CN106661538A

CN106661538A - Compositions and methods for the treatment of HER2/NEU over-expressing tumors

Info

Publication number: CN106661538A
Application number: CN201580010568.4A
Authority: CN
Inventors: V·谢哈比; A·瓦勒查; P·C·马西亚格; Y·佩特森; N·梅森; M·西维
Original assignee: University of Pennsylvania Penn; Advaxis Inc
Current assignee: University of Pennsylvania Penn; Ayala Pharmaceuticals Inc
Priority date: 2014-02-25
Filing date: 2015-02-25
Publication date: 2017-05-10
Also published as: AU2015223136A1; SG11201607036XA; RU2016137834A; KR20160122829A; EP3110942A4; CA2940646A1; MX2016011114A; BR112016019534A2; IL247436A0; EP3110942A2; WO2015130810A2; JP2017507943A; NZ723750A; WO2015130810A3; KR20240038103A

Abstract

This invention provides compositions and methods for treating and vaccinating against a HER2/neu antigen-expressing tumor and inducing an immune response against the same in a subject.

Description

For treating the composition and method of HER2/NEU overexpression tumours

Technical field

The present invention is provided to the immunity to HER2/neu antigen presentation tumours for inducing people and canid experimenter is answered Answer and for treat the tumour and be inoculated with the tumour vaccine composition and method.In another embodiment, people is tested Person is child or adolescent.

Background technology

Listerisa monocytogenes in mjme (Listeria monocytogenes) is main infection antigen presenting cell And the intracellular pathogen lived in the cytoplasm of these cells has been adapted to.Host cell such as macrophage, actively gulp down Phagocyte listerisa monocytogenes in mjme and most of bacterium are degraded in phagolysosome.Some bacteriums pass through haemolysis Element, the effect of Listeriolysin O (LLO) are pierced through phagocytosis lysosome membrane and are escaped into host's cytosol.Once it is in kytoplasm In colloidal sol, listerisa monocytogenes in mjme can make host's actin polymerization, and directly be delivered to cell from cell, So as to further invading host immune system and producing the insignificant antibody response to listerisa monocytogenes in mjme.

HER2/neu (referred to herein as " Her-2 ") is the glycoprotein of 185kDa, and it is the epidermis of EGFR-TK Growth factor receptors (EGFR) family member, and by extracellular domain, membrane-spanning domain and Intracellular domain (known to be related to cell signalling) group Into.In people, Her2 antigens overexpression in the 25 to 40% of all breast cancer, and also many bones (osteosarcoma-OSA), Overexpression in ovary, lung, pancreas, brain and gastrointestinal cancer.The overexpression of Her-2 and uncontrolled cell growth and signal transduction Correlation, both of which each contributes to the development of tumour.The cancer patient of overexpression Her-2 shows tolerance, can even if existing Direct body fluid, the CD8 to Her-2 of detection⁺T cell and CD4⁺T cell response.

Large-scale cultivation dog is spontaneous to develop into OSA, has reproduced many aspects of children OSA, including histology heterogeneity, invasion and attack Property local disease and early stage shift.In dog, OSA may occur in which in any bone, but limb bone only accounts for all impacted bones 75%-85%, and wherein, OSA is referred to as " limbs osteosarcoma ".Remaining OSA impact axial skeleton, including maxilla, Mandibular, spine, cranium, rib, nasal cavity bone, paranasal sinus bone and pelvis.In diagnosis, 95% dog suffers from micrometastasis disease Disease, although and carried out amputation and chemotherapy, Median survival time also only has 10 months, and most of dog is shifted due to progressive Property disease and be carried out euthanasia.Lung metastases disease is two species morbidities and main causes of death.

The Primary Malignant Bone Tumor of children to young adult colony is not relