCAGCTGGCTCTCACACTGATAGACACCAACCGCTCTCGGGCCTGCCACCCCTGTTCTCCGATGTGTAAGGGCTCCCGCTGCTGGGGAGAGAGTTCTGAGGATTGTCAGAGCCTGACGCGCACTGTCTGTGCCGGTGGCTGTGCCCGCTGCAAGGGGCCACTGCCCACTGACTGCTGCCATGAGCAGTGTGCTGCCGGCTGCACGGGCCCCAAGCACTCTGACTGCCTGGCCTGCCTCCACTTCAACCACAGTGGCATCTGTGAGCTGCACTGCCCAGCCCTGGTCACCTACAACACAGACACGTTTGAGTCCATGCCCAATCCCGAGGGCCGGTATACATTCGGCGCCAGCTGTGTGACTGCCTGTCCCTACAACTACCTTTCTACGGACGTGGGATCCTGCACCCTCGTCTGCCCCCTGCACAACCAAGAGGTGACAGCAGAGGAT(SEQ ID NO:54)。

In another embodiment, the nucleic acid sequence encoding the human her2/neu EC2 fragment for use in the chimera spans 1077-1554bp of the human her2/neu EC2 fragment and includes a 50bp extension, and is as set forth in (SEQ ID NO: 51).

AATATCCAGGAGTTTGCTGGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTCTGCCGGAGAGCTTTGATGGGGACCCAGCCTCCAACACTGCCCCGCTCCAGCCAGAGCAGCTCCAAGTGTTTGAGACTCTGGAAGAGATCACAGGTTACCTATACATCTCAGCATGGCCGGACAGCCTGCCTGACCTCAGCGTCTTCCAGAACCTGCAAGTAATCCGGGGACGAATTCTGCACAATGGCGCCTACTCGCTGACCCTGCAAGGGCTGGGCATCAGCTGGCTGGGGCTGCGCTCACTGAGGGAACTGGGCAGTGGACTGGCCCTCATCCACCATAACACCCACCTCTGCTTCGTGCACACGGTGCCCTGGGACCAGCTCTTTCGGAACCCGCACCAAGCTCTGCTCCACACTGCCAACCGGCCAGAGGACGAGTGTGTGGGCGAGGGCCTGGCCTGCCACCAGCTGTGCGCCCGAGGG(SEQ ID NO:51)。

In one embodiment, the entire EC2 human her2/neu fragment spans 907-1504bp of the human her2/neu gene and is as shown (SEQ ID NO: 55).

TACCTTTCTACGGACGTGGGATCCTGCACCCTCGTCTGCCCCCTGCACAACCAAGAGGTGACAGCAGAGGATGGAACACAGCGGTGTGAGAAGTGCAGCAAGCCCTGTGCCCGAGTGTGCTATGGTCTGGGCATGGAGCACTTGCGAGAGGTGAGGGCAGTTACCAGTGCCAATATCCAGGAGTTTGCTGGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTCTGCCGGAGAGCTTTGATGGGGACCCAGCCTCCAACACTGCCCCGCTCCAGCCAGAGCAGCTCCAAGTGTTTGAGACTCTGGAAGAGATCACAGGTTACCTATACATCTCAGCATGGCCGGACAGCCTGCCTGACCTCAGCGTCTTCCAGAACCTGCAAGTAATCCGGGGACGAATTCTGCACAATGGCGCCTACTCGCTGACCCTGCAAGGGCTGGGCATCAGCTGGCTGGGGCTGCGCTCACTGAGGGAACTGGGCAGTGGACTGGCCCTCATCCACCATAACACCCACCTCTGCTTCGTGCACACGGTGCCCTGGGACCAGCTCTTTCGGAACCCGCACCAAGCTCTGCTCCACACTGCCAACCGGCCAGAG(SEQ ID NO:55)。

In another embodiment, the nucleic acid sequence encoding the human her2/neu IC1 fragment used in the chimera is set forth in (SEQ ID NO: 52).

CAGCAGAAGATCCGGAAGTACACGATGCGGAGACTGCTGCAGGAAACGGAGCTGGTGGAGCCGCTGACACCTAGCGGAGCGATGCCCAACCAGGCGCAGATGCGGATCCTGAAAGAGACGGAGCTGAGGAAGGTGAAGGTGCTTGGATCTGGCGCTTTTGGCACAGTCTACAAGGGCATCTGGATCCCTGATGGGGAGAATGTGAAAATTCCAGTGGCCATCAAAGTGTTGAGGGAAAACACATCCCCCAAAGCCAACAAAGAAATCTTAGACGAAGCATACGTGATGGCTGGTGTGGGCTCCCCATATGTCTCCCGCCTTCTGGGCATCTGCCTGACATCCACGGTGCAGCTGGTGACACAGCTTATGCCCTATGGCTGCCTCTTAGACT(SEQ ID NO:52)。

In another embodiment, the nucleic acid sequence encoding the entire human her2/neu IC1 fragment spans 2034-3243 of the human her2/neu gene and is as set forth in (SEQ ID NO: 56).

CAGCAGAAGATCCGGAAGTACACGATGCGGAGACTGCTGCAGGAAACGGAGCTGGTGGAGCCGCTGACACCTAGCGGAGCGATGCCCAACCAGGCGCAGATGCGGATCCTGAAAGAGACGGAGCTGAGGAAGGTGAAGGTGCTTGGATCTGGCGCTTTTGGCACAGTCTACAAGGGCATCTGGATCCCTGATGGGGAGAATGTGAAAATTCCAGTGGCCATCAAAGTGTTGAGGGAAAACACATCCCCCAAAGCCAACAAAGAAATCTTAGACGAAGCATACGTGATGGCTGGTGTGGGCTCCCCATATGTCTCCCGCCTTCTGGGCATCTGCCTGACATCCACGGTGCAGCTGGTGACACAGCTTATGCCCTATGGCTGCCTCTTAGACCATGTCCGGGAAAACCGCGGACGCCTGGGCTCCCAGGACCTGCTGAACTGGTGTATGCAGATTGCCAAGGGGATGAGCTACCTGGAGGATGTGCGGCTCGTACACAGGGACTTGGCCGCTCGGAACGTGCTGGTCAAGAGTCCCAACCATGTCAAAATTACAGACTTCGGGCTGGCTCGGCTGCTGGACATTGACGAGACAGAGTACCATGCAGATGGGGGCAAGGTGCCCATCAAGTGGATGGCGCTGGAGTCCATTCTCCGCCGGCGGTTCACCCACCAGAGTGATGTGTGGAGTTATGGTGTGACTGTGTGGGAGCTGATGACTTTTGGGGCCAAACCTTACGATGGGATCCCAGCCCGGGAGATCCCTGACCTGCTGGAAAAGGGGGAGCGGCTGCCCCAGCCCCCCATCTGCACCATTGATGTCTACATGATCATGGTCAAATGTTGGATGATTGACTCTGAATGTCGGCCAAGATTCCGGGAGTTGGTGTCTGAATTCTCCCGCATGGCCAGGGACCCCCAGCGCTTTGTGGTCATCCAGAATGAGGACTTGGGCCCAGCCAGTCCCTTGGACAGCACCTTCTACCGCTCACTGCTGGAGGACGATGACATGGGGGACCTGGTGGATGCTGAGGAGTATCTGGTACCCCAGCAGGGCTTCTTCTGTCCAGACCCTGCCCCGGGCGCTGGGGGCATGGTCCACCACAGGCACCGCAGCTCATCTACCAGGAGTGGCGGTGGGGACCTGACACTAGGGCTGGAGCCCTCTGAAGAGGAGGCCCCCAGGTCTCCACTGGCACCCTCCGAAGGGGCT(SEQ ID NO:56)。

In one embodiment, the LLO used in the methods and compositions provided herein is a listeria LLO. In one embodiment, the listeria from which the LLO is derived is Listeria Monocytogenes (LM). In another embodiment, the Listeria is Listeria monocytogenes (Listeria ivanovii). In another embodiment, the Listeria is Listeria welshimeri (Listeria welshimeri). In another embodiment, the Listeria is Listeria monocytogenes (Listeria seeligeri). In another embodiment, the LLO protein is a non-listerial LLO protein. In another embodiment, the LLO protein is a synthetic LLO protein. In another embodiment, it is a recombinant LLO protein.

In one embodiment, the LLO protein is encoded by the following nucleic acid sequence (SEQ ID NO:3)

atgaaaaaaataatgctagtttttattacacttatattagttagtctaccaattgcgcaacaaactgaagcaaaggatgcatctgcattcaataaagaaaattcaatttcatccatggcaccaccagcatctccgcctgcaagtcctaagacgccaatcgaaaagaaacacgcggatgaaatcgataagtatatacaaggattggattacaataaaaacaatgtattagtataccacggagatgcagtgacaaatgtgccgccaagaaaaggttacaaagatggaaatgaatatattgttgtggagaaaaagaagaaatccatcaatcaaaataatgcagacattcaagttgtgaatgcaatttcgagcctaacctatccaggtgctctcgtaaaagcgaattcggaattagtagaaaatcaaccagatgttctccctgtaaaacgtgattcattaacactcagcattgatttgccaggtatgactaatcaagacaataaaatagttgtaaaaaatgccactaaatcaaacgttaacaacgcagtaaatacattagtggaaagatggaatgaaaaatatgctcaagcttatccaaatgtaagtgcaaaaattgattatgatgacgaaatggcttacagtgaatcacaattaattgcgaaatttggtacagcatttaaagctgtaaataatagcttgaatgtaaacttcggcgcaatcagtgaagggaaaatgcaagaagaagtcattagttttaaacaaatttactataacgtgaatgttaatgaacctacaagaccttccagatttttcggcaaagctgttactaaagagcagttgcaagcgcttggagtgaatgcagaaaatcctcctgcatatatctcaagtgtggcgtatggccgtcaagtttatttgaaattatcaactaattcccatagtactaaagtaaaagctgcttttgatgctgccgtaagcggaaaatctgtctcaggtgatgtagaactaacaaatatcatcaaaaattcttccttcaaagccgtaatttacggaggttccgcaaaagatgaagttcaaatcatcgacggcaacctcggagacttacgcgatattttgaaaaaaggcgctacttttaatcgagaaacaccaggagttcccattgcttatacaacaaacttcctaaaagacaatgaattagctgttattaaaaacaactcagaatatattgaaacaacttcaaaagcttatacagatggaaaaattaacatcgatcactctggaggatacgttgctcaattcaacatttcttgggatgaagtaaattatgat(SEQ ID NO:3)。

In another embodiment, the LLO protein has the sequence SEQ ID NO 4

MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSMAPPASPPASPKTPIEKKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVERWNEKYAQAYPNVSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNYD(SEQ ID NO:4)

The first 25 amino acids of the preprotein corresponding to the sequence are the signal sequence and are cleaved from the LLO when it is secreted by the bacteria. Thus, in this embodiment, the full length active LLO protein is 504 residues long. In another embodiment, the LLO protein has the sequence shown in GenBank accession No. DQ054588, DQ054589, AY878649, U25452 or U25452. In another embodiment, the LLO protein is a variant of the LLO protein. In another embodiment, the LLO protein is a homolog of the LLO protein. Each possibility represents a separate embodiment of the invention.

In another embodiment, "truncated LLO" or "tLLO" refers to a fragment of an LLO comprising a PEST-like domain. In another embodiment, the term refers to an LLO fragment that does not contain an amino-terminal activation domain and does not include cystine at position 484. In another embodiment, the LLO fragment consists of a PEST sequence. In another embodiment, the LLO fragment comprises a PEST sequence. In another embodiment, the LLO fragment consists of approximately the first 400 to 441 amino acids of a 529 amino acid full length LLO protein. In another embodiment, the LLO fragment is a non-hemolytic form of the LLO protein.

In another embodiment of the methods and compositions of the invention, the polypeptide encoded by the nucleic acid sequence of the methods and compositions of the invention is a fusion protein comprising the chimeric Her-2/neu antigen and a further polypeptide, while in another embodiment the fusion protein comprises inter alia the LM non-hemolytic LLO protein or the like (examples herein).

In one embodiment, the LLO fragment consists approximately of residues 1-25. In another embodiment, the LLO fragment consists approximately of residues 1-50. In another embodiment, the LLO fragment consists approximately of residues 1-75. In another embodiment, the LLO fragment consists of approximately residues 1-100. In another embodiment, the LLO fragment consists approximately of residues 1-125. In another embodiment, the LLO fragment consists approximately of residues 1-150. In another embodiment, the LLO fragment consists approximately of residue 1175. In another embodiment, the LLO fragment consists approximately of residues 1-200. In another embodiment, the LLO fragment consists approximately of residues 1-225. In another embodiment, the LLO fragment consists approximately of residues 1-250. In another embodiment, the LLO fragment consists approximately of residues 1-275. In another embodiment, the LLO fragment consists approximately of residues 1-300. In another embodiment, the LLO fragment consists approximately of residues 1-325. In another embodiment, the LLO fragment consists approximately of residues 1-350. In another embodiment, the LLO fragment consists approximately of residues 1-375. In another embodiment, the LLO fragment consists approximately of residues 1-400. In another embodiment, the LLO fragment consists approximately of residues 1-425. Each possibility represents a separate embodiment of the invention.

In another embodiment, the fusion protein of the methods and compositions of the invention comprises a PEST sequence, either from an LLO protein or from another organism, such as a prokaryote.

In another embodiment, the PEST-like AA sequence has a sequence selected from SEQ ID NOS 5-9. In another embodiment, the PEST-like sequence is a PEST-like sequence from a LM ActA protein. In another embodiment, the PEST-like sequence is KTEEQPSEVNTGPR (SEQ ID NO:5), KASVTDTSEGDLDSSMQSADESTPQPLK (SEQ ID NO:6), KNEEVNASDFPPPPTDEELR (SEQ ID NO:7), or RGGIPTSEEFSSLNSGDFTDDENSETTEEEIDR (SEQ ID NO: 8). In another embodiment, the PEST-like sequence is a streptolysin O protein from Streptococcus (Streptococcus sp.). In another embodiment, the PEST-like sequence is streptolysin O from Streptococcus pyogenes (Streptococcus pyogenes), such as KQNTASTETTTTNEQPK (SEQ ID NO:9) at AA 35-51. In another embodiment, the PEST-like sequence is derived from streptolysin O of Streptococcus equisimilis (Streptococcus equisimilis), e.g., KQNTANTETTTTNEQPK (SEQ ID NO:10) at AA 38-54. In another embodiment, the PEST-like sequence is another PEST-like AA sequence derived from a prokaryote. In another embodiment, the PEST-like sequence is any other PEST-like sequence known in the art. Each possibility represents a separate embodiment of the invention.

In one embodiment, fusion of the antigen to the PEST-like sequence of LM enhances cell-mediated and anti-tumor immunity of the antigen. Thus, fusion of the antigen with other PEST-like sequences derived from other prokaryotes also enhances the immunogenicity of the antigen. PEST-like sequences of other prokaryotes are identified, for example, according to the method for LM as described by Rechsteiner and Rogers (1996, Trends biochem. Sci.21: 267-271). Alternatively, PEST-like AA sequences from other prokaryotes may also be identified based on this method. Other prokaryotes (where it is desirable to have PEST-like AA sequences) include, but are not limited to, other listeria species. In another embodiment, the PEST-like sequence is embedded in an antigenic protein. Thus, in another embodiment, a "fusion" relates to an antigenic protein comprising an antigen and a PEST-like amino acid sequence attached to one end of or embedded in the antigen.

In another embodiment, provided herein is a vaccine comprising a recombinant polypeptide of the invention. In another embodiment, provided herein is a vaccine consisting of a recombinant polypeptide of the invention.

In another embodiment, provided herein are nucleotide molecules encoding the recombinant polypeptides of the invention. In another embodiment, provided herein is a vaccine comprising a nucleotide molecule.

In another embodiment, provided herein are nucleotide molecules encoding the recombinant polypeptides of the invention.

In another embodiment, provided herein is a recombinant polypeptide encoded by a nucleotide molecule of the invention.

In another embodiment, provided herein is a vaccine comprising a nucleotide molecule or recombinant polypeptide of the invention.

In another embodiment, provided herein are immunogenic compositions comprising a nucleotide molecule or recombinant polypeptide of the invention.

In another embodiment, provided herein is a vector comprising a nucleotide molecule or recombinant polypeptide of the invention.

In another embodiment, provided herein is a recombinant form of listeria comprising a nucleotide molecule of the present invention.

In another embodiment, provided herein are vaccines comprising recombinant forms of the listeria of the present invention.

In another embodiment, provided herein are cultures of recombinant forms of the listeria of the present invention.

In one embodiment, the vaccine for use in the methods of the invention comprises a recombinant listeria monocytogenes strain, in any form or embodiment as described herein. In one embodiment, the vaccine for use according to the invention consists of the recombinant listeria monocytogenes of the present invention in any of the forms or embodiments as described herein. In another embodiment, the vaccine for use in the methods of the invention consists essentially of the recombinant listeria monocytogenes of the present invention in any of the forms or embodiments as described herein. In one embodiment, the term "comprising" refers to a vaccine including recombinant listeria monocytogenes, as well as including other vaccines or treatments as may be known in the art. In another embodiment, the term "consisting essentially of … …" relates to a vaccine whose functional component is recombinant listeria monocytogenes, however, it may include other components of the vaccine that are not directly involved in the therapeutic effect of the vaccine, and may relate to components that, for example, facilitate the action (e.g., stabilization, preservation, etc.) of the recombinant listeria monocytogenes. In another embodiment, the term "consisting of … …" relates to a vaccine comprising recombinant listeria monocytogenes.

In another embodiment, the methods of the invention comprise the step of administering a recombinant listeria monocytogenes in any form or embodiment as described herein. In one embodiment, the method of the invention consists of the step of administering the recombinant listeria monocytogenes of the invention in any form or embodiment as described herein. In another embodiment, the methods of the invention consist essentially of the step of administering the recombinant listeria monocytogenes of the invention in any of the forms or embodiments as described herein. In one embodiment, the term "comprising" refers to a method comprising the step of administering recombinant listeria monocytogenes, as well as other methods or treatments known in the art. In another embodiment, the term "consisting essentially of … …" relates to a method whose functional step is the administration of recombinant listeria monocytogenes, however, it may comprise other steps of the method that do not directly participate in the therapeutic effect of the method, and may for example relate to steps that facilitate the administration of the effect of recombinant listeria monocytogenes. In one embodiment, the term "consisting of … …" relates to a method of administering recombinant listeria monocytogenes without additional steps.

In another embodiment, the listeria of the methods and compositions of the present invention is listeria monocytogenes. In another embodiment, the listeria is listeria monocytogenes. In another embodiment, the listeria is listeria welshimeri. In another embodiment, the listeria is listeria monocytogenes. Each type of listeria represents an independent embodiment of the present invention.

In one embodiment, the listeria strain of the methods and compositions of the present invention is the ADXS31-164 strain. In another embodiment, ADXS31-164 stimulates secretion of IFN- γ from splenocytes from wild type FVB/N mice. Further, the data provided herein show that ADXS31-164 is capable of eliciting anti-Her 2/neu specific immune responses against human epitopes located in different domains of the target antigen.

In another embodiment, the invention provides a recombinant form of listeria comprising a nucleotide molecule encoding a Her-2 chimeric protein or a fragment thereof.

In one embodiment, the invention provides a method of inducing an anti-Her-2 immune response in a subject, comprising administering to the subject a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to a Her-2 chimeric protein or to a fragment thereof, thereby inducing an anti-Her-2 immune response in the subject.

In one embodiment, the fusion protein of the methods and compositions of the invention comprises an LLO signal sequence from an LLO. In another embodiment, the proteins of the two molecules (the LLO fragment and the antigen) are directly linked. In another embodiment, the two molecules are linked by a short spacer peptide consisting of one or more amino acids. In one embodiment, the spacers have no specific biological activity, but rather are linked to the proteins or maintain some minimum distance or other spatial relationship between them. In another embodiment, the amino acids making up the spacer are selected to affect some property of the molecule, such as folding, net charge, or hydrophobicity. In another embodiment, the two molecules of protein (LLO fragment and antigen) are separately synthesized or unfused. In another embodiment, the two molecules of protein are each synthesized from the same nucleic acid. In yet another embodiment, the two molecules are synthesized separately from separate nucleic acids. Each possibility represents a separate embodiment of the invention.

In another embodiment, provided herein is a method of inducing an anti-Her-2 immune response in a subject, comprising administering to the subject a recombinant nucleotide encoding a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to a Her-2 chimeric protein or to a fragment thereof, thereby inducing an anti-Her-2 immune response in the subject.

In one embodiment, provided herein is a method of eliciting an enhanced immune response in a subject against a Her 2/neu-expressing tumor, while in another embodiment, the method comprises administering to the subject a composition comprising a recombinant listeria vaccine strain provided herein. In another embodiment, the immune response against a Her-2 expressing tumor comprises an immune response to a subdominant epitope of a Her-2 protein. In another embodiment, the immune response against a Her-2 expressing tumor comprises an immune response to several subdominant epitopes of the Her-2 protein. In another embodiment, the immune response against a Her-2 expressing tumor comprises an immune response to at least 1-5 subdominant epitopes of the Her-2 protein. In another embodiment, the immune response against a Her-2 expressing tumor comprises an immune response to at least 1-10 subdominant epitopes of the Her-2 protein. In another embodiment, the immune response against a Her-2 expressing tumor comprises an immune response to at least 1-17 subdominant epitopes of the Her-2 protein. In another embodiment, the immune response against a Her-2 expressing tumor comprises an immune response to at least 17 subdominant epitopes of the Her-2 protein.

It has been reported that point mutations or amino acid deletions in the oncogenic protein Her2/neu mediate treatment of resistant tumor cells when a small fragment listeria-based vaccine or trastuzumab (trastuzumab) (a monoclonal antibody directed against an epitope located in the extracellular domain of Her2/neu antigen) targets a resistant tumor. Described herein are chimeric Her 2/neu-based compositions having two extracellular and one intracellular fragments of Her2/neu antigen, displaying MHC-class I epitope clusters of oncogenes. This chimeric protein with 3H 2Dq and at least 17 located human MHC-type I epitopes of Her2/neu antigen was fused to the first 441 amino acids of the listeria monocytogenes listeriolysin O protein and expressed and secreted by the attenuated strain of listeria monocytogenes LmddA.

Previous reports have shown that when Her2/neu transgenic mice are immunized with listeria-based vaccines that express and secrete small fragments of Her2/neu antigen (each with only one H2Dq epitope of the Her2/neu oncogene), respectively, tumors that overexpress Her2/neu can escape due to mutations in those epitopes of Her2/neu antigen that are targeted by each vaccine (see Singh R, Paterson y. immunological discovery tumors tissues and mice and cancer Res2007;67: 1887-92). The following unexpected results are shown herein: when three or more epitopes of Her2/neu protein are incorporated into a chimeric vaccine, it can eliminate the selection and escape of these tumors due to escape mutations. Immunization with the novel Her2/neu chimeric listeria vaccine did not generate any escape mutations that may be associated with point mutations or amino acid deletions in the Her2/neu antigen (see example 4 herein).

In one embodiment, provided herein is a method of engineering a listeria vaccine strain to express a Her-2 chimeric protein or to express a recombinant polypeptide of a chimeric protein, the method comprising transforming a listeria strain with a nucleic acid molecule. In another embodiment, the nucleic acid molecule comprises a first open reading frame encoding a polypeptide, wherein the polypeptide comprises a Her2/neu chimeric antigen. In another embodiment, the nucleic acid molecule further comprises a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene not present in the chromosome of the recombinant listeria strain, thereby engineering the listeria vaccine strain to express the Her-2 chimeric protein.

In one embodiment, the methods and compositions provided herein further comprise an adjuvant, while in another embodiment, the adjuvant comprises a granulocyte/macrophage colony-stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, monophosphoryl lipid A, or an unmethylated CpG-containing oligonucleotide.

In one embodiment, attenuated Listeria strains, such as the LM delta-actA mutant (Brundage et al, 1993, Proc. Natl. Acad. Sci., USA,90: 11890-. In another embodiment, an attenuated listeria strain is constructed by introducing one or more attenuating mutations, as understood by one of skill in the art in possession of the present disclosure. Examples of such strains include, but are not limited to, Listeria strains auxotrophic for aromatic amino acids (Alexander et al, 1993, Infection and Immunity1061: 2245-.

In another embodiment, the nucleic acid molecules of the methods and compositions of the present invention are operably linked to a promoter/regulatory sequence. In another embodiment, the first open reading frame of the methods and compositions of the present invention is operably linked to a promoter/regulatory sequence. In another embodiment, the second open reading frame of the methods and compositions of the present invention is operably linked to a promoter/regulatory sequence. In another embodiment, each open reading frame is operably linked to a promoter/regulatory sequence. Each possibility represents a separate embodiment of the invention.

When armed with the present disclosure and the methods provided herein, one of skill in the art will readily appreciate that different transcription promoters, terminators, delivery vectors, or specific gene sequences (such as those in commercially available cloning vectors) may be successfully used in the methods and compositions of the present invention. As contemplated herein, these functions are provided, for example, in commercially available vectors known as the pUC series. In another embodiment, non-essential DNA sequences (e.g., antibiotic resistance genes) are removed. Each possibility represents a separate embodiment of the invention. In another embodiment, commercially available plasmids are used in the present invention. These plasmids can be obtained from various sources, e.g., Invitrogen (La Jolla, CA), Stratagene (La Jolla, CA), Clontech (Palo Alto, CA), or can be constructed using methods well known in the art.

Another embodiment is a plasmid such as pcr2.1(Invitrogen, La Jolla, CA), which is a prokaryotic expression vector with a prokaryotic origin of replication and promoter/regulatory elements that facilitate expression in prokaryotes. In another embodiment, the exogenous nucleotide sequence is removed to reduce the size of the plasmid and increase the size of the cassette that can be placed therein.

Such methods are well known in the art and are described, for example, in Sambrook et al (1989, Molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York) and Ausubei et al (1997, Current Protocols in Molecular Biology, Green & Wiley, New York).

Antibiotic resistance genes are used in conventional selection and cloning methods commonly employed in molecular biology and vaccine preparation. Antibiotic resistance genes contemplated by the present invention include, but are not limited to, gene products that confer resistance to ampicillin, penicillin, methicillin, streptomycin, erythromycin, kanamycin, tetracycline, Chloramphenicol (CAT), neomycin, hygromycin, gentamicin, and other antibiotics known in the art. Each gene represents a separate embodiment of the present invention.

Methods for transforming bacteria are well known in the art and include competent Cell-based calcium chloride, electroporation, phage-mediated transduction, chemical and physical transformation techniques (de Boer et al, 1989, Cell56:641-649; Miller et al, 1995, FASEB J.,9:190-199; Sambrook et al, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York; Ausubel et al, 1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York; Gerhardt et al, eds.,1994, Methods for General and Molecular bacteriology, American Society for Microbiology, Micrology, Wash., Wash et al, 1992, phage-mediated transduction, chemical and physical transformation techniques (Pre. carbide, C.C.C.. In another embodiment, the listeria vaccine strain of the present invention is transformed by electroporation. Each representing a separate embodiment of the invention.

In another embodiment, conjugation is used to introduce genetic material and/or plasmids into the bacteria. Methods of conjugation are well known in the art and are described, for example, in Nikodinovic J et al (A differentiation sn p-derived Escherichia coli-Streptomyces shuttle expression vector generation by plasmid 2006Nov;56(3):223-7) and Autochning JM et al (Regulation of a Bacillus subtilis mobile genetic element by interactive cellular signaling and the viral DNA damageresponse. Proc Natl Acad Sci U S.2005Aug30; 102(35): 12554-9). Each representing a separate embodiment of the invention.

In one embodiment, "transformation" is used equivalently to the term "transfection" and refers to the engineering of bacterial cells to take up plasmids or other heterologous DNA molecules. In another embodiment, "transformation" refers to engineering a bacterial cell to express genes of a plasmid or other heterologous DNA molecule. Each possibility represents a separate embodiment of the invention.

Plasmids and other expression vectors useful in the present invention are described elsewhere herein, and may include such features as: promoter/regulatory sequences, origins of replication of gram-negative and gram-positive bacteria, isolated nucleic acids encoding fusion proteins and isolated nucleic acids encoding genes for amino acid metabolism. Further, an isolated nucleic acid encoding a fusion protein and an amino acid metabolism gene has a promoter suitable for driving expression of such an isolated nucleic acid. Promoters useful for driving expression in bacterial systems are well known in the art and include bacteriophage lambda, the bla promoter of the beta-lactamase gene of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene of pBR 325. Further examples of prokaryotic promoters include The major right and left promoters (PL and PR) of bacteriophage lambda 5, The trp, recA, lacZ, lad and gal promoters of E.coli (E.coli), The alpha-amylase (Ulmenen et al, 1985.J.Bacteriol.162:176-182) and S28-specific promoters (Gilman et al, 1984Gene32:11-20) of Bacillus, The promoters of bacteriophage of Bacillus (Gryczan, 1982, In: The molecular biology of The Bacillus, Academic, Inc., New York), and The Streptomyces promoter (Ward et al, 1986, mol.Gen.Genet.203: 468-478). Additional prokaryotic promoters contemplated by the present invention are reviewed, for example, in Glick (1987, J.Ind.Microbiol.1: 277-); cenatiempo, (1986, Biochimie,68: 505-); and Gottesman, (1984, Ann. Rev. Genet.18: 415-442). Further examples of promoter/regulatory elements contemplated by the present invention include, but are not limited to, the listeria prfA promoter, the listeria hly promoter, the listeria p60 promoter, and the listeria ActA promoter (GenBank acc. No. nc _003210), or fragments thereof.

In another embodiment, the plasmids of the methods and compositions of the invention comprise a gene encoding a fusion protein. In another embodiment, the subsequence is cloned and the appropriate subsequence is cleaved using an appropriate restriction enzyme. In another embodiment, the fragments are then ligated to produce the desired DNA sequence. In another embodiment, DNA encoding the antigen is produced using a DNA amplification method, such as Polymerase Chain Reaction (PCR). First, segments of native DNA on either side of the new end are amplified separately. The 5 'end of one amplified sequence encodes a peptide linker, while the 3' end of the other amplified sequence also encodes a peptide linker. Since the 5 'end of the first fragment is complementary to the 3' end of the second fragment, both fragments (after partial purification, e.g., on LMP agarose) can be used as overlapping templates in the third PCR reaction. The amplified sequence will contain the codon, a segment on the carboxy side of the open site (now forming an amino sequence), a linker and a sequence on the amino side of the open site (now forming a carboxy sequence). The antigen is ligated into a plasmid. Each representing a separate embodiment of the invention.

In another embodiment, the invention further comprises a phage-based chromosomal integration system for clinical use. Host strains that are auxotrophic for enzymes essential to use, including, but not limited to, d-alanine racemase, such as Lmdal (-) dat (-). In another embodiment, to avoid the "phage healing step", a PSA-based phage integration system (Lauer et al, 2002J Bacteriol, 184: 4177-. In another embodiment, this requires continuous selection by antibiotics to maintain the integrated gene. Thus, in another embodiment, the present invention enables the establishment of a phage-based chromosomal integration system that does not require selection with antibiotics. But rather complements an auxotrophic host strain.

In another embodiment, recombinant proteins of the invention are synthesized using recombinant DNA methods. In one embodiment, this comprises generating a DNA sequence encoding the fusion protein, placing the DNA in an expression cassette, such as a plasmid of the invention, under the control of specific promoter/regulatory elements, and expressing the protein. In another embodiment, DNA encoding a fusion protein of the invention (e.g., a non-hemolytic LLO/antigen) is prepared by any suitable method including, for example, cloning and restriction of the appropriate sequence or direct chemical synthesis by methods such as the phosphotriester method of Narang et al (1979, meth.Enzymol.68: 90-99); the phosphodiester method of Brown et al (1979, meth. enzymol68: 109-151); the diethylphosphoramidate acid method of Beaucage et al (1981, tetra. Lett., 22: 151859-1862); and U.S. Pat. No. 4,458,066.

In another embodiment, chemical synthesis is used to generate single stranded oligonucleotides. In various embodiments, the single-stranded oligonucleotide is converted to double-stranded DNA by hybridization to a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One skilled in the art will recognize that although chemical synthesis of DNA is limited to sequences of about 100 bases, longer sequences can be obtained by ligating shorter sequences. In another embodiment, the subsequence is cloned and the appropriate subsequence is cleaved using an appropriate restriction enzyme. The fragments are then ligated to produce the desired DNA sequence.

In another embodiment, the DNA encoding the fusion protein or recombinant protein of the invention is cloned using DNA amplification methods such as Polymerase Chain Reaction (PCR). Thus, it PCR amplifies the gene of the non-hemolytic LLO using a sense primer comprising a suitable restriction site and an antisense primer comprising another restriction site, e.g. a different restriction site facilitating cloning. The same procedure is repeated for isolated nucleic acids encoding the antigen. Connecting the nonhemolytic LLO and the antigen sequence and inserting into a plasmid or vector, resulting in a vector encoding the nonhemolytic LLO linked to the antigen terminus. The two molecules are linked directly or by a short spacer introduced at a restriction site.

In another embodiment, the molecules are separated by a peptide spacer consisting of one or more amino acids, typically without specific biological activity, but rather to link the proteins or maintain some minimum distance or other spatial relationship between them. In another embodiment, the composition AA of the spacer is selected to affect some property of the molecule, such as folding, net charge, or hydrophobicity. In another embodiment, the nucleic acid sequence encoding the fusion or recombinant protein is transformed into a variety of host cells, including E.coli, other bacterial hosts such as Listeria, yeast, and various higher eukaryotic cells such as COS, CHO and HeLa cell lines and myeloma cell lines. The recombinant fusion protein gene is operably linked to the appropriate expression control sequences of each host. Promoter/regulatory sequences are described in detail elsewhere herein. In another embodiment, the plasmid further comprises additional promoter regulatory elements, as well as a ribosome binding site and a transcription termination signal. For eukaryotic cells, the control sequences will include promoters and enhancers from, for example, immunoglobulin genes, SV40, cytomegalovirus, and the like, and polyadenylation sequences. In another embodiment, the sequences include splice donor and acceptor sequences.

In one embodiment, the term "operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence is "operably linked" to a coding sequence in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

In another embodiment, to select an auxotrophic bacterium comprising a plasmid, the transformed auxotrophic bacterium is grown on a medium that selects for expression of an amino acid metabolic gene. In another embodiment, a bacterium auxotrophic for D-glutamic acid synthesis is transformed with a plasmid comprising a D-glutamic acid synthesis gene, and the auxotrophic bacterium will grow in the absence of D-glutamic acid, without transformation with the plasmid, or without expression of a plasmid encoding a protein for D-glutamic acid synthesis. In another embodiment, when a D-alanine synthesis auxotrophic bacterium is transformed and expresses a plasmid of the invention (if the plasmid comprises an isolated nucleic acid encoding an amino acid metabolizing enzyme for D-alanine synthesis), it will grow in the absence of D-alanine. Such methods of making appropriate media containing or lacking the necessary growth factors, supplements, amino acids, vitamins, antibiotics, etc. are well known in the art and are commercially available (Becton-Dickinson, Franklin lakes, N.J.). Each representing a separate embodiment of the invention.

In another embodiment, after an auxotrophic bacterium comprising a plasmid of the invention is selected on an appropriate medium, the bacterium is propagated in the presence of selection pressure. Such proliferation involves the growth of the bacteria in a medium without the auxotrophic factor. The presence of a plasmid expressing an amino acid metabolizing enzyme in an auxotrophic bacterium allows the plasmid to replicate together with the bacterium, thereby continuously selecting bacteria having the plasmid. When provided with the present disclosure and the methods herein, one of skill in the art can readily scale up the production of listeria vaccine vectors by adjusting the volume of medium in which the auxotrophic bacterium comprising the plasmid is grown.

In another embodiment, one skilled in the art will appreciate that other auxotrophic strains and supplementation systems are used with the present invention.

In one embodiment, provided herein is a method of impeding the growth of Her-2 expressing tumors in a subject, wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising a recombinant listeria vaccine strain described herein.

In another embodiment, provided herein is a method of impeding the growth of Her-2 expressing tumors in a subject, wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising a recombinant listeria vaccine strain described herein.

In another embodiment, provided herein is a method of eliciting an enhanced immune response to a Her 2/neu-expressing tumor in a subject, wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising the recombinant listeria vaccine strain described herein. In yet another embodiment, the immune response against a Her 2/neu-expressing tumor comprises an immune response to at least one subdominant epitope of a Her2/neu protein.

In one embodiment, provided herein is a method of preventing escape mutations in the treatment of Her2/neu overexpressing tumors, wherein and in another embodiment, the method comprises the step of administering to said subject a composition comprising a recombinant listeria vaccine strain provided herein.

In another embodiment, provided herein is a method of preventing the onset of a Her2/neu antigen-expressing tumor in a subject, wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising a recombinant listeria vaccine strain provided herein.

In one embodiment, provided herein is a method of reducing the frequency of T regulatory cells within a tumor, wherein and in another embodiment, the method comprises the step of administering to a subject a composition comprising a recombinant listeria vaccine strain provided herein.

In another embodiment, provided herein is a method of reducing the frequency of T regulatory cells within a tumor, wherein and in another embodiment, the method comprises the step of administering to a subject a composition comprising a recombinant listeria vaccine strain provided herein.

In one embodiment, provided herein is a method of reducing the frequency of myeloid-derived suppressor cells within a tumor, wherein and in another embodiment, the method comprises the step of administering to a subject a composition comprising a recombinant listeria vaccine strain provided herein.

In another embodiment, provided herein is a method of reducing the frequency of myeloid derived suppressor cells, wherein and in another embodiment, the method comprises the step of administering to a subject a composition comprising a recombinant listeria vaccine strain provided herein.

In one embodiment, provided herein is a method of preventing the formation of Her 2/neu-expressing tumors in a subject, wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising a recombinant listeria vaccine strain provided herein.

In another embodiment, provided herein is a method of preventing the formation of Her 2/neu-expressing tumors in a subject, wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising a recombinant listeria vaccine strain provided herein.

In one embodiment, provided herein is a method of treating a Her 2/neu-expressing tumor in a subject, wherein and in another embodiment, the method comprises the step of administering to the subject a composition comprising a recombinant listeria vaccine strain provided herein.

In one embodiment, provided herein is a method of administering a composition of the invention. In another embodiment, provided herein is a method of administering a vaccine of the invention. In another embodiment, provided herein is a method of administering a recombinant polypeptide or recombinant nucleotide of the invention. In another embodiment, the step of administering a composition, vaccine, recombinant polypeptide or recombinant nucleotide of the invention is performed using a listeria comprising the composition, vaccine, recombinant nucleotide or an attenuated recombinant form of expressing the recombinant polypeptide, each in its own separate embodiment. In another embodiment, administration is performed using a different attenuated bacterial vector. In another embodiment, administration is performed using a DNA vaccine (e.g., a naked DNA vaccine). In another embodiment, administration of a recombinant polypeptide of the invention is performed by recombinantly producing the protein and then administering the recombinant protein to the subject. Each possibility represents a separate embodiment of the invention.

In another embodiment, the immune response elicited by the methods and compositions of the present invention comprises CD8⁺T cell mediated responses. In another embodiment, the immune response consists essentially of CD8⁺T cell mediated responses. In another embodiment, the only component of the detectable immune response is CD8⁺T cell mediated responses.

In another embodiment, the immune response elicited by the methods and compositions provided herein comprises CD4⁺T cell mediated responses. In another embodiment, the immune response consists essentially of CD4⁺T cell mediated responses. In another embodiment, the only component of the detectable immune response is CD4⁺T cell mediated responses. In another embodiment, CD4⁺The T cell mediated response is accompanied by a detectable antibody response against the antigen. In another embodiment, CD4⁺The T cell-mediated response is not accompanied by a detectable antibody response to the antigen.

In another embodiment, the invention provides for inducing suboptimal CD8 for an antigen in a subject⁺CD8 of T cell epitope⁺A method of T cell mediated immune response comprising the steps of: (a) fusing a nucleotide molecule encoding Her2-neu chimeric antigen or a fragment thereof with a nucleotide molecule encoding an N-terminal fragment of an LLO protein, thereby generating recombinant nucleotides encoding an LLO-antigen fusion protein; and (b) administering to the subject the recombinant nucleotide or the LLO-antigen fusion; thereby inducing suboptimal CD8 for antigen⁺CD8 of T cell epitope⁺T cell mediated immune responses.

In one embodiment, provided herein is a method of increasing the intratumoral ratio of CD8+/T regulatory cells, wherein and in another embodiment, the method comprises the step of administering to a subject a composition comprising a recombinant polypeptide, recombinant listeria, or recombinant vector of the present invention.

In another embodiment, provided herein is a method of increasing the intratumoral ratio of CD8+/T regulatory cells, wherein and in another embodiment, the method comprises the step of administering to a subject a composition comprising a recombinant polypeptide, recombinant listeria, or recombinant vector of the present invention.

In another embodiment, the immune response elicited by the methods and compositions provided herein includes an immune response to at least one subdominant epitope of an antigen. In another embodiment, the immune response comprises an immune response to at least one dominant epitope. In another embodiment, the immune response consists essentially of an immune response to at least one subdominant epitope. In another embodiment, the only detectable component of the immune response is the immune response to at least one subdominant epitope. Each type of immune response represents a separate embodiment of the present invention.

Methods of measuring immune responses are well known in the art and include, for example, measuring inhibition of tumor growth, flow cytometry, target cell lysis assays (e.g., chromium release assays), the use of tetramers, and the like. Each representing a separate embodiment of the invention.

In another embodiment, the present invention provides a method of impeding the growth of Her-2 expressing tumors in a subject, wherein and in another embodiment, the method comprises administering to the subject a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to a Her-2 chimeric protein or a fragment thereof, or a recombinant nucleotide encoding the recombinant polypeptide, wherein the subject increases the immune response against a Her-2 expressing tumor, thereby impeding the growth of Her-2 expressing tumors in the subject.

In another embodiment, the invention provides a method of improving the antigenicity of a Her-2 chimeric protein, wherein and in another embodiment, the method comprises the steps of: fusing nucleotides encoding an N-terminal fragment of an LLO protein with nucleotides encoding a Her-2 protein or a fragment thereof to produce recombinant nucleotides, thereby improving the antigenicity of the Her-2 chimeric protein.

In another embodiment, provided herein is a method of improving the antigenicity of a Her-2 chimeric protein, wherein and in another embodiment, the method comprises engineering a listeria strain to express a recombinant nucleotide. In another embodiment, a different bacterial vector is used to express the recombinant nucleotide. In another embodiment, the bacterial vector is attenuated. In another embodiment, a DNA vaccine (e.g., a naked DNA vaccine) is used to express the recombinant nucleotide. In another embodiment, administration of the LLO-Her-2 chimeric fusion peptide encoded by nucleotides is performed by recombinantly producing the protein and then administering the recombinant protein to the subject. Each possibility represents a separate embodiment of the invention.

In one embodiment, the invention provides a method of "epitope spreading" of a tumor. In another embodiment, immunization using the compositions and methods provided herein induces epitope spreading on other tumors that have antigens other than those carried in the vaccines of the present invention.

In another embodiment, the dominant or subdominant epitope is dominant or subdominant, respectively, in the subject being treated. In another embodiment, the dominant or subdominant epitope is dominant or subdominant in the population being treated.

In one embodiment, provided herein is a method of treating, impeding or inhibiting cancer or tumor growth in a subject by epitope spreading, wherein and in another embodiment, the cancer is associated with the expression of an antigen or fragment thereof comprised in a composition of the invention. In another embodiment, the method comprises administering to the subject a composition comprising a recombinant polypeptide, recombinant listeria, or recombinant vector of the present invention. In yet another embodiment, the subject increases the immune response against a cancer expressing an antigen or a tumor expressing an antigen, thereby treating, impeding, or inhibiting cancer or tumor growth in the subject.

In one embodiment, "dominant CD8⁺T cell epitope "refers to antigen-specific CD8 that is more than 30% bound⁺Epitopes recognized by T cells, said antigen being specific for CD8⁺T cells are initiated by vaccination with a protein, or by infection by a pathogen containing the protein, or by malignant growth of cancer cells containing the protein. In another embodiment, the term refers to antigen-specific CD8 so primed being greater than 35%⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is greater than 40% bound⁺Epitopes recognized by T cells. In another embodiment, the term refers toIs more than 45% antigen-specific CD8⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is greater than 50% bound⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is greater than 55% bound⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is greater than 60% bound⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is greater than 65% bound⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is greater than 70% bound⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is greater than 75% bound⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is greater than 80% bound⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is greater than 85% bound⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is greater than 90% bound⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is greater than 95% bound⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is greater than 96% bound⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is greater than 97% protected⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is greater than 98% bound⁺Epitopes recognized by T cells.

In one embodiment, "sub-optimal CD8⁺T cell epitope "refers to antigen-specific CD8 that is less than 30% bound⁺Epitopes recognized by T cells, said antigen being specific for CD8⁺T cells are initiated by vaccination with a protein, or by infection by a pathogen containing the protein, or by malignant growth of cancer cells containing the protein. In another embodiment, the term refers to antigen-specific CD8 that is less than 28% bound⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigenic peptides that are more than 26% protectedAnisotropic CD8⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is less than 24% antigen-specific⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is greater than 22% bound⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is less than 20% bound⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is greater than 18% bound⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is less than 16% bound⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is greater than 14% bound⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is greater than 12% bound⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is less than 10% bound⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is greater than 8% bound⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is less than 6% bound⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is less than 5% bound⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is greater than 4% bound⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is less than 3% antigen-specific⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is less than 2% bound⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is less than 1% antigen-specific⁺Epitopes recognized by T cells. In another embodiment, the term refers to antigen-specific CD8 that is less than 0.5% bound⁺Epitopes recognized by T cells.

Each type of dominant and subdominant epitope represents a separate embodiment of the invention.

In one embodiment, the antigen in the methods and compositions of the invention is expressed at a detectable level on non-tumor cells of the subject. In another embodiment, the antigen is expressed at a detectable level on at least a percentage (e.g., 0.01%, 0.03%, 0.1%, 0.3%, 1%, 2%, 3%, or 5%) of non-tumor cells in the subject. In one embodiment, "non-tumor cell" refers to a cell outside of the tumor body. In another embodiment, "non-tumor cell" refers to a non-malignant cell. In another embodiment, "non-tumor cell" refers to a non-transformed cell. In another embodiment, the non-tumor cell is a somatic cell. In another embodiment, the non-tumor cell is a germ cell. Each possibility represents a separate embodiment of the invention.

In one embodiment, a "detectable level" refers to a level detectable by a standard assay. In one embodiment, the assay is an immunological assay. In one embodiment, the assay is an enzyme-linked immunoassay (ELISA). In another embodiment, the assay is a western blot. In another embodiment, the assay is FACS. One skilled in the art understands that any other assay available in the art can be used in the methods provided herein. In another embodiment, the detectable level is determined relative to a background level of the particular assay. Methods of performing each of these techniques are well known to those of skill in the art and each represents a separate embodiment of the present invention.

In one embodiment, vaccination with LM expressing a recombinant antigen induces epitope spreading. In another embodiment, vaccination with LLO-antigen fusions induces epitope spreading even outside the context of Her 2. Each possibility represents a separate embodiment of the invention.

In another embodiment, the present invention provides a method of impeding the growth of Her-2 expressing tumors in a subject comprising administering to the subject a recombinant polypeptide comprising an N-terminal fragment of an LLO protein fused to a Her-2 chimeric antigen, wherein the antigen has one or more subdominant CD8⁺T cell epitopes, wherein the subject increases the immune response against a tumor expressing the antigen, therebyPreventing the growth of Her-2 expressing tumors in the subject. In another embodiment, the antigen does not contain any dominant CD8⁺A T cell epitope. In another embodiment, provided herein is a method of preventing the growth of Her-2 expressing tumors in a subject, comprising administering to the subject a recombinant form of listeria comprising recombinant nucleotides encoding a recombinant polypeptide provided herein.

In another embodiment, the present invention provides a method of inducing the formation of cytotoxic T cells in a host having cancer, comprising administering to the host a composition of the present invention, thereby inducing the formation of cytotoxic T cells in a host having cancer.

In another embodiment, the present invention provides a method of reducing the incidence of cancer comprising administering a composition of the present invention. In another embodiment, the present invention provides a method of alleviating cancer comprising administering a composition of the present invention. Each possibility represents a separate embodiment of the invention.

In one embodiment, the composition is administered to cells of the subject ex vivo; in another embodiment, the composition is administered to a cell of a donor ex vivo; in another embodiment, the composition is administered to a cell of a donor in vivo and then transferred to a subject. Each possibility represents a separate embodiment of the invention.

In one embodiment, the cancer treated by the methods of the invention is breast cancer. In another embodiment, the cancer is a Her 2-containing cancer. In another embodiment, the cancer is melanoma. In another embodiment, the cancer is pancreatic cancer. In another embodiment, the cancer is ovarian cancer. In another embodiment, the cancer is gastric cancer. In another embodiment, the cancer is a cancerous lesion of the pancreas. In another embodiment, the cancer is lung adenocarcinoma. In another embodiment, the cancer is colorectal adenocarcinoma. In another embodiment, the cancer is a squamous cell lung carcinoma. In another embodiment, the cancer is gastric adenocarcinoma. In another embodiment, the cancer is an ovarian surface epithelial tumor (e.g., a benign, proliferative, or malignant variant thereof). In another embodiment, the cancer is oral squamous cell carcinoma. In another embodiment, the cancer is non-small cell lung cancer. In another embodiment, the cancer is a CNS cancer. In another embodiment, the cancer is endometrial cancer. In another embodiment, the cancer is bladder cancer. In another embodiment, the cancer is a head and neck cancer. In another embodiment, the cancer is prostate cancer. Each possibility represents a separate embodiment of the invention.

In another embodiment of the methods of the invention, the subject increases the immune response against the tumor expressing the antigen or the target antigen, thereby mediating an anti-tumor effect.

In another embodiment, the invention provides an immunogenic composition for the treatment of cancer, the composition comprising a fusion of a truncated LLO and a Her-2 chimeric protein. In another embodiment, the immunogenic composition further comprises a listeria strain expressing the fusion. Each possibility represents a separate embodiment of the invention. In another embodiment, the invention provides an immunogenic composition for the treatment of cancer, the composition comprising a Her-2 chimeric protein-expressing listeria strain.

In one embodiment, the treatment regimen of the present invention is therapeutic. In another embodiment, the regimen is prophylactic. In another embodiment, the vaccine of the present invention is used to protect people at risk for cancer (such as breast cancer or other types of Her 2-containing tumors) because of family inheritance or other circumstances that predispose them to these types of diseases, as will be appreciated by those skilled in the art. In another embodiment, the vaccine is used for cancer immunotherapy following tumor growth by surgical resection (debulking), conventional chemotherapy, or radiation therapy. Following these treatments, the vaccine of the present invention is administered, whereby the CTL response of the vaccine to the tumor antigen destroys the remaining metastases and prolongs the remission stage of the cancer. In another embodiment, the vaccine of the present invention is used to affect the growth of a previously established tumor and kill existing tumor cells. Each possibility represents a separate embodiment of the invention.

In another embodiment, the vaccines and immunogenic compositions used in any of the methods described above have any of the features of the vaccines and immunogenic compositions of the invention. Each feature represents a separate embodiment of the invention.

The present invention contemplates various embodiments of dosage ranges. In one embodiment, in the case of a vaccine vector, the dose range is 0.4LD₅₀(ii)/dose. In another embodiment, the dose is from about 0.4-4.9LD₅₀(ii)/dose. In another embodiment, the dose is from about 0.5-0.59LD₅₀(ii)/dose. In another embodiment, the dose is from about 0.6-0.69LD₅₀(ii)/dose. In another embodiment, the dose is from about 0.7-0.79LD₅₀(ii)/dose. In another embodiment, the dose is about 0.8LD₅₀(ii)/dose. In another embodiment, the dose is 0.4LD₅₀Dose to 0.8LD₅₀(ii)/dose.

In another embodiment, the dose is 10⁷Bacteria/dose. In another embodiment, the dose is 1.5x10⁷Bacteria/dose. In another embodiment, the dose is 2x10⁷Bacteria/dose. In another embodiment, the dose is 3x10⁷Bacteria/dose. In another embodiment, the dose is 4x10⁷Bacteria/dose. In another embodiment, the dose is 6x10⁷Bacteria/dose. In another embodiment, the dose is 8x10⁷Bacteria/dose. In another embodiment, the dose is 1x10⁸Bacteria/dose. In another embodiment, the dose is 1.5x10⁸Bacteria/dose. In another embodiment, the dose is 2x10⁸Bacteria/dose. In another embodiment, the dose is 3x10⁸Bacteria/dose. In another embodiment, the dose is 4x10⁸Bacteria/dose. In another embodiment, the dose is 6x10⁸Bacteria/dose. In another embodiment, the dose is 8x10⁸Bacteria/dose. In another embodiment, the dose is 1x10⁹Bacteria/dose. In another embodiment, the dosageIs 1.5x10⁹Bacteria/dose. In another embodiment, the dose is 2x10⁹Bacteria/dose. In another embodiment, the dose is 3x10⁹Bacteria/dose. In another embodiment, the dose is 5x10⁹Bacteria/dose. In another embodiment, the dose is 6x10⁹Bacteria/dose. In another embodiment, the dose is 8x10⁹Bacteria/dose. In another embodiment, the dose is 1x10¹⁰Bacteria/dose. In another embodiment, the dose is 1.5x10¹⁰Bacteria/dose. In another embodiment, the dose is 2x10¹⁰Bacteria/dose. In another embodiment, the dose is 3x10¹⁰Bacteria/dose. In another embodiment, the dose is 5x10¹⁰Bacteria/dose. In another embodiment, the dose is 6x10¹⁰Bacteria/dose. In another embodiment, the dose is 8x10¹⁰Bacteria/dose. In another embodiment, the dose is 8x10⁹Bacteria/dose. In another embodiment, the dose is 1x10¹¹Bacteria/dose. In another embodiment, the dose is 1.5x10¹¹Bacteria/dose. In another embodiment, the dose is 2x10¹¹Bacteria/dose. In another embodiment, the dose is 3x10¹¹Bacteria/dose. In another embodiment, the dose is 5x10¹¹Bacteria/dose. In another embodiment, the dose is 6x10¹¹Bacteria/dose. In another embodiment, the dose is 8x10¹¹Bacteria/dose. Each possibility represents a separate embodiment of the invention.

In one embodiment, the vaccine or immunogenic composition of the invention is administered to a subject separately. In another embodiment, the vaccine or immunogenic composition is administered with another cancer therapy. Each possibility represents a separate embodiment of the invention.

In one embodiment, the recombinant listeria of the methods and compositions of the present invention is stably transformed with a construct encoding a Her-2 chimeric antigen or a LLO-Her-2 chimeric antigen fusion. In one embodiment, the construct contains a polylinker to facilitate further subcloning. Several techniques are known for producing recombinant listeria.

In one embodiment, the construct or nucleic acid molecule is integrated into the listeria chromosome using homologous recombination. Techniques for homologous recombination are well known in the art and are described, for example, in Baloglu S, BoyleSM, et al (Immune responses of mice to vaccinia virus organisms expressing Listeria monocytogenes partial stereoslysin or Brucella abortus ribosomal L7/L12protein. vet Microbiol2005,109(1-2): 11-7); and Jiang LL, Song HH, et al, (Characterization of a mutant Listeria monocytogenes strain expressing greenfluorescent protein. acta Biochim Biophys Sin (Shanghai)2005,37(1): 19-24). In another embodiment, homologous recombination is performed according to the description provided in U.S. patent No. 6,855,320. In this case, a recombinant LM strain expressing E7 was made by chromosomal integration of the E7 gene, yielding a recombinant termed Lm-AZ/E7, in which the E7 gene is under the control of the hly promoter and contains the hly signal sequence to ensure secretion of the gene product. In another embodiment, recombinants are selected using a temperature sensitive plasmid. Each representing a separate embodiment of the invention.

In another embodiment, the construct or nucleic acid molecule is integrated into the listeria chromosome using transposon insertion. The technique of transposon insertion is well known in the art and is described in particular by Sun et al (Infection and Immunity1990, 58: 3770-3778) in the construction of DP-L967. In another embodiment, transposon mutagenesis has the advantage that stable genomic insertion mutants can be formed, but has the disadvantage that the location of the foreign gene insertion in the genome is unknown.

In another embodiment, the construct or nucleic acid molecule is integrated into the Listeria chromosome using a phage integration site (Lauer P, Chow MY et al, Construction, chromatography, and reduce of two Listeria monocytogenes sites-specific phase integration vectors JBacteriol2002;184(15): 4177-86). In certain embodiments of this method, the integrase gene and attachment site of a bacteriophage (e.g., U153 or PSA Listeria phage) are used to insert a heterologous gene into the corresponding attachment site, which can be any suitable site in the genome (e.g., the 3' end of the comK or arg RNA gene). In another embodiment, the endogenous prophages are self-healed from the attachment site used and then integrate the construct or heterologous gene. In another embodiment, the method produces a single copy integrant. Each possibility represents a separate embodiment of the invention.

In another embodiment, one of the various promoters is used to express an antigen or a fusion protein containing the antigen. In one embodiment, the LM promoter is used, for example, the promoters of the hly, actA, plca, plcB and mpl genes used to encode listerial proteins, hemolysin, actA, phosphatidylinositol-specific phospholipase, phospholipase C and metalloprotease, respectively. Each possibility represents a separate embodiment of the invention.

In another embodiment, the methods and compositions of the invention employ Her-2 chimeric proteins or homologs of the LLO sequences of the invention. In another embodiment, the methods and compositions of the invention use Her-2 chimeric proteins from non-human mammals. In one embodiment, the terms "homology", "homologous", and the like, when referring to any protein or peptide, refer to the percentage of amino acid residues in a candidate sequence that are identical to the residues of the corresponding native polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Methods and computer programs for alignment are well known in the art.

In another embodiment, the term "homology", when referring to any nucleic acid sequence, similarly indicates the percentage of nucleotides in the candidate sequence that are identical to the nucleotides of the corresponding native nucleic acid sequence.

In one embodiment, homology is determined by computer algorithms for sequence alignment, by methods well described in the art. For example, computer algorithmic analysis of nucleic acid sequence homology may include the use of any number of available software packages, such as, for example, BLAST, DOMAIN, bauity (BLAST enhanced alignment utility), genppept, and TREMBL packages.

In another embodiment, "homology" refers to greater than 70% identity to a sequence selected from SEQ ID NOS: 1-4. In another embodiment, "homology" refers to greater than 72% identity to a sequence selected from SEQ ID NOS: 1-4. In another embodiment, the identity is greater than 75%. In another embodiment, the identity is greater than 78%. In another embodiment, the identity is greater than 80%. In another embodiment, the identity is greater than 82%. In another embodiment, the identity is greater than 83%. In another embodiment, the identity is greater than 85%. In another embodiment, the identity is greater than 87%. In another embodiment, the identity is greater than 88%. In another embodiment, the identity is greater than 90%. In another embodiment, the identity is greater than 92%. In another embodiment, the identity is greater than 93%. In another embodiment, the identity is greater than 95%. In another embodiment, the identity is greater than 96%. In another embodiment, the identity is greater than 97%. In another embodiment, the identity is greater than 98%. In another embodiment, the identity is greater than 99%. In another embodiment, identity is 100%. Each possibility represents a separate embodiment of the invention.

In another embodiment, homology is determined by determining candidate sequence Hybridization, a method well described in the art (see, e.g., "Nucleic Acid Hybridization" Hames, B.D., and Higgins S.J., Eds. (1985); Sambrook et al, 2001, Molecular Cloning, analytical Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al, 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y.). For example, methods of hybridization to the complement of DNA encoding a native caspase peptide may be performed under moderate to stringent conditions. Hybridization conditions are, for example, incubation at 42 ℃ overnight at a temperature comprising: 10-20% formamide, 5 XSSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5 XDenhardt's solution, 10% dextran sulfate, and 20. mu.g/ml denatured, sheared salmon sperm DNA.

In one embodiment of the invention, a "nucleic acid" refers to a strand of at least two base-sugar-phosphate combinations. In one embodiment, the term includes DNA and RNA. In one embodiment, "nucleotide" refers to a monomeric unit of a nucleic acid polymer. In one embodiment, the RNA can be in the form of tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), antisense RNA, small inhibitory RNA (sirna), micro-RNA (mirna), and ribozymes. The use of siRNA and miRNA has been described (Caudy AA et al, Genes & Dedevel 16: 2491-96 and references therein). The DNA may be in the form of plasmid DNA, viral DNA, linear DNA or chromosomal DNA or derivatives of these groups. In addition, these forms of DNA and RNA can be single-stranded, double-stranded, triple-stranded, or quadruple-stranded. In another embodiment, the term also includes artificial nucleic acids, which may contain other types of backbones but are identical in base. In one embodiment, the artificial nucleic acid is PNA (peptide nucleic acid). PNAs contain a peptide backbone and nucleotide bases and, in one embodiment, are capable of binding to DNA and RNA molecules. In another embodiment, the nucleotide is oxetane (oxolane) modified. In another embodiment, the nucleotide is modified by replacing one or more phosphodiester linkages with phosphorothioate linkages. In another embodiment, the artificial nucleic acid comprises any other variant of the phosphate backbone of a natural nucleic acid known in the art. The use of phosphorothioate nucleic acids and PNA is known to those skilled in the art and is described, for example, in Neilsen PE, Curr Opin Struct Biol9:353-57 and Raz NK et al Biochem Biophys Res Commun.297: 1075-84. The production and use of nucleic acids is known to those skilled in the art and is described, for example, in Molecular Cloning, (2001), Sambrook and Russell, eds. and Methods in Enzymology: Methods for Molecular Cloning in eukaryotic cells (2003) purification and G.C.Fared. Each nucleic acid derivative represents a separate embodiment of the present invention.

In one embodiment, the homology of the proteins and/or peptides of any of the amino acid sequences listed herein is determined by methods well described in the art, including immunoblot analysis, or by computational algorithm analysis of the amino acid sequence (using any of a number of software packages available), by established methods. Some of these packages may include FASTA, BLAST, mpsrc or Scanps packages, and may be analyzed using, for example, the use of Smith and Waterman algorithms, and/or global/local or BLOCKS alignments. Each method of determining homology represents a separate embodiment of the invention.

In another embodiment, the invention provides a kit comprising reagents for performing the methods of the invention. In another embodiment, the present invention provides a kit comprising a composition, tool or device of the present invention.

In one embodiment, the term "contacting" or "administering" refers to the direct contact of a cancer cell or tumor with a composition of the invention. In another embodiment, the term refers to indirect contact of a cancer cell or tumor with a composition of the invention. In another embodiment, the methods of the invention include methods wherein the subject is contacted with the compositions of the invention, and thereafter the compositions are contacted with the cancer cells or tumors by diffusion or any other active or passive transport method known in the art that circulates the compounds in the body. Each possibility represents a separate embodiment of the invention.

In another embodiment, the terms "gene" and "recombinant gene" refer to a nucleic acid molecule comprising an open reading frame encoding a polypeptide of the present invention. Typically, such natural allelic variants may produce 1-5% variation in the nucleotide sequence of a given gene. Alternative alleles can be identified by sequencing the gene of interest in many different individuals or organisms. This can be readily carried out by using hybridization probes to identify the same locus in many individuals or organisms. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations that are the result of natural allelic variation and that do not alter functional activity are within the scope of the invention.

Pharmaceutical composition

In another embodiment, the pharmaceutical compositions containing the vaccines and compositions of the present invention are administered to a subject by any method known to those skilled in the art, such as parenterally, peri-carcinostically (paracancerally), transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, intracranially, intravaginally, or intratumorally.

In another embodiment of the methods and compositions provided herein, the vaccine or composition is administered orally, and thus, is formulated in a form suitable for oral administration, i.e., as a solid or liquid formulation. Suitable solid oral dosage forms include tablets, capsules, pills, granules, pellets and the like. Suitable liquid oral dosage forms include solutions, suspensions, dispersions, emulsions, oils, and the like. In another embodiment of the invention, the active ingredient is formulated in a capsule. According to this embodiment, the composition of the invention comprises, in addition to the active compound and the inert carrier or diluent, a hard gel capsule.

In another embodiment, the vaccine or composition is administered by intravenous, intra-arterial, or intramuscular injection of a liquid formulation. Suitable liquid dosage forms include solutions, suspensions, dispersions, emulsions, oils, and the like. In one embodiment, the pharmaceutical composition is administered intravenously, and thus is formulated in a form suitable for intravenous administration. In another embodiment, the pharmaceutical composition is administered intra-arterially, and is therefore formulated in a form suitable for intra-arterial administration. In another embodiment, the pharmaceutical composition is administered intramuscularly, and thus it is formulated in a form suitable for intramuscular administration.

In one embodiment, the term "treating" refers to curing a disease. In another embodiment, "treating" or "treatment" refers to preventing a disease. In another embodiment, "treating" or "treatment" refers to reducing the incidence of disease. In another embodiment, "treating" or "treatment" refers to ameliorating the symptoms of a disease. In another embodiment, "treating" refers to increasing the non-performance survival or overall survival of a patient. In another embodiment, "treating" or "treatment" refers to stabilizing the progression of the disease. In another embodiment, "treating" or "treatment" refers to inducing remission. In another embodiment, "treating" or "treatment" refers to slowing the progression of the disease. In another embodiment, the terms "reduce", "suppress" and "inhibiting" refer to alleviating or reducing. Each possibility represents a separate embodiment of the invention.

As used herein, the term "about" refers in numerical terms to plus or minus 5%, or plus or minus 10% in another embodiment, or plus or minus 15% in another embodiment, or plus or minus 20% in another embodiment.

In one embodiment, the term "subject" refers to a mammal, including a canine, in need of treatment or susceptible to a condition or sequela thereof. Subjects may include dogs of various breeds, zoo animals including wolves, cats, pigs, cows, sheep, goats, horses, rats and mice, and humans. The term "subject" does not exclude individuals who are normal in all respects.

In one embodiment, for therapeutic purposes, the term "mammal" refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as canines (including dogs) and horses, cats, cows, pigs, sheep, and the like.

In one embodiment, the canine may be an adult or juvenile canine.

In reference to treatment of a tumor, a "therapeutically effective amount" refers to an amount capable of causing one or more of the following effects: (1) inhibit tumor growth to some extent, including, slowing growth and completely inhibiting growth; (2) reducing the number of tumor cells; (3) reducing tumor size; (4) inhibit (i.e., reduce, slow, or completely stop) tumor cell infiltration into peripheral organs; (5) inhibit (i.e., reduce, slow, or completely stop) metastasis; (6) enhancing an anti-tumor immune response, which may, but need not, result in regression or rejection of the tumor; and/or (7) relieve to some extent one or more symptoms associated with the disease. For the purpose of treating tumors, a "therapeutically effective amount" of a vaccine provided herein can be determined empirically or in a routine manner.

The following examples are presented in order to more fully illustrate the preferred embodiments of the present invention. However, they should in no way be considered as limiting the scope of the invention.

Examples

Materials and methods

Oligonucleotides were synthesized by Invitrogen (Carlsbad, CA) and DNA sequencing was performed by Genewiz Inc, southpainfield, NJ. Flow cytometry reagents were purchased from Becton dickinson biosciences (BD, San Diego, CA). Cell culture medium, supplements and all other reagents, unless indicated, were from Sigma (st. Her2/neu HLA-A2 peptide was synthesized by EZbiolabs (Westfield, IN). Complete RPMI1640(C-RPMI) medium contained 2mM glutamine, 0.1mM non-essential amino acids and 1mM sodium pyruvate, 10% fetal bovine serum, penicillin/streptomycin, Hepes (25 mM). Polyclonal anti-LLO antibodies were previously described, while anti-Her 2/neu antibodies were purchased from Sigma.

Mouse and cell lines

All animal experiments were conducted at university of pennsylvania or university of regce according to IACUC approved protocol. FVB/N mice were purchased from Jackson laboratories (Bar Harbor, ME). FVB/NHer2/neu transgenic mice overexpressing rat Her2/neu tumor protein were housed and raised in the animal center at the university of Pennsylvania. The NT-2 tumor cell line expressed high levels of rat Her2/neu protein, derived from spontaneous breast tumors of these mice, cultured as described previously. DHFR-G8(3T3/neu) cells were obtained from the ATCC and cultured according to the ATCC recommendations. The EMT6-Luc cell line was generous from dr. john Ohlfest (University of Minnesota, MN) and cultured in complete C-RPMI medium. Bioluminescence work was conducted under the direction of the Small Animal Imaging Facility (SAIF) at the university of pennsylvania (philiadelphia, PA).

Listeria constructs and antigen expression

Mark Greene, at university of pennsylvania, friendly provides Her2/neu-pGEM7Z containing the full-length human Her2/neu (hHer2) gene cloned into the pGEM7Z plasmid (Promega, Madison WI). This plasmid was used as a template to amplify three segments of hHer-2/neu, i.e., EC1, EC2, and IC1, by PCR using pfx DNA polymerase (Invitrogen) and oligonucleotides shown in table 1.

Table 1: primers for cloning human her-2 chimera

The Her-2/neu chimeric construct was generated by direct fusion of the SOEing PCR method and each individual hHer-2/neu segment as template. The primers are shown in table 2.

Sequences of primers for amplifying different regions of human Her2

The ChHer2 gene was excised from pAdv138 using XhoI and SpeI restriction enzymes and cloned in the framework of a truncated, non-hemolytic fragment with LLO in an Lmdd shuttle vector (pAdv 134). The sequences of the insert, LLO and hly promoters were confirmed by DNA sequencing analysis. The plasmid was electroporated into an electro-competent actA, dal, dat mutant listeria monocytogenes strain and LmddA and positive clones were selected on Brain Heart Infusion (BHI) agar plates containing streptomycin (250 μ g/ml). In some experiments, similar Listeria strains expressing a hHer2/neu (Lm-hHer2) fragment were used for comparative purposes. These have been described previously. In all studies, an unrelated listeria construct (Lm-control) was included to take into account the antigen independent effect of listeria on the immune system. Lm-control is based on the same Listeria platform as ADXS31-164, but expresses different antigens such as HPV16-E7 or NY-ESO-1. The expression and secretion of fusion proteins from listeria was tested. Each construct was passaged twice in vivo.

Cytotoxicity assays

3-5 FVB/N mice per group, each group at 1X10 weekly intervals⁸Lm-LLO-ChHer2, ADXS31-164, Lm-hHer2ICI or Lm-control (expressing irrelevant antigens) of Colony Forming Units (CFU) were immunized three times or not. NT-2 cells were grown in vitro, detached by trypsin and treated with mitomycin C (250. mu.g/ml in serum-free C-RPMI medium) for 45 min at 37 ℃. After 5 washes, they were combined with splenocytes harvested from immunized or non-immunized animals at a ratio of 1:5 (stimulator: effector) at 37 ℃ and 5% CO₂The mixture was incubated for 5 days. Standard cytotoxicity assays were performed according to the methods described previously, using europium-labeled 3T3/neu (DHFR-G8) cells as targets. After 4 hours incubation, a spectrophotometer (Perkin Elmer, Victor) was used²) Europium release from killed target cells was measured at 590 nm. The percentage of specific lysis was defined as (lysis-spontaneous lysis)/(maximum lysis-spontaneous lysis of the experimental group).

Secretion of interferon-gamma from splenocytes from immunized mice

3-5 FVB/N or HLA-A2 transgenic mice per group, each group treated with 1X10 at weekly intervals⁸CFU ADXS31-164, negative listeria control (expressing an unrelated antigen) were immunized three times or not. One week after the last immunization, splenocytes were isolated from FVB/N mice and plated at 5 × 10⁶Individual cells/well were co-cultured with mitomycin C-treated NT-2 cells in C-RPMI medium in 24-well plates. Splenocytes from HLA-a2 transgenic mice were incubated in the presence of 1 μ M HLA-a2 specific peptide or 1 μ g/ml of recombinant His-tagged ChHer 2protein (produced in e.coli and purified by a nickel-based affinity chromatography system). Samples from supernatants were obtained after 24 or 72 hours and mouse interferon-gamma (IFN-. gamma.) was usedEnzyme-linked immunosorbent assay (ELISA) kits were tested for the presence of IFN-. gamma.according to the manufacturer's recommendations.

Tumor study in Her2 transgenic animals

With 5x10⁸CFU Lm-LLO-ChHer2, ADXS31-164 or Lm-control immunized six weeks old FVB/N rat Her2/neu transgenic mice (9-14/group) 6 times. Their appearance of spontaneous breast tumors was observed twice a week, measured using electronic calipers, for up to 52 weeks. When the escaped (escaped) tumor reached an average diameter of 1cm²Size (C) it was excised and stored in RNAlater at-20 ℃. To determine the effect of mutations in Her2/neu protein on the escape of these tumors, genomic DNA was extracted using a genomic DNA isolation kit and sequenced.

Effect of ADXS31-164 on regulatory T cells in spleen and tumors

Mice were implanted subcutaneously (s.c.) with 1x10⁶NT-2 cells. On days 7, 14 and 21, they were treated with 1X10⁸ADXS31-164, LmddA-control of CFU, or immunization. Tumors and spleens were extracted at 28 days and tested for CD3 by FACS analysis⁺/CD4⁺/FoxP3⁺The presence of tregs. Briefly, splenocytes were isolated by homogenizing the spleen between two slides in C-RPMI medium. Tumors were minced using a sterile razor blade and digested with buffer containing DNase (12U/ml) and collagenase (2mg/ml) in PBS. After incubation with stirring at RT for 60min, cells were isolated by vigorous aspiration. Erythrocytes were lysed with RBC lysis buffer followed by several washes with complete RPMI-1640 medium containing 10% FBS. After filtration through a nylon mesh screen, tumor cells and splenocytes were resuspended in FACS buffer (2% FBS/PBS) and stained with anti-CD 3-PerCP-Cy5.5, CD4-FITC, CD25-APC antibodies, followed by permeabilization and staining with anti-Foxp 3-PE. Flow cytometry analysis was performed using 4-color FACS calibur (BD) and data were analyzed using cell-finding software (BD).

Statistical analysis

The log-rank Chi-Squared test was used for survival data and student t-tests were used for CTL and ELISA assays, which were performed in triplicate. P-values (marked by x) of less than 0.05 were considered statistically significant in these analyses. All statistical analyses were performed using Prism software, v.4.0a (2006) or SPSS software, v.15.0 (2006). For all FVB/N rat Her2/neu transgenic studies, we used 8-14 mice per group unless otherwise stated, and at least 8 mice per group for all wild type FVB/N studies. All studies were repeated at least once, except for long-term tumor studies in Her2/neu transgenic mouse models.

Example 1: listeria monocytogenes strains producing an LLO fragment that secretes fusion with Her-2 fragment: construction of ADXS 31-164.

The construction of a chimeric Her2/neu gene (ChHer2) was previously described. Briefly, the ChHer2 gene was generated by direct fusion of two extracellular (aa40-170 and aa359-433) and one intracellular fragment (aa678-808) of the Her2/neu protein by the SOEingPCR method. Chimeric proteins have most of the known epitopes of human MHC class I proteins. The ChHer2 gene was excised from plasmid pAdv138 (which was used to construct Lm-LLO-ChHer2) and cloned into the LmddA shuttle plasmid, resulting in plasmid pAdv164 (fig. 1A). There are two major differences between these two plasmid backbones. 1) While pAdv138 used the chloramphenicol resistance marker (cat) for in vitro selection of recombinant bacteria, pAdv164 has the D-alanine racemase gene (dal) from bacillus subtilis, which uses a metabolic complementation pathway for in vitro selection and in vivo plasmid maintenance in the LmddA strain without the dal-dat gene. This vaccine platform was designed and developed to address concerns of FDA antibiotic resistance to engineered listeria vaccine strains. 2) Unlike pAdv138, pAdv164 had no prfA gene copy in the plasmid (see sequence below and figure 1A), as this was not necessary for in vivo supplementation with the Lmdd strain. The LmddA vaccine strain also lacks the actA gene (responsible for intracellular movement and cell-to-cell spread of Listeria), so the recombinant vaccine strain derived from this backbone is 100 times less pathogenic than the Lmdd derived from its parent strain. The LmddA-based vaccine also cleared faster (less than 48 hours) than the Lmdd-based vaccine from the spleens of immunized mice. The expression and secretion of the fusion protein tLLO-ChHer2 from this strain was comparable to the expression and secretion of Lm-LLO-ChHer2 in cell culture supernatants of TCA pellet after 8 hours of in vitro growth (fig. 1B), since a band of-104 KD was detected by anti-LLO antibody using western blot analysis. A listeria backbone strain expressing only tLLO was used as a negative control.

pAdv164 sequence (7075 base pairs) (see fig. 1):

cggagtgtatactggcttactatgttggcactgatgagggtgtcagtgaagtgcttcatgtggcaggagaaaaaaggctgcaccggtgcgtcagcagaatatgtgatacaggatatattccgcttcctcgctcactgactcgctacgctcggtcgttcgactgcggcgagcggaaatggcttacgaacggggcggagatttcctggaagatgccaggaagatacttaacagggaagtgagagggccgcggcaaagccgtttttccataggctccgcccccctgacaagcatcacgaaatctgacgctcaaatcagtggtggcgaaacccgacaggactataaagataccaggcgtttccccctggcggctccctcgtgcgctctcctgttcctgcctttcggtttaccggtgtcattccgctgttatggccgcgtttgtctcattccacgcctgacactcagttccgggtaggcagttcgctccaagctggactgtatgcacgaaccccccgttcagtccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggaaagacatgcaaaagcaccactggcagcagccactggtaattgatttagaggagttagtcttgaagtcatgcgccggttaaggctaaactgaaaggacaagttttggtgactgcgctcctccaagccagttacctcggttcaaagagttggtagctcagagaaccttcgaaaaaccgccctgcaaggcggttttttcgttttcagagcaagagattacgcgcagaccaaaacgatctcaagaagatcatcttattaatcagataaaatatttctagccctcctttgattagtatattcctatcttaaagttacttttatgtggaggcattaacatttgttaatgacgtcaaaaggatagcaagactagaataaagctataaagcaagcatataatattgcgtttcatctttagaagcgaatttcgccaatattataattatcaaaagagaggggtggcaaacggtatttggcattattaggttaaaaaatgtagaaggagagtgaaacccatgaaaaaaataatgctagtttttattacacttatattagttagtctaccaattgcgcaacaaactgaagcaaaggatgcatctgcattcaataaagaaaattcaatttcatccatggcaccaccagcatctccgcctgcaagtcctaagacgccaatcgaaaagaaacacgcggatgaaatcgataagtatatacaaggattggattacaataaaaacaatgtattagtataccacggagatgcagtgacaaatgtgccgccaagaaaaggttacaaagatggaaatgaatatattgttgtggagaaaaagaagaaatccatcaatcaaaataatgcagacattcaagttgtgaatgcaatttcgagcctaacctatccaggtgctctcgtaaaagcgaattcggaattagtagaaaatcaaccagatgttctccctgtaaaacgtgattcattaacactcagcattgatttgccaggtatgactaatcaagacaataaaatagttgtaaaaaatgccactaaatcaaacgttaacaacgcagtaaatacattagtggaaagatggaatgaaaaatatgctcaagcttatccaaatgtaagtgcaaaaattgattatgatgacgaaatggcttacagtgaatcacaattaattgcgaaatttggtacagcatttaaagctgtaaataatagcttgaatgtaaacttcggcgcaatcagtgaagggaaaatgcaagaagaagtcattagttttaaacaaatttactataacgtgaatgttaatgaacctacaagaccttccagatttttcggcaaagctgttactaaagagcagttgcaagcgcttggagtgaatgcagaaaatcctcctgcatatatctcaagtgtggcgtatggccgtcaagtttatttgaaattatcaactaattcccatagtactaaagtaaaagctgcttttgatgctgccgtaagcggaaaatctgtctcaggtgatgtagaactaacaaatatcatcaaaaattcttccttcaaagccgtaatttacggaggttccgcaaaagatgaagttcaaatcatcgacggcaacctcggagacttacgcgatattttgaaaaaaggcgctacttttaatcgagaaacaccaggagttcccattgcttatacaacaaacttcctaaaagacaatgaattagctgttattaaaaacaactcagaatatattgaaacaacttcaaaagcttatacagatggaaaaattaacatcgatcactctggaggatacgttgctcaattcaacatttcttgggatgaagtaaattatgatctcgagacccacctggacatgctccgccacctctaccagggctgccaggtggtgcagggaaacctggaactcacctacctgcccaccaatgccagcctgtccttcctgcaggatatccaggaggtgcagggctacgtgctcatcgctcacaaccaagtgaggcaggtcccactgcagaggctgcggattgtgcgaggcacccagctctttgaggacaactatgccctggccgtgctagacaatggagacccgctgaacaataccacccctgtcacaggggcctccccaggaggcctgcgggagctgcagcttcgaagcctcacagagatcttgaaaggaggggtcttgatccagcggaacccccagctctgctaccaggacacgattttgtggaagaatatccaggagtttgctggctgcaagaagatctttgggagcctggcatttctgccggagagctttgatggggacccagcctccaacactgccccgctccagccagagcagctccaagtgtttgagactctggaagagatcacaggttacctatacatctcagcatggccggacagcctgcctgacctcagcgtcttccagaacctgcaagtaatccggggacgaattctgcacaatggcgcctactcgctgaccctgcaagggctgggcatcagctggctggggctgcgctcactgagggaactgggcagtggactggccctcatccaccataacacccacctctgcttcgtgcacacggtgccctgggaccagctctttcggaacccgcaccaagctctgctccacactgccaaccggccagaggacgagtgtgtgggcgagggcctggcctgccaccagctgtgcgcccgagggcagcagaagatccggaagtacacgatgcggagactgctgcaggaaacggagctggtggagccgctgacacctagcggagcgatgcccaaccaggcgcagatgcggatcctgaaagagacggagctgaggaaggtgaaggtgcttggatctggcgcttttggcacagtctacaagggcatctggatccctgatggggagaatgtgaaaattccagtggccatcaaagtgttgagggaaaacacatcccccaaagccaacaaagaaatcttagacgaagcatacgtgatggctggtgtgggctccccatatgtctcccgccttctgggcatctgcctgacatccacggtgcagctggtgacacagcttatgccctatggctgcctcttagactaatctagacccgggccactaactcaacgctagtagtggatttaatcccaaatgagccaacagaaccagaaccagaaacagaacaagtaacattggagttagaaatggaagaagaaaaaagcaatgatttcgtgtgaataatgcacgaaatcattgcttatttttttaaaaagcgatatactagatataacgaaacaacgaactgaataaagaatacaaaaaaagagccacgaccagttaaagcctgagaaactttaactgcgagccttaattgattaccaccaatcaattaaagaagtcgagacccaaaatttggtaaagtatttaattactttattaatcagatacttaaatatctgtaaacccattatatcgggtttttgaggggatttcaagtctttaagaagataccaggcaatcaattaagaaaaacttagttgattgccttttttgttgtgattcaactttgatcgtagcttctaactaattaattttcgtaagaaaggagaacagctgaatgaatatcccttttgttgtagaaactgtgcttcatgacggcttgttaaagtacaaatttaaaaatagtaaaattcgctcaatcactaccaagccaggtaaaagtaaaggggctatttttgcgtatcgctcaaaaaaaagcatgattggcggacgtggcgttgttctgacttccgaagaagcgattcacgaaaatcaagatacatttacgcattggacaccaaacgtttatcgttatggtacgtatgcagacgaaaaccgttcatacactaaaggacattctgaaaacaatttaagacaaatcaataccttctttattgattttgatattcacacggaaaaagaaactatttcagcaagcgatattttaacaacagctattgatttaggttttatgcctacgttaattatcaaatctgataaaggttatcaagcatattttgttttagaaacgccagtctatgtgacttcaaaatcagaatttaaatctgtcaaagcagccaaaataatctcgcaaaatatccgagaatattttggaaagtctttgccagttgatctaacgtgcaatcattttgggattgctcgtataccaagaacggacaatgtagaattttttgatcccaattaccgttattctttcaaagaatggcaagattggtctttcaaacaaacagataataagggctttactcgttcaagtctaacggttttaagcggtacagaaggcaaaaaacaagtagatgaaccctggtttaatctcttattgcacgaaacgaaattttcaggagaaaagggtttagtagggcgcaatagcgttatgtttaccctctctttagcctactttagttcaggctattcaatcgaaacgtgcgaatataatatgtttgagtttaataatcgattagatcaacccttagaagaaaaagaagtaatcaaaattgttagaagtgcctattcagaaaactatcaaggggctaatagggaatacattaccattctttgcaaagcttgggtatcaagtgatttaaccagtaaagatttatttgtccgtcaagggtggtttaaattcaagaaaaaaagaagcgaacgtcaacgtgttcatttgtcagaatggaaagaagatttaatggcttatattagcgaaaaaagcgatgtatacaagccttatttagcgacgaccaaaaaagagattagagaagtgctaggcattcctgaacggacattagataaattgctgaaggtactgaaggcgaatcaggaaattttctttaagattaaaccaggaagaaatggtggcattcaacttgctagtgttaaatcattgttgctatcgatcattaaattaaaaaaagaagaacgagaaagctatataaaggcgctgacagcttcgtttaatttagaacgtacatttattcaagaaactctaaacaaattggcagaacgccccaaaacggacccacaactcgatttgtttagctacgatacaggctgaaaataaaacccgcactatgccattacatttatatctatgatacgtgtttgtttttctttgctggctagcttaattgcttatatttacctgcaataaaggatttcttacttccattatactcccattttccaaaaacatacggggaacacgggaacttattgtacaggccacctcatagttaatggtttcgagccttcctgcaatctcatccatggaaatatattcatccccctgccggcctattaatgtgacttttgtgcccggcggatattcctgatccagctccaccataaattggtccatgcaaattcggccggcaattttcaggcgttttcccttcacaaggatgtcggtccctttcaattttcggagccagccgtccgcatagcctacaggcaccgtcccgatccatgtgtctttttccgctgtgtactcggctccgtagctgacgctctcgccttttctgatcagtttgacatgtgacagtgtcgaatgcagggtaaatgccggacgcagctgaaacggtatctcgtccgacatgtcagcagacgggcgaaggccatacatgccgatgccgaatctgactgcattaaaaaagccttttttcagccggagtccagcggcgctgttcgcgcagtggaccattagattctttaacggcagcggagcaatcagctctttaaagcgctcaaactgcattaagaaatagcctctttctttttcatccgctgtcgcaaaatgggtaaatacccctttgcactttaaacgagggttgcggtcaagaattgccatcacgttctgaacttcttcctctgtttttacaccaagtctgttcatccccgtatcgaccttcagatgaaaatgaagagaaccttttttcgtgtggcgggctgcctcctgaagccattcaacagaataacctgttaaggtcacgtcatactcagcagcgattgccacatactccgggggaaccgcgccaagcaccaatataggcgccttcaatccctttttgcgcagtgaaatcgcttcatccaaaatggccacggccaagcatgaagcacctgcgtcaagagcagcctttgctgtttctgcatcaccatgcccgtaggcgtttgctttcacaactgccatcaagtggacatgttcaccgatatgttttttcatattgctgacattttcctttatcgcggacaagtcaatttccgcccacgtatctctgtaaaaaggttttgtgctcatggaaaactcctctcttttttcagaaaatcccagtacgtaattaagtatttgagaattaattttatattgattaatactaagtttacccagttttcacctaaaaaacaaatgatgagataatagctccaaaggctaaagaggactataccaactatttgttaattaa(SED ID NO：53)

example 2: ADXS31-164 is as immunogenic as LM-LLO-ChHER 2.

In a standard CTL assay, the immunogenic properties of ADXS31-164 to generate anti-Her 2/neu specific cytotoxic T cells were compared to the immunogenic properties of the Lm-LLO-ChHer2 vaccine. Both vaccines elicited strong but comparable cytotoxic T cell responses to Her2/neu antigen expressed by 3T3/neu target cells. Therefore, mice immunized with listeria expressing only Her2 intracellular fragment fused to LLO showed lower lytic activity than chimeras containing more MHC class I epitopes. No CTL activity was detected in non-immunized animals or mice injected with an unrelated listeria vaccine (fig. 2A). ADXS31-164 was also able to stimulate IFN-. gamma.secretion from splenocytes from wild type FVB/N mice (FIG. 2B). This was detected in the culture supernatants of these cells co-cultured with mitomycin C treated NT-2 cells expressing high levels of Her2/neu antigen (FIG. 5C).

After immunization with ADXS31-164, appropriate processing and presentation of human MHC class I epitopes was detected in HLA-A2 mice. Spleen cells from the immunized HLA-A2 transgene were incubated for 72 hours with peptides corresponding to the restricted epitope of HLA-A2 located in the Her2/neu molecule, either extracellular (HLYQGCQVV SEQ ID NO:11 or KIFGSLAFL SEQ ID NO:12) or intracellular (RLLQETELV SEQ ID NO:13) (FIG. 2C). Recombinant ChHer 2protein was used as a positive control, with no or no peptide as a negative control. Data from this experiment show that ADXS31-164 is capable of eliciting anti-Her 2/neu specific immune responses against human epitopes located in different domains of the target antigen.

Example 3: ADXS31-164 is more effective than LM-LLO-ChHER2 in preventing the onset of spontaneous breast tumors.

Comparison of the antitumor effects of ADXS31-164 and Lm-LLO-ChHer2 in Her2/neu transgenic animals that develop slow-growing, spontaneous breast tumors at 20-25 weeks of age. All animals immunized with the irrelevant listeria-control vaccine developed breast tumors within 21-25 weeks and were killed before 33 weeks. In contrast, the recombinant Listeria-Her 2/neu vaccine caused a significant delay in breast tumor formation. At week 45, more than 50% of mice vaccinated with ADXS31-164 (5 out of 9) remained tumor-free, compared to 25% of mice immunized with Lm-LLO-ChHer 2. At week 52, 2 of the 8 mice immunized with ADXS31-164 remained tumor-free, while all mice from the other experimental groups had died of disease (fig. 3). These results indicate that although ADXS31-164 is more attenuated, it is more effective than Lm-LLO-ChHer2 in preventing the onset of spontaneous breast tumors in Her2/neu transgenic animals.

Example 4: mutation of the HER2/NEU gene when immunized with ADXS 31-164.

Mutations in MHC class I epitopes of Her2/neu are thought to be responsible for tumor escape when immunized with small fragment vaccines or trastuzumab (Herceptin, a monoclonal antibody targeting an epitope in the extracellular domain of Her 2/neu). To assess this, genomic material was extracted from the escaped tumors of transgenic animals and the corresponding fragments of the neu gene in tumors immunized with chimeric or control vaccines were sequenced. No mutations were observed in the Her-2/neu gene of any of the vaccinated tumor samples, suggesting additional escape mechanisms (data not shown).

Example 5: ADXS31-164 caused a significant decrease in T regulatory cells within the tumor.

To illustrate the effect of ADXS31-164 on regulatory T cell frequency in spleen and tumors, mice were implanted with NT-2 tumor cells. Splenocytes and intratumoral lymphocytes were isolated after three immunizations and Treg staining was performed, which was defined as CD3⁺/CD4⁺/CD25⁺/FoxP3⁺Cells, although when analyzed alone, were labeled with FoxP3 or CD25 with comparable results. The results show that immunization with ADXS31-164 had no effect on the frequency of tregs in the spleen compared to an unrelated listeria vaccine or non-immunized animals (see fig. 4). In contrast, immunization with the listeria vaccine had a significant effect on the presence of tregs in the tumor (fig. 5A). All CD3 in untreated tumors⁺On average 19.0% of T cells were tregs, the frequency decreased to 4.2% for unrelated vaccines and 3.4% for ADXS31-164, with a 5-fold decrease in the frequency of intratumoral tregs (fig. 5B). The decrease in frequency of intratumoral tregs in mice treated with the LmddA vaccine was not attributable to differences in tumor size. In a representative experiment, tumors from mice immunized with ADXS31-164 [ mean diameter (mm) ± SD, 6.71 ± 0.43, n =5 ± ]]Significantly less than from untreated mice (8.69 ± 0.98, n =5, p<0.01) or mice treated with an irrelevant vaccine (8.41 ± 1.47, n =5, p =0.04), whereas a comparison of these latter two groups shows no tumor sizeThere was a statistically significant difference (p = 0.73). The lower frequency of tregs in tumors treated with the LmddA vaccine resulted in an increased intratumoral CD8/Treg ratio, suggesting that a more favorable tumor microenvironment could be obtained after immunization with the LmddA vaccine. However, only the vaccine expressing the target antigen HER2/neu (ADXS31-164) was able to reduce tumor growth, indicating that the decline in tregs was only effective in this presence of an antigen-specific response in the tumor.

Example 6: HER-2 chimera expressing listeria vaccines without introducing escape mutations

Tumor samples of mice immunized with different vaccines such as Lm-LLO-138, LmddA164 and the unrelated vaccine Lm-LLO-NY were harvested. DNA was purified from these samples, DNA fragments corresponding to the Her-2/neu regions IC1, EC1, and EC2 were amplified, and sequenced to determine if there were immune escape mutations. Alignment of sequences from each DNA was performed using CLUSTALW. The results of the analysis showed no mutations in the DNA sequences harvested from the tumors. The detailed analysis of these sequences is shown below.

Alignment EC2 (975-1029 bp for Her-2-neu)

Reference to

GGTCACAGCTGAGGACGGAACACAGCGTTGTGAGAAATGCAGCAAGCCCTGTGCT

(SEQ ID NO:14)

Lm-LLO-138-2 GGTCACAGCTGAGGACGGAACACAGCGTTGTGAGAAATGCAGCAAGCCCTGTGCT

Lm-LLO-138-3 GGTCACAGCTGAGGACGGAACACAGCGTTGTGAGAAATGCAGCAAGCCCTGTGCT

Lm-ddA-164-1 GGTCACAGCTGAGGACGGAACACAGCGTTGTGAGAAATGCAGCAAGCCCTGTGCT

LmddA164-2 GGTCACAGCTGAGGACGGAACACAGCGTTGTGAGAAATGCAGCAAGCCCTGTGCT

Lm-ddA-164-3 GGTCACAGCTGAGGACGGAACACAGCGTTGTGAGAAATGCAGCAAGCCCTGTGCT

LmddA164-4 GGTCACAGCTGAGGACGGAACACAGCGTTGTGAGAAATGCAGCAAGCCCTGTGCT

Lm-ddA-164-5 GGTCACAGCTGAGGACGGAACACAGCGTTGTGAGAAATGCAGCAAGCCCTGTGCT

LmddA-164-6 GGTCACAGCTGAGGACGGAACACAGCGTTCTGAGAAATGCAGCAAGCCCTGTGCT

Reference to

CGAGTGTGCTATGGTCTGGGCATGGAGCACCTTCGAGGGGCGAGGGCCATCACCAGTGAC

(SEQ ID NO:15)

Lm-LLO-138-2

CGAGTGTGCTATGGTCTGGGCATGGAGCACCTTCGAGGGGCGAGGGCCATCACCAGTGAC

Lm-LLO-138-3

CGAGTGTGCTATGGTCTGGGCATGGAGCACCTTCGAGGGGCGAGGGCCATCACCAGTGAC

Lm-ddA-164-1

CGAGTGTGCTATGGTCTGGGCATGGAGCACCTTCGAGGGGCGAGGGCCATCACCAGTGAC

LmddA164-2

CGAGTGTGCTATGGTCTGGGCATGGAGCACCTTCGAGGGGCGAGGGCCATCACCAGTGAC

Lm-ddA-164-3

CGAGTGTGCTATGGTCTGGGCATGGAGCACCTTCGAGGGGCGAGGGCCATCACCAGTGAC

LmddA164-4

CGAGTGTGCTATGGTCTGGGCATGGAGCACCTTCGAGGGGCGAGGGCCATCACCAGTGAC

Lm-ddA-164-5

CGAGTGTGCTATGGTCTGGGCATGGAGCACCTTCGAGGGGCGAGGGCCATCACCAGTGAC

LmddA-164-6

CGAGTGTGCTATGGTCTGGGCATGGAGCACCTTCGAGGGGCGAGGGCCATCACCAGTGAC

Reference to

AATGTCCAGGAGTTTGATGGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTTTGCCGGAG

(SEQ ID No:16)

Lm-LLO-138-2

AATGTCCAGGAGTTTGATGGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTTTGCCGGAG

Lm-LLO-138-3

AATGTCCAGGAGTTTGATGGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTTTGCCGGAG

Lm-ddA-164-1

AATGTCCAGGAGTTTGATGGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTTTGCCGGAG

LmddA164-2

AATGTCCAGGAGTTTGATGGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTTTGCCGGAG

Lm-ddA-164-3

AATGTCCAGGAGTTTGATGGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTTTGCCGGAG

LmddA164-4

AATGTCCAGGAGTTTGATGGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTTTGCCGGAG

Lm-ddA-164-5

AATGTCCAGGAGTTTGATGGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTTTGCCGGAG

LmddA-164-6

AATGTCCAGGAGTTTGATGGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTTTGCCGGAG

Reference to

AGCTTTGATGGGGACCCCTCCTCCGGCATTGCTCCGCTGAGGCCTGAGCAGCTCCAAGTG

(SEQ ID No:17)

Lm-LLO-138-2

AGCTTTGATGGGGACCCCTCCTCCGGCATTGCTCCGCTGAGGCCTGAGCAGCTCCAAGTG

Lm-LLO-138-3

AGCTTTGATGGGGACCCCTCCTCCGGCATTGCTCCGCTGAGGCCTGAGCAGCTCCAAGTG

Lm-ddA-164-1

AGCTTTGATGGGGACCCCTCCTCCGGCATTGCTCCGCTGAGGCCTGAGCAGCTCCAAGTG

LmddA164-2

AGCTTTGATGGGGACCCCTCCTCCGGCATTGCTCCGCTGAGGCCTGAGCAGCTCCAAGTG

Lm-ddA-164-3

AGCTTTGATGGGGACCCCTCCTCCGGCATTGCTCCGCTGAGGCCTGAGCAGCTCCAAGTG

LmddA164-4

AGCTTTGATGGGGACCCCTCCTCCGGCATTGCTCCGCTGAGGCCTGAGCAGCTCCAAGTG

Lm-ddA-164-5

AGCTTTGATGGGGACCCCTCCTCCGGCATTGCTCCGCTGAGGCCTGAGCAGCTCCAAGTG

LmddA-164-6

AGCTTTGATGGGGACCCCTCCTCCGGCATTGCTCCGCTGAGGCCTGAGCAGCTCCAAGTG reference

TTCGAAACCCTGGAGGAGATCACAGGTTACCTGTACATCTCAGCATGGCCAGACAGTCTC

(SEQ ID NO:18)

Lm-LLO-138-2

TTCGAAACCCTGGAGGAGATCACAGGTTACCTGTACATCTCAGCATGGCCAGACAGTCTC

Lm-LLO-138-3

TTCGAAACCCTGGAGGAGATCACAGGTTACCTGTACATCTCAGCATGGCCAGACAGTCTC

Lm-ddA-164-1

TTCGAAACCCTGGAGGAGATCACAGGTTACCTGTACATCTCAGCATGGCCAGACAGTCTC

LmddA164-2

TTCGAAACCCTGGAGGAGATCACAGGTTACCTGTACATCTCAGCATGGCCAGACAGTCTC

Lm-ddA-164-3

TTCGAAACCCTGGAGGAGATCACAGGTTACCTGTACATCTCAGCATGGCCAGACAGTCTC

LmddA164-4

TTCGAAACCCTGGAGGAGATCACAGGTTACCTGTACATCTCAGCATGGCCAGACAGTCTC

Lm-ddA-164-5

TTCGAAACCCTGGAGGAGATCACAGGTTACCTGTACATCTCAGCATGGCCANACAGTCTC

LmddA-164-6

TTCGAAACCCTGGAGGAGATCACAGGTTACCTGTACATCTCAGCATGGCCAGACAGTCT

Reference to

CGTGACCTCAGTGTCTTCCAGAACCTTCGAATCATTCGGGGACGGATTCTCCACGATGGC

(SEQ ID NO:19)

Lm-LLO-138-2

CGTGACCTCAGTGTCTTCCAGAACCTTCGAATCATTCGGGGACGGATTCTCCACGATGGC

Lm-LLO-138-3

CGTGACCTCAGTGTCTTCCAGAACCTTCGAATCATTCGGGGACGGATTCTCCACGATGGC

Lm-ddA-164-1

CGTGACCTCAGTGTCTTCCAGAACCTTCGAATCATTCGGGGACGGATTCTCCACGATGGC

LmddA164-2

CGTGACCTCAGTGTCTTCCAGAACCTTCGAATCATTCGGGGACGGATTCTCCACGATGGC

Lm-ddA-164-3

CGTGACCTCAGTGTCTTCCAGAACCTTCGAATCATTCGGGGACGGATTCTCCACGATGGC

LmddA164-4

CGTGACCTCAGTGTCTTCCAAAACCTTCGAATCATTCGGGGACGGATTCTCCACGATGGC

Lm-ddA-164-5

CGTGACCTCAGTGTCTTCCAAAACCTTCGAATCATTCGGGGACGGATTCTCCACGATGGC

LmddA-164-6

CGTGACCTCAGTGTCTTCCAAAACCTTCGAATCATTCGGGGACGGATTCTCCACGATGGC

Reference to

GCGTACTCATTGACACTGCAAGGCCTGGGGATCCACTCGCTGGGGCTGCGCTCACTGCGG

(SEQ ID NO:20)

Lm-LLO-138-2

GCGTACTCATTGACACTGCAAGGCCTGGGGATCCACTCGCTGGGGCTGCGCTCACTGCGG

Lm-LLO-138-3

GCGTACTCATTGACACTGCAAGGCCTGGGGATCCACTCGCTGGGGCTGCGCTCACTGCGG

Lm-ddA-164-1

GCGTACTCATTGACACTGCAAGGCCTGGGGATCCACTCGCTGGGGCTGCGCTCACTGCGG

LmddA164-3

GCGTACTCATTGACACTGCAAGGCCTGGGGATCCACTCGCTGGGGCTGCGCTCACTGCGG

Lm-ddA-164-5

GCGTACTCATTGACACTGCAAGGCCTGGGGATCCACTCGCTGGGGCTGCGCTCACTGCGG

Lm-ddA-164-6

GCGTACTCATTGACACTGCAAGGCCTGGGGATCCACTCGCTGGGGCTGCGCTCACTGCGG

Reference to

GAGCTGGGCAGTGGATTGGCTCTGATTCACCGCAACGCCCATCTCTGCTTTGTACACACT

(SEQ ID NO:21)

Lm-LLO-138-2

GAGCTGGGCAGTGGATTGGCTCTGATTCACCGCAACGCCCATCTCTGCTTTGTACACACT

Lm-LLO-138-3

GAGCTGGGCAGTGGATTGGCTCTGATTCACCGCAACGCCCATCTCTGCTTTGTACACACT

Lm-ddA-164-1

GAGCTGGGCAGTGGATTGGCTCTGATTCACCGCAACGCCCATCTCTGCTTTGTACACACT

LmddA164-3

GAGCTGGGCAGTGGATTGGCTCTGATTCACCGCAACGCCCATCTCTGCTTTGTACACACT

Lm-ddA-164-5

GAGCTGGGCAGTGGATTGGCTCTGATTCACCGCAACGCCCATCTCTGCTTTGTACACACT

Lm-ddA-164-6

GAGCTGGGCAGTGGATTGGCTCTGATTCACCGCAACGCCCATCTCTGCTTTGTACACACT

Reference to

GTACCTTGGGACCAGCTCTTCCGGAACCCACATCAGGCCCTGCTCCACAGTGGGAACCGG

(SEQ ID NO:22)

Lm-LLO-138-2

GTACCTTGGGACCAGCTCTTCCGGAACCCACATCAGGCCCTGCTCCACAGTGGGAACCGG

Lm-LLO-138-3

GTACCTTGGGACCAGCTCTTCCGGAACCCACATCAGGCCCTGCTCCACAGTGGGAACCGG

Lm-ddA-164-1

GTACCTTGGGACCAGCTCTTCCGGAACCCACATCAGGCCCTGCTCCACAGTGGGAACCGG

LmddA164-3

GTACCTTGGGACCAGCTCTTCCGGAACCCACATCAGGCCCTGCTCCACAGTGGGAACCGG

Lm-ddA-164-5

GTACCTTGGGACCANCTCTTCCGGAACCCACATCAGGCCCTGCTCCACAGTGGGAACCGG

Lm-ddA-164-6

GTACCTTGGGACCAGCTCTTCCGGAACCCACATCAGGCCCTGCTCCACAGTGGGAACCGG

Reference to

CCGGAAGAGGATTGTGGTCTCGAGGGCTTGGTCTGTAACTCACTGTGTGCCCACGGGCAC

(SEQ ID NO:23)

Lm-LLO-138-2

CCGGAAGAGGATTGTGGTCTCGAGGGCTTGGTCTGTAACTCACTGTGTGCCCACGGGCAC

Lm-LLO-138-3

CCGGAAGAGGATTGTGGTCTCGAGGGCTTGGTCTGTAACTCACTGTGTGCCCACGGGCAC

Lm-ddA-164-1

CCGGAAGAGGATTGTGGTCTCGAGGGCTTGGTCTGTAACTCACTGTGTGCCCACGGGCAC

LmddA164-3

CCGGAAGAGGATTGTGGTCTCGAGGGCTTGGTCTGTAACTCACTGTGTGCCCACGGGCAC

Lm-ddA-164-6

CCGGAAGAGGATTGTGGTCTCGAGGGCTTGGTCTGTAACTCACTGTGTGCCCACGGGCAC

Reference to

TGCTGGGGGCCAGGGCCCACCCAGTGTGTCAACTGCAGTCATTTCCTTCGGGGCCAGGAG

(SEQ ID NO:24)

Lm-LLO-138-2

TGCTGGGGGCCAGGGCCCACCCAGTGTGTCAACTGCAGTCATTTCCTTCGGGGCCAGGAG

Lm-LLO-138-3

TGCTGGGGGCCAGGGCCCACCCAGTGTGTCAACTGCAGTCATTTCCTTCGGGGCCAGGAG

Lm-ddA-164-1

TGCTGGGGGCCAGGGCCCACCCAGTGTGTCAACTGCAGTCATTTCCTTCGGGGCCAGGAG

LmddA164-3

TGCTGGGGGCCAGGGCCCACCCAGTGTGTCAACTGCAGTCATTTCCTTCGGGGCCAGGAG

Lm-ddA-164-6

TGCTGGGGGCCAGGGCCCACCCA-------------------------------------

Alignment IC1 (2114-3042 bp from Her-2-neu)

Reference to

CGCCCAGCGGAGCAATGCCCAACCAGGCTCAGATGCGGATCCTAAAAGAGACGGAGC

(SEQ ID NO:25)

Lm-LLO-NY-2 CGCCCAGCGGAGCAATGCCCAACCAGGCTCAGATGCGGATCCTAAAAGAGACGGAGC

Lm-LLO-138-4 CGCCCAGCGGAGCAATGCCCAACCAGGCTCAGATGCGGATCCTAAAAGAGACGGAGC

Lm-ddA-164-2 CGCCCAGCGGAGCAATGCCCAACCAGGCTCAGATGCGGATCCTAAAAGAGACGGAGC

Lm-ddA-164-3 CGCCCAGCGGAGCAATGCCCAACCAGGCTCAGATGCGGATCCTAAAAGAGACGGAGC

Lm-ddA164-6 CGCCCAGCGGAGCAATGCCCAACCAGGCTCAGATGCGGATCCTAAAAGAGACGGAGC

Reference to

TAAGGAAGGTGAAGGTGCTTGGATCAGGAGCTTTTGGCACTGTCTACAAGGGCATCTGGA

(SEQ ID NO:26)

Lm-LLO-NY-1

TAAGGAAGGTGAAGGTGCTTGGATCAGGAGCTTTTGGCACTGTCTACAAGGGCATCTGGA

Lm-LLO-NY-2

TAAGGAAGGTGAAGGTGCTTGGATCAGGAGCTTTTGGCACTGTCTACAAGGGCATCTGGA

Lm-LLO-138-1

TAAGGAAGGTGAACGTGCTTGGATCAGGAGCTTTTGGCACTGTCTACAAGGGCATCTGGA

Lm-LLO-138-2

TAAGGAAGGTGAAGGTGCTTGGATCAGGAGCTTTTGGCACTGTCTACAAGGGCATCTGGA

Lm-LLO-138-3

TAAGGAAGGTGAAGGTGCTTGGATCAGGAGCTTTTGGCACTGTCTACAAGGGCATCTGGA

Lm-LLO-138-4

TAAGGAAGGTGAAGGTGCTTGGATCAGGAGCTTTTGGCACTGTCTACAAGGGCATCTGGA

Lm-ddA-164-1

TAAGGAAGGTGAAGGTGCTTGGATCAGGAGCTTTTGGCACTGTCTACAAGGGCATCTGGA

Lm-ddA-164-2

TAAGGAAGGTGAAGGTGCTTGGATCAGGAGCTTTTGGCACTGTCTACAAGGGCATCTGGA

Lm-ddA-164-3

TAAGGAAGGTGAAGGTGCTTGGATCAGGAGCTTTTGGCACTGTCTACAAGGGCATCTGGA

Lm-ddA-164-4

TAAGGAAGGTGAAGGTGCTTGGATCAGGAGCTTTTGGCACTGTCTACAAGGGCATCTGGA

Lm-ddA-164-5

TAAGGAAGGTGAAGGTGCTTGGATCAGGAGCTTTTGGCACTGTCTACAAGGGCATCTGGA

Lm-ddA164-6

TAAGGAAGGTGAAGGTGCTTGGATCAGGAGCTTTTGGCACTGTCTACAAGGGCATCTGGA

Reference to

TCCCAGATGGGGAGAATGTGAAAATCCCCGTGGCTATCAAGGTGTTGAGAGAAAACACAT

(SEQ ID NO:27)

Lm-LLO-NY-1

TCCCAGATGGGGAGAATGTGAAAATCCCCGTGGCTATCAAGGTGTTGAGAGAAAACACAT

Lm-LLO-NY-2

TCCCAGATGGGGAGAATGTGAAAATCCCCGTGGCTATCAAGGTGTTGAGAGAAAACACAT

Lm-LLO-138-1

TCCCAGATGGGGAGAATGTGAAAATCCCCGTGGCTATCAAGGTGTTGAGAGAAAACACAT

Lm-LLO-138-2

TCCCAGATGGGGAGAATGTGAAAATCCCCGTGGCTATCAAGGTGTTGAGAGAAAACACAT

Lm-LLO-138-3

TCCCAGATGGGGAGAATGTGAAAATCCCCGTGGCTATCAAGGTGTTGAGAGAAAACACAT

Lm-LLO-138-4

TCCCAGATGGGGAGAATGTGAAAATCCCCGTGGCTATCAAGGTGTTGAGAGAAAACACAT

Lm-ddA-164-1

TCCCAGATGGGGAGAATGTGAAAATCCCCGTGGCTATCAAGGTGTTGAGAGAAAACACAT

Lm-ddA-164-2

TCCCAGATGGGGAGAATGTGAAAATCCCCGTGGCTATCAAGGTGTTGAGAGAAAACACAT

Lm-ddA-164-3

TCCCAGATGGGGAGAATGTGAAAATCCCCGTGGCTATCAAGGTGTTGAGAGAAAACACAT

Lm-ddA-164-4

TCCCAGATGGGGAGAATGTGAAAATCCCCGTGGCTATCAAGGTGTTGAGAGAAAACACAT

Lm-ddA-164-5

TCCCAGATGGGGAGAATGTGAAAATCCCCGTGGCTATCAAGGTGTTGAGAGAAAACACAT

Lm-ddA164-6

TCCCAGATGGGGAGAATGTGAAAATCCCCGTGGCTATCAAGGTGTTGAGAGAAAACACAT

Reference to

CTCCTAAAGCCAACAAAGAAATTCTAGATGAAGCGTATGTGATGGCTGGTGTGGGTTCTC

(SEQ ID NO:28)

Lm-LLO-NY-1

CTCCTAAAGCCAACAAAGAAATTCTAGATGAAGCGTATGTGATGGCTGGTGTGGGTTCTC

Lm-LLO-NY-2

CTCCTAAAGCCAACAAAGAAATTCTAGATGAAGCGTATGTGATGGCTGGTGTGGGTTCTC

Lm-LLO-138-1

CTCCTAAAGCCAACAAAGAAATTCTAGATGAAGCGTATGTGATGGCTGGTGTGGGTTCTC

Lm-LLO-138-2

CTCCTAAAGCCAACAAAGAAATTCTAGATGAAGCGTATGTGATGGCTGGTGTGGGTTCTC

Lm-LLO-138-3

CTCCTAAAGCCAACAAAGAAATTCTAGATGAAGCGTATGTGATGGCTGGTGTGGGTTCTC

lm-LLO-138-4

CTCCTAAAGCCAACAAAGAAATTCTAGATGAAGCGTATGTGATGGCTGGTGTGGGTTCTC

Lm-ddA-164-1

CTCCTAAAGCCAACAAAGAAATTCTAGATGAAGCGTATGTGATGGCTGGTGTGGGTTCTC

Lm-ddA-164-2

CTCCTAAAGCCAACAAAGAAATTCTAGATGAAGCGTATGTGATGGCTGGTGTGGGTTCTC

Lm-ddA-164-3

CTCCTAAAGCCAACAAAGAAATTCTAGATGAAGCGTATGTGATGGCTGGTGTGGGTTCTC

Lm-ddA-164-4

CTCCTAAAGCCAACAAAGAAATTCTAGATGAAGCGTATGTGATGGCTGGTGTGGGTTCTC

Lm-ddA-164-5

CTCCTAAAGCCAACAAAGAAATTCTAGATGAAGCGTATGTGATGGCTGGTGTGGGTTCTC

Lm-ddA164-6

CTCCTAAAGCCAACAAAGAAATTCTAGATGAAGCGTATGTGATGGCTGGTGTGGGTTCTC

Reference to

CGTATGTGTCCCGCCTCCTGGGCATCTGCCTGACATCCACAGTACAGCTGGTGACACAGC

(SEQ ID NO:29)

Lm-LLO-NY-1

CGTATGTGTCCCGCCTCCTGGGCATCTGCCTGACATCCACAGTACAGCTGGTGACACAGC

Lm-LLO-NY-2

CGTATGTGTCCCGCCTCCTGGGCATCTGCCTGACATCCACAGTACAGCTGGTGACACAGC

Lm-LLO-138-1

CGTATGTGTCCCGCCTCCTGGGCATCTGCCTGACATCCACAGTACAGCTGGTGACACAGC

Lm-LLO-138-2

CGTATGTGTCCCGCCTCCTGGGCATCTGCCTGACATCCACAGTACAGCTGGTGACACAGC

Lm-LLO-138-3

CGTATGTGTCCCGCCTCCTGGGCATCTGCCTGACATCCACAGTACAGCTGGTGACACAGC

Lm-LLO-138-4

CGTATGTGTCCCGCCTCCTGGGCATCTGCCTGACATCCACAGTACAGCTGGTGACACAGC

Lm-ddA-164-1

CGTATGTGTCCCGCCTCCTGGGCATCTGCCTGACATCCACAGTACAGCTGGTGACACAGC

Lm-ddA-164-2

CGTATGTGTCCCGCCTCCTGGGCATCTGCCTGACATCCACAGTACAGCTGGTGACACAGC

Lm-ddA-164-3

CGTATGTGTCCCGCCTCCTGGGCATCTGCCTGACATCCACAGTACAGCTGGTGACACAGC

Lm-ddA-164-4

CGTATGTGTCCCGCCTCCTGGGCATCTGCCTGACATCCACAGTACAGCTGGTGACACAGC

Lm-ddA-164-5

CGTATGTGTCCCGCCTCCTGGGCATCTGCCTGACATCCACAGTACAGCTGGTGACACAGC

Lm-ddA164-6

CGTATGTGTCCCGCCTCCTGGGCATCTGCCTGACATCCACAGTACAGCTGGTGACACAGC

Reference to

TTATGCCCTACGGCTGCCTTCTGGACCATGTCCGAGAACACCGAGGTCGCCTAGGCTCCC

(SEQ ID NO:30)

Lm-LLO-NY-1

TTATGCCCTACGGCTGCCTTCTGGACCATGTCCGAGAACACCGAGGTCGCCTAGGCTCCC

Lm-LLO-NY-2

TTATGCCCTACGGCTGCCTTCTGGACCATGTCCGAGAACACCGAGGTCGCCTAGGCTCCC

Lm-LLO-138-1

TTATGCCCTACGGCTGCCTTCTGGACCATGTCCGAGAACACCGAGGTCGCCTAGGCTCCC

Lm-LLO-138-2

TTATGCCCTACGGCTGCCTTCTGGACCATGTCCGAGAACACCGAGGTCGCCTAGGCTCCC

Lm-LLO-138-3

TTATGCCCTACGGCTGCCTTCTGGACCATGTCCGAGAACACCGAGGTCGCCTAGGCTCCC

Lm-LLO-138-4

TTATGCCCTACGGCTGCCTTCTGGACCATGTCCGAGAACACCGAGGTCGCCTAGGCTCCC

Lm-ddA-164-1

TTATGCCCTACGGCTGCCTTCTGGACCATGTCCGAGAACACCGAGGTCGCCTAGGCTCCC

Lm-ddA-164-2

TTATGCCCTACGGCTGCCTTCTGGACCATGTCCGAGAACACCGAGGTCGCCTAGGCTCCC

Lm-ddA-164-3

TTATGCCCTACGGCTGCCTTCTGGACCATGTCCGAGAACACCGAGGTCGCCTAGGCTCCC

Lm-ddA-164-4

TTATGCCCTACGGCTGCCTTCTGGACCATGTCCGAGAACACCGAGGTCGCCTAGGCTCCC

Lm-ddA-164-5

TTATGCCCTACGGCTGCCTTCTGGACCATGTCCGAGAACACCGAGGTCGCCTAGGCTCCC

Lm-ddA164-6

TTATGCCCTACGGCTGCCTTCTGGACCATGTCCGAGAACACCGAGGTCGCCTAGGCTCCC

Reference to

AGGACCTGCTCAACTGGTGTGTTCAGATTGCCAAGGGGATGAGCTACCTGGAGGACGTGC

(SEQ ID NO:31)

Lm-LLO-NY-1

AGGACCTGCTCAACTGGTGTGTTCAGATTGCCAAGGGGATGAGCTACCTGGAGGACGTGC

Lm-LLO-NY-2

AGGACCTGCTCAACTGGTGTGTTCAGATTGCCAAGGGGATGAGCTACCTGGAGGACGTGC

Lm-LLO-138-1

AGGACCTGCTCAACTGGTGTGTTCAGATTGCCAAGGGGATGAGCTACCTGGAGGACGTGC

Lm-LLO-138-2

AGGACCTGCTCAACTGGTGTGTTCAGATTGCCAAGGGGATGAGCTACCTGGAGGACGTGC

Lm-LLO-138-3

AGGACCTGCTCAACTGGTGTGTTCAGATTGCCAAGGGGATGAGCTACCTGGAGGACGTGC

Lm-LLO-138-4

AGGACCTGCTCAACTGGTGTGTTCAGATTGCCAAGGGGATGAGCTACCTGGAGGACGTGC

Lm-ddA-164-1

AGGACCTGCTCAACTGGTGTGTTCAGATTGCCAAGGGGATGAGCTACCTGGAGGACGTGC

Lm-ddA-164-2

AGGACCTGCTCAACTGGTGTGTTCAGATTGCCAAGGGGATGAGCTACCTGGAGGACGTGC

Lm-ddA-164-3

AGGACCTGCTCAACTGGTGTGTTCAGATTGCCAAGGGGATGAGCTACCTGGAGGACGTGC

Lm-ddA-164-4

AGGACCTGCTCAACTGGTGTGTTCAGATTGCCAAGGGGATGAGCTACCTGGAGGACGTGC

Lm-ddA-164-5

AGGACCTGCTCAACTGGTGTGTTCAGATTGCCAAGGGGATGAGCTACCTGGAGGACGTGC

Lm-ddA164-6

AGGACCTGCTCAACTGGTGTGTTCAGATTGCCAAGGGGATGAGCTACCTGGAGGACGTGC

Reference to

GGCTTGTACACAGGGACCTGGCTGCCCGGAATGTGCTAGTCAAGAGTCCCAACCACGTCA

(SEQ ID NO:32)

Lm-LLO-NY-1

GGCTTGTACACAGGGACCTGGCTGCCCGGAATGTGCTAGTCAAGAGTCCCAACCACGTCA

Lm-LLO-NY-2

GGCTTGTACACAGGGACCTGGCTGCCCGGAATGTGCTAGTCAAGAGTCCCAACCACGTCA

Lm-LLO-138-1

GGCTTGTACACAGGGACCTGGCTGCCCGGAATGTGCTAGTCAAGAGTCCCAACCACGTCA

Lm-LLO-138-2

GGCTTGTACACAGGGACCTGGCTGCCCGGAATGTGCTAGTCAAGAGTCCCAACCACGTCA

Lm-LLO-138-3

GGCTTGTACACAGGGACCTGGCTGCCCGGAATGTGCTAGTCAAGAGTCCCAACCACGTCA

Lm-LLO-138-4

GGCTTGTACACAGGGACCTGGCTGCCCGGAATGTGCTAGTCAAGAGTCCCAACCACGTCA

Lm-ddA-164-1

GGCTTGTACACAGGGACCTGGCTGCCCGGAATGTGCTAGTCAAGAGTCCCAACCACGTCA

Lm-ddA-164-2

GGCTTGTACACAGGGACCTGGCTGCCCGGAATGTGCTAGTCAAGAGTCCCAACCACGTCA

Lm-ddA-164-4

GGCTTGTACACAGGGACCTGGCTGCCCGGAATGTGCTAGTCAAGAGTCCCAACCACGTCA

Lm-ddA-164-3

GGCTTGTACACAGGGACCTGGCTGCCCGGAATGTGCTAGTCAAGAGTCCCAACCACGTCA

Lm-ddA-164-5

GGCTTGTACACAGGGACCTGGCTGCCCGGAATGTGCTAGTCAAGAGTCCCAACCACGTCA

Lm-ddA164-6

GGCTTGTACACAGGGACCTGGCTGCCCGGAATGTGCTAGTCAAGAGTCCCAACCACGTCA

Reference to

AGATTACAGATTTCGGGCTGGCTCGGCTGCTGGACATTGATGAGACAGAGTACCATGCAG

(SEQ ID NO:33)

Lm-LLO-NY-1

AGATTACAGATTTCGGGCTGGCTCGGCTGCTGGACATTGATGAGACAGAGTACCATGCAG

Lm-LLO-NY-2

AGATTACAGATTTCGGGCTGGCTCGGCTGCTGGACATTGATGAGACAGAGTACCATGCAG

Lm-LLO-138-1

AGATTACAGATTTCGGGCTGGCTCGGCTGCTGGACATTGATGAGACAGAGTACCATGCAG

Lm-LLO-138-2

AGATTACAGATTTCGGGCTGGCTCGGCTGCTGGACATTGATGAGACAGAGTACCATGCAG

Lm-LLO-138-3

AGATTACAGATTTCGGGCTGGCTCGGCTGCTGGACATTGATGAGACAGAGTACCATGCAG

Lm-LLO-138-4

AGATTACAGATTTCGGGCTGGCTCGGCTGCTGGACATTGATGAGACAGAGTACCATGCAG

Lm-ddA-164-1

AGATTACAGATTTCGGGCTGGCTCGGCTGCTGGACATTGATGAGACAGAGTACCATGCAG

Lm-ddA-164-2

AGATTACAGATTTCGGGCTGGCTCGGCTGCTGGACATTGATGAGACAGAGTACCATGCAG

Lm-ddA-164-3

AGATTACAGATTTCGGGCTGGCTCGGCTGCTGGACATTGATGAGACAGAGTACCATGCAG

Lm-ddA-164-4

AGATTACAGATTTCGGGCTGGCTCGGCTGCTGGACATTGATGAGACAGAGTACCATGCAG

Lm-ddA-164-5

AGATTACAGATTTCGGGCTGGCTCGGCTGCTGGACATTGATGAGACAGAGTACCATGCAG

Lm-ddA164-6

AGATTACAGATTTCGGGCTGGCTCGGCTGCTGGACATTGATGAGACAGAGTACCATGCAG

Reference to

ATGGGGGCAAGGTGCCCATCAAATGGATGGCATTGGAATCTATTCTCAGACGCCGGTTCA

(SEQ ID NO:34)

Lm-LLO-NY-1

ATGGGGGCAAGGTGCCCATCAAATGGATGGCATTGGAATCTATTCTCAGACGCCGGTTCA

Lm-LLO-NY-2

ATGGGGGCAAGGTGCCCATCAAATGGATGGCATTGGAATCTATTCTCAGACGCCGGTTCA

Lm-LLO-138-1

ATGGGGGCAAGGTGCCCATCAAATGGATGGCATTGGAATCTATTCTCAGACGCCGGTTCA

Lm-LLO-138-2

ATGGGGGCAAGGTGCCCATCAAATGGATGGCATTGGAATCTATTCTCAGACGCCGGTTCA

Lm-LLO-138-3

ATGGGGGCAAGGTGCCCATCAAATGGATGGCATTGGAATCTATTCTCAGACGCCGGTTCA

Lm-LLO-138-4

ATGGGGGCAAGGTGCCCATCAAATGGATGGCATTGGAATCTATTCTCAGACGCCGGTTCA

Lm-ddA-164-1

ATGGGGGCAAGGTGCCCATCAAATGGATGGCATTGGAATCTATTCTCAGACGCCGGTTCA

Lm-ddA-164-2

ATGGGGGCAAGGTGCCCATCAAATGGATGGCATTGGAATCTATTCTCAGACGCCGGTTCA

Lm-ddA-164-3

ATGGGGGCAAGGTGCCCATCAAATGGATGGCATTGGAATCTATTCTCAGACGCCGGTTCA

Lm-ddA-164-4

ATGGGGGCAAGGTGCCCATCAAATGGATGGCATTGGAATCTATTCTCAGACGCCGGTTCA

Lm-ddA-164-5

ATGGGGGCAAGGTGCCCATCAAATGGATGGCATTGGAATCTATTCTCAGACGCCGGTTCA

Lm-ddA-164-6

ATGGGGGCAAGGTGCCCATCAAATGGATGGCATTGGAATCTATTCTCAGACGCCGGTTCA

Reference to

CCCATCAGAGTGATGTGTGGAGCTATGGAGTGACTGTGTGGGAGCTGATGACTTTTGGGG

(SEQ ID NO:35)

Lm-LLO-NY-1

CCCATCAGAGTGATGTGTGGAGCTATGGAGTGACTGTGTGGGAGCTGATGACTTTTGGGG

Lm-LLO-NY-2

CCCATCAGAGTGATGTGTGGAGCTATGGAGTGACTGTGTGGGAGCTGATGACTTTTGGGG

Lm-LLO-138-1

CCCATCAGAGTGATGTGTGGAGCTATGGAGTGACTGTGTGGGAGCTGATGACTTTTGGGG

Lm-LLO-138-2

CCCATCAGAGTGATGTGTGGAGCTATGGAGTGACTGTGTGGGAGCTGATGACTTTTGGGG

Lm-LLO-138-3

CCCATCAGAGTGATGTGTGGAGCTATGGAGTGACTGTGTGGGAGCTGATGACTTTTGGGG

Lm-LLO-138-4

CCCATCAGAGTGATGTGTGGAGCTATGGAGTGACTGTGTGGGAGCTGATGACTTTTGGGG

Lm-ddA-164-1

CCCATCAGAGTGATGTGTGGAGCTATGGAGTGACTGTGTGGGAGCTGATGACTTTTGGGG

Lm-ddA-164-2

CCCATCAGAGTGATGTGTGGAGCTATGGAGTGACTGTGTGGGAGCTGATGACTTTTGGGG

Lm-ddA-164-3

CCCATCAGAGTGATGTGTGGAGCTATGGAGTGACTGTGTGGGAGCTGATGACTTTTGGGG

Lm-ddA-164-4

CCCATCAGAGTGATGTGTGGAGCTATGGAGTGACTGTGTGGGAGCTGATGACTTTTGGGG

Lm-ddA-164-5

CCCATCAGAGTGATGTGTGGAGCTATGGAGTGACTGTGTGGGAGCTGATGACTTTTGGGG

Lm-ddA164-6

CCCATCAGAGTGATGTGTGGAGCTATGGAGTGACTGTGTGGGAGCTGATGACTTTTGGGG

Reference to

CCAAACCTTACGATGGAATCCCAGCCCGGGAGATCCCTGATTTGCTGGAGAAGGGAGAA

(SEQ ID NO:36)

Lm-LLO-NY-1CCAAACCTTACGATGGAATCCCAGCCCGGGAGATCCCTGATTTGCTGGAGAAGGGAGAA

Lm-LLO-NY-2 CCAAACCTTACGATGGAATCCCAGCCCGGGAGATCCCTGATTTGCTGGAGAAGGGAGAA

Lm-LLO-138-1 CCAAACCTTACGATGGAATCCCAGCCCGGGAGATCCCTGATTTGCTGGAGAAGGGAGAA

Lm-LLO-138-3 CCAAACCTTACGATGGAATCCCAGCCCGGGAGATCCCTGATTTGCTGGAGAAGGGAGAA

Lm-LLO-138-4 CCAAACCTTACGATGNAATCCCAGCCCGGGAGATCCCTGATTTGCTGGAGAAGGGAGAA

Lm-ddA164-6 CCAAACCTTACGATGGAATCCCAGCCCGGGAGATCCCTGATTTGCTGGAGAAGGGAGAA

Lm-ddA-164-2 CCAAACCTTACGATGGAATCCCAGCCCGGGAGATCCCTGATTTGCTGGAGAAGGGAGAA

Lm-LLO-138-2 CCAAACCTTACGATGGAATCCCAGCCCGGGAGATCCCTGATTTGCTGGAGAAGGGAGAA

Lm-ddA-164-3 CCAAACCTTACGATGGAATCCCAGCCCGGGAGATCCCTGATTTGCTGGAGAAGGGAGAA

Lm-ddA-164-5 CCAAACCTTACGATGGAATCCCAGCCCGGGAGATCCCTGATTTGCTGGAGAAGGGAGAA

Lm-ddA-164-1 CCAAACCTTACGATGGAATCCCAGCCCGGGAGATCCCTGATTTGCTGGAGAAGGGAGAA

Lm-ddA-164-4 CCAAACCTTACGATGGAATCCCAGCCCGGGAGATCCCTGATTTGCTGGAGAAGGGAGAA

Reference to

CGCCTACCTCAGCCTCCAATCTGCACCATTGATGTCTACATGATTATGGTCAAATGTT

(SEQ ID NO:37)

Lm-LLO-NY-1 CGCCTACCTCAGCCTCCAATCTGCACCATTGATGTCTACATGATTATGGTCAAATGTT

Lm-LLO-NY-2 CGCCTACCTCAGCCTCCAATCTGCACCATTGATGTCTACATGATTATGGTCAAATGTT

Lm-LLO-138-1 CGCCTACCTCAGCCTCCAATCTGCACCATTGATGTCTACATGATTATGGTCAAATGTT

Lm-LLO-138-2 CGCCTACCTCAGCCTCCAATCTGCACCATTGATGTCTACATGATTATGGTCAAATGTT

Lm-LLO-138-3 CGCCTACCTCAGCCTCCAATCTGCACCATTGATGTCTACATGATTATGGTCAAATGTT

Lm-LLO-138-4 CGCCTACCTCAGCCTCCAATCTGCACCATTGATGTCTACATGATTATGGTCAAATGTT

Lm-ddA-164-1 CGCCTACCTCAGCCTCCAATCTGCACCATTGATGTCTACATGATTATGGTCAAATGTT

Lm-ddA-164-2 CGCCTACCTCAGCCTCCAATCTGCACCATTGATGTCTACATGATTATGGTCAAATGTT

Lm-ddA-164-3 CGCCTACCTCAGCCTCCAATCTGCACCATTGATGTCTACATGATTATGGTCAAATGTT

Lm-ddA-164-4 CGCCTACCTCAGCCTCCAATCTGCACCATTGATGTCTACATGATTATGGTCAAATGTT

Lm-ddA-164-5 CGCCTACCTCAGCCTCCAATCTGCACCATTGATGTCTACATGATTATGGTCAAATGTT

Lm-ddA164-6 CGCCTACCTCAGCCTCCAATCTGCACCATTGATGTCTACATGATTATGGTCAAATGTT

Reference to

GGATGATTGACTCTGAATGTCGCCCGAGATTCCGGGAGTTGGTGTCAGAATTTT

(SEQ ID NO:38)

Lm-LLO-NY-1

GGATGATTGACTCTGAATGTCGCCCGAGATTCCGGGAGTTGGTGTCAGAATTTT

Lm-LLO-NY-2

GGATGATTGACTCTGAATGTCGCCCGAGATTCCGGGAGTTGGTGTCAGAATTTT

Lm-LLO-138-2

GGATGATTGACTCTGAATGTCCCCCGAGATTCCGGGAGTTGGTGTCAAAATTTT

Lm-LLO-138-3

GGATGATTGACTCTGAATGTCGCCCGAGATTCCGGGAGTTGGTGTCAGAATTTT

Lm-LLO-138-4

GGATGATTGACTCTGAATGTCGCCCGAGATTCCGGGAGTTGGTGTCAGAATTTT

Lm-ddA-164-1

GGATGATTGACTCTGAATGTCGCCCGAGATTCCGGGAGTTGGTGTCAGAATTTT

Lm-ddA-164-2

GGATGATTGACTCTGAATGTCGCCCGAGATTCCGGGAGTTGGTGTCAGAATTTT

Lm-ddA-164-3

GGATGATTGACTCTGAATGTCGCCCGAGATTCCGGGAGTTGGTGTCAGAATTTT

Lm-ddA-164-5

GGATGATTGACTCTGAATGTCGCCCGAGATTCCGGGAGTTGGTGTCAGAATTTT

Lm-ddA-164-4

GGATGATTGACTCTGAATGTCGCCCGAGATTCCGGGAGTTGGTGTCAGAATTTT

Lm-ddA164-6

GGATGATTGACTCTGAATGTCGCCCGAGATTCCGGGAGTTGGTGTCAGAATTTT

Reference to

CACGTATGGCGAGGGACCCCCAGCGTTTTGTGGTCATCCAGAACGAGGACTT

(SEQ ID NO:39)

Lm-LLO-NY-1

CACGTATGGCGAGGGACCCCCAGCGTTTTGTGGTCATCCAGAACGAGGACTT

Lm-LLO-NY-2

CACGTATGGCGAGGGACCCCCAGCGTTTTGTGGTCATCCAGAACGAGGACTT

Lm-LLO-138-2

CACGTATGGCGAGGGACCCCCAGCGTTTTGTGGTCATCCAGAACGAGGACTT

Lm-LLO-138-3

CACGTATGGCGAGGGACCCCCAGCGTTTTGTGGTCATCCAGAACGAGGACTT

Lm-LLO-138-4

CACGTATGGCGAGGGACCCCCAGCGTTTTGTGGTCATCCAGAACGAGGACTT

Lm-ddA-164-1

CACGTATGGCGAGGGACCCCCAGCGTTTTGTGGTCATCCAGAACGAGGACTT

Lm-ddA-164-2

CACGTATGGCGAGGGACCCCCAGCGTTTTGTGGTCATCCAGAACGAGGACTT

Lm-ddA-164-3

CACGTATGGCGAGGGACCCCCAGCGTTTTGTGGTCATCCAGAACGAGGACTT

Lm-ddA-164-5

CACGTATGGCGAGGGACCCCCAGCGTTTTGTGGTCATCCAGAACGAGGACTT

Lm-ddA-164-6

CACGTATGGCGAGGGACCCCCAGCGTTTTGTGGTCATCCAGAACGAGGACTT

Alignment EC1 (399-758 bp for Her-2-neu)

Reference to

CCCAGGCAGAACCCCAGAGGGGCTGCGGGAGCTGCAGCTTCGAAGTCTCACAGAGATCCT

(SEQ ID NO:40)

Lm-LLO-138-1

CCCAGGCAGAACCCCAGAGGGGCTGCGGGAGCTGCAGCTTCGAAGTCTCACAGAGATCCT

Lm-LLO-138-2

CCCAGGCAGAACCCCAGAGGGGCTGCGGGAGCTGCAGCTTCGAAGTCTCACAGAGATCCT

Lm-ddA-164-1

CCCAGGCAGAACCCCAGAGGGGCTGCGGGAGCTGCAGCTTCGAAGTCTCACAGAGATCCT

LmddA-164-2

CCCAGGCAGAACCCCAGAGGGGCTGCGGGAGCTGCAGCTTCGAAGTCTCACAGAGATCCT

LmddA-164-3

CCCAGGCAGAACCCCAGAGGGGCTGCGGGAGCTGCAGCTTCGAAGTCTCACAGAGATCCT

LmddA164-4

CCCAGGCAGAACCCCAGAGGGGCTGCGGGAGCTGCAGCTTCGAAGTCTCACAGAGATCCT

Reference to

GAAGGGAGGAGTTTTGATCCGTGGGAACCCTCAGCTCTGCTACCAGGACATGGTTTTGTG

(SEQ ID NO:41)

Lm-LLO-138-1

GAAGGGAGGAGTTTTGATCCGTGGGAACCCTCAGCTCTGCTACCAGGACATGGTTTTGTG

Lm-LLO-138-2

GAAGGGAGGAGTTTTGATCCGTGGGAACCCTCAGCTCTGCTACCAGGACATGGTTTTGTG

Lm-ddA-164-1

GAAGGGAGGAGTTTTGATCCGTGGGAACCCTCAGCTCTGCTACCAGGACATGGTTTTGTG

LmddA-164-2

GAAGGGAGGAGTTTTGATCCGTGGGAACCCTCAGCTCTGCTACCAGGACATGGTTTTGTG

LmddA-164-3

GAAGGGAGGAGTTTTGATCCGTGGGAACCCTCAGCTCTGCTACCAGGACATGGTTTTGTG

LmddA164-4

GAAGGGAGGAGTTTTGATCCGTGGGAACCCTCAGCTCTGCTACCAGGACATGGTTTTGTG

Reference to

CCGGGCCTGTCCACCTTGTGCCCCCGCCTGCAAAGACAATCACTGTTGGGGTGAGAGTCC

(SEQ ID NO:42)

Lm-LLO-138-1

CCGGGCCTGTCCACCTTGTGCCCCCGCCTGCAAAGACAATCACTGTTGGGGTGAGAGTCC

Lm-LLO-138-2

CCGGGCCTGTCCACCTTGTGCCCCCGCCTGCAAAGACAATCACTGTTGGGGTGAGAGTCC

Lm-ddA-164-1

CCGGGCCTGTCCACCTTGTGCCCCCGCCTGCAAAGACAATCACTGTTGGGGTGAGAGTCC

LmddA-164-2

CCGGGCCTGTCCACCTTGTGCCCCCGCCTGCAAAGACAATCACTGTTGGGGTGAGAGTCC

LmddA-164-3

CCGGGCCTGTCCACCTTGTGCCCCCGCCTGCAAAGACAATCACTGTTGGGGTGAGAGTCC

LmddA164-4

CCGGGCCTGTCCACCTTGTGCCCCCGCCTGCAAAGACAATCACTGTTGGGGTGAGAGTCC

Reference to

GGAAGACTGTCAGATCTTGACTGGCACCATCTGTACCAGTGGTTGTGCCCGGTGCAAGGG

(SEQ ID NO:43)

Lm-LLO-138-1

GGAAGACTGTCAGATCTTGACTGGCACCATCTGTACCAGTGGTTGTGCCCGGTGCAAGGG

Lm-LLO-138-2

GGAAGACTGTCAGATCTTGACTGGCACCATCTGTACCAGTGGTTGTGCCCGGTGCAAGGG

Lm-ddA-164-1

GGAAGACTGTCAGATCTTGACTGGCACCATCTGTACCAGTGGTTGTGCCCGGTGCAAGGG

LmddA-164-2

GGAAGACTGTCAGATCTTGACTGGCACCATCTGTACCAGTGGTTGTGCCCGGTGCAAGGG

LmddA-164-3

GGAAGACTGTCAGATCTTGACTGGCACCATCTGTACCAGTGGTTGTGCCCGGTGCAAGGG

LmddA164-4

GGAAGACTGTCAGATCTTGACTGGCACCATCTGTACCAGTGGTTGTGCCCGGTGCAAGGG

Reference to

CCGGCTGCCCACTGACTGCTGCCATGAGCAGTGTGCCGCAGGCTGCACGGGCCCCAAGCA

(SEQ ID NO:44)

Lm-LLO-138-1

CCGGCTGCCCACTGACTGCTGCCATGAGCAGTGTGCCGCAGGCTGCACGGGCCCCAAGCA

Lm-LLO-138-2

CCGGCTGCCCACTGACTGCTGCCATGAGCAGTGTGCCGCAGGCTGCACGGGCCCCAAGCA

Lm-ddA-164-1

CCGGCTGCCCACTGACTGCTGCCATGAGCAGTGTGCCGCAGGCTGCACGGGCCCCAAGCA

LmddA-164-2

CCGGCTGCCCACTGACTGCTGCCATGAGCAGTGTGCCGCAGGCTGCACGGGCCCCAAGTA

LmddA-164-3

CCGGCTGCCCACTGACTGCTGCCATGAGCAGTGTGCCGCAGGCTGCACGGGCCCCAAGTA

LmddA164-4

CCGGCTGCCCACTGACTGCTGCCATGAGCAGTGTGCCGCAGGCTGCACGGGCCCCAAGTA

Example 7: peripheral immunization with ADXS31-164 delayed the growth of metastatic breast cancer cell lines in the brain.

IP mice were immunized with either ADXS31-164 or an unrelated Lm-control vaccine, and then implanted intracranially with 5,000EMT6-Luc tumor cells, which expressed luciferase and low levels of Her2/neu (fig. 6C). Tumors were monitored by ex vivo imaging of anesthetized mice at various times post-inoculation. On day 8 post tumor inoculation, tumors were detected in all control animals, but mice in the ADXS31-164 group did not show any detectable tumors (fig. 6A and B). ADXS31-164 can significantly delay the onset of these tumors, as on day 11 post tumor inoculation, all mice in the negative control group had died of tumors, but all mice in the ADXS31-164 group were still alive and showed only small signs of tumor growth. These results strongly suggest that the immune response obtained by peripheral administration of ADXS31-164 may reach the central nervous system, and that LmddA-based vaccines may have potential use for treatment of CNS tumors.

Example 8: canine osteosarcoma was treated by immunization with ADXS 31-164.

Canine osteosarcoma is a cancer of the long (leg) bone, which is the major killer in dogs over 10 years of age. The standard treatment is amputation immediately after diagnosis, followed by chemotherapy. However, cancer always metastasizes to the lungs. With chemotherapy, dogs survive approximately 18 months, compared to 6-12 months without treatment. The HER2 antigen is thought to be present in up to 50% of osteosarcomas. ADXS31-164 produces an immune attack on cells expressing this antigen and has been developed for human breast cancer.

Dogs histologically diagnosed with osteosarcoma and demonstrated HER2/neu expression by malignant cells met the recruitment criteria.

Canine osteosarcoma assay

In the first batch, the limb was excised, followed by a chemotherapy treatment round. Subsequent 3 doses of Her-2 vaccine were administered with or without boosting at 6 month intervals.

All dogs received 4 weeks of carboplatin treatment. Four weeks after the last carboplatin administration, dogs received ADXS-HER2 for a total of 3 doses once every three weeks. Group 1(3 dogs) received 1x10⁸CFU per dose, group 2(3 dogs) each received 5x10⁸CFU received 1x10 per dose and group 3(3 dogs)⁹CFU per dose. Additional dogs can be added to the group to gather more data if potential doses to limit toxicity are observed. Therefore, 9-18 dogs were treated in the initial study.

In the second batch, the same treatment as in the first batch was repeated, except that only a single dose of the vaccine was administered, followed by chemotherapy (after 1 month) for a total of 4 doses.

Further, in both batches, a single dose was administered one month after chemotherapy.

While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

1. A method of treating Her-2/neu-expressing tumor growth or cancer in a non-human animal, the method comprising the step of administering a recombinant listeria comprising a nucleic acid molecule encoding a fusion polypeptide, wherein said fusion polypeptide comprises a Her2/neu chimeric antigen fused to an additional adjuvant polypeptide.

2. The method of claim 1, wherein the non-human animal is a dog.

3. The method of claim 1, wherein administering said fusion polypeptide to a subject having an Her 2/neu-expressing tumor prevents escape mutations in said tumor.

4. The method of claim 1, wherein said nucleic acid molecule comprises a first open reading frame encoding said immunogenic composition, wherein said nucleic acid molecule is located in said recombinant Listeria vaccine strain.

5. The method of claim 4, wherein said nucleic acid molecule further comprises a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is lacking in the chromosome of said recombinant Listeria vaccine strain.

6. The method of claim 1, wherein said Her2/neu chimeric antigen comprises at least 5, 9, 13, 14, or 17 of the mapped human MHC-class I epitopes.

7. The method of claim 4, wherein said nucleic acid molecule is integrated into the Listeria genome.

8. The method of claim 4, wherein said nucleic acid molecule is located in a plasmid within said recombinant Listeria vaccine strain.

9. The method of claim 8, wherein said plasmid is stably maintained in said recombinant Listeria vaccine strain in the absence of antibiotic selection.

10. The method of claim 8, wherein said plasmid does not confer antibiotic resistance upon said recombinant Listeria.

11. The method of claim 1, wherein said recombinant Listeria strain is attenuated.

12. The method of claim 1, wherein said recombinant Listeria lacks the ActA virulence gene.

13. The method of claim 1, wherein the additional polypeptide is a nonhemolytic LLO protein or an N-terminal fragment.

14. The method of claim 1, wherein said additional polypeptide is a PEST sequence.

15. The method of claim 1, wherein the additional polypeptide is an ActA N-terminal fragment.

16. The method of any one of claims 13-15, wherein said additional polypeptide is fused to said Her2/neu chimeric antigen.

17. The method of claim 5, wherein said metabolic enzyme encoded by said second open reading frame is an amino acid metabolic enzyme.

18. The method of claim 5, wherein said metabolic enzyme encoded by said second open reading frame is an alanine racemase enzyme or a D-amino acid transferase enzyme.

19. The method of claim 5, wherein said nucleic acid molecule further comprises a third open reading frame.

20. The method of claim 19, wherein said metabolic enzyme encoded by said third open reading frame is a D-amino acid transferase enzyme or an alanine racemase enzyme.

21. The method of claim 1, wherein said recombinant Listeria strain has been passaged through an animal host.

22. The method of claim 1, further comprising an independent adjuvant.

23. The method of claim 22, wherein said adjuvant comprises a granulocyte/macrophage colony-stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, monophosphoryl lipid a, or an unmethylated CpG-containing oligonucleotide.

24. The method of claim 1, wherein said tumor is an Her2/neu positive tumor.

25. The method of claim 1, wherein said cancer is an Her 2/neu-expressing cancer.

26. The method of claim 1, wherein the cancer is osteosarcoma, ovarian cancer, gastric cancer, or a Central Nervous System (CNS) cancer.

27. The method of claim 1, wherein the osteosarcoma cancer is a canine osteosarcoma.

28. A method of preventing Her-2/neu-expressing tumor growth or cancer in a non-human animal, the method comprising the step of administering a recombinant listeria comprising a nucleic acid encoding a fusion polypeptide, wherein said fusion polypeptide comprises a Her2/neu chimeric antigen fused to an additional adjuvant polypeptide.

29. The method of claim 28, wherein the non-human animal is a dog.

30. The method of claim 28, wherein administering said fusion polypeptide to a subject having an Her 2/neu-expressing tumor prevents escape mutations in said tumor.

31. The method of claim 28, wherein said nucleic acid molecule comprises a first open reading frame encoding said immunogenic composition, wherein said nucleic acid molecule is located in said recombinant listeria vaccine strain.

32. The method of claim 31, wherein said nucleic acid molecule further comprises a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is lacking in the chromosome of said recombinant listeria vaccine strain.

33. The method of claim 28, wherein said Her2/neu chimeric antigen comprises at least 5, 9, 13, 14, or 17 of the mapped human MHC-class I epitopes.

34. The method of claim 32, wherein said nucleic acid molecule is integrated into the genome of said listeria.

35. The method of claim 32, wherein said nucleic acid molecule is located in a plasmid within said recombinant Listeria vaccine strain.

36. The method of claim 35, wherein said plasmid is stably maintained in said recombinant listeria vaccine strain in the absence of antibiotic selection.

37. The method of claim 35, wherein said plasmid does not confer antibiotic resistance upon said recombinant listeria.

38. The method of claim 28, wherein said recombinant listeria strain is attenuated.

39. The method of claim 28, wherein said recombinant listeria lacks the ActA virulence gene.

40. The method of claim 28, wherein the additional polypeptide is a nonhemolytic LLO protein or an N-terminal fragment.

41. The method of claim 28, wherein said additional polypeptide is a PEST sequence.

42. The method according to claim 28, wherein the further polypeptide is an ActA N-terminal fragment.

43. The method of any one of claims 40-42, wherein said additional polypeptide is fused to said Her2/neu chimeric antigen.

44. The method of claim 32, wherein said metabolic enzyme encoded by said second open reading frame is an amino acid metabolic enzyme.

45. The method of claim 32, wherein said metabolic enzyme encoded by said second open reading frame is an alanine racemase enzyme or a D-amino acid transferase enzyme.

46. The method of claim 32, wherein said nucleic acid molecule further comprises a third open reading frame.

47. The method of claim 46, wherein said metabolic enzyme encoded by said third open reading frame is a D-amino acid transferase enzyme or an alanine racemase enzyme.

48. The method of claim 28, wherein said recombinant Listeria strain has been passaged through an animal host.

49. The method of claim 28, further comprising an independent adjuvant.

50. The method of claim 49, wherein said adjuvant comprises a granulocyte/macrophage colony-stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, monophosphoryl lipid A, or an unmethylated CpG-containing oligonucleotide.

51. The method of claim 28, wherein said tumor is an Her2/neu positive tumor.

52. The method of claim 28, wherein said cancer is an Her 2/neu-expressing cancer.

53. The method of claim 28, wherein the cancer is osteosarcoma, ovarian cancer, gastric cancer, or a Central Nervous System (CNS) cancer.

54. The method of claim 28, wherein said osteosarcoma cancer is a canine osteosarcoma.

55. A method of eliciting an enhanced immune response against Her-2/neu-expressing tumor growth or cancer in a non-human animal, said method comprising the step of administering a recombinant listeria comprising a nucleic acid encoding a fusion polypeptide, wherein said fusion polypeptide comprises a Her2/neu chimeric antigen fused to an additional adjuvant polypeptide.

56. The method of claim 55, wherein the non-human animal is a dog.

57. The method of claim 55, wherein administering said fusion polypeptide to a subject having an Her 2/neu-expressing tumor prevents escape mutations in said tumor.

58. The method of claim 55, wherein said nucleic acid molecule comprises a first open reading frame encoding said immunogenic composition, wherein said nucleic acid molecule is located in said recombinant Listeria vaccine strain.

59. The method of claim 58, wherein said nucleic acid molecule further comprises a second open reading frame encoding a metabolic enzyme, wherein said metabolic enzyme complements an endogenous gene that is lacking in the chromosome of said recombinant Listeria vaccine strain.

60. The method of claim 55, wherein said Her2/neu chimeric antigen comprises at least 5, 9, 13, 14, or 17 of the mapped human MHC-class I epitopes.

61. The method of claim 58, wherein said nucleic acid molecule is integrated into the Listeria genome.

62. The method of claim 58, wherein said nucleic acid molecule is located in a plasmid within said recombinant Listeria vaccine strain.

63. The method of claim 62, wherein said plasmid is stably maintained in said recombinant Listeria vaccine strain in the absence of antibiotic selection.

64. The method of claim 62, wherein said plasmid does not confer antibiotic resistance upon said recombinant Listeria.

65. The method of claim 55, wherein said recombinant Listeria strain is attenuated.

66. The method of claim 55, wherein said recombinant Listeria lacks the ActA virulence gene.

67. The method of claim 55, wherein the additional polypeptide is a nonhemolytic LLO protein or an N-terminal fragment.

68. The method of claim 55, wherein said additional polypeptide is a PEST sequence.

69. The method of claim 55, wherein the additional polypeptide is an ActA N-terminal fragment.

70. The method of any one of claims 67-69, wherein said additional polypeptide is fused to said Her2/neu chimeric antigen.

71. The method of claim 59, wherein said metabolic enzyme encoded by said second open reading frame is an amino acid metabolic enzyme.

72. The method of claim 59, wherein said metabolic enzyme encoded by said second open reading frame is an alanine racemase enzyme or a D-amino acid transferase enzyme.

73. The method of claim 59, wherein said nucleic acid molecule further comprises a third open reading frame.

74. The method of claim 73, wherein said metabolic enzyme encoded by said third open reading frame is a D-amino acid transferase enzyme or an alanine racemase enzyme.

75. The method of claim 55, wherein said recombinant Listeria strain has been passaged through an animal host.

76. The method of claim 55, further comprising an independent adjuvant.

77. The method of claim 76, wherein said adjuvant comprises a granulocyte/macrophage colony-stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, monophosphoryl lipid A, or an unmethylated CpG-containing oligonucleotide.

78. The method of claim 55, wherein said tumor is an Her2/neu positive tumor.

79. The method of claim 55, wherein said cancer is an Her 2/neu-expressing cancer.

80. The method of claim 55, wherein said cancer is osteosarcoma, ovarian cancer, gastric cancer, or a Central Nervous System (CNS) cancer.

81. The method of claim 55, wherein said osteosarcoma cancer is a canine osteosarcoma.

82. The method of any one of claims 1, 28, or 55, wherein said immune response against said Her 2/neu-expressing tumor comprises an immune response to a subdominant epitope of said Her2/neu protein.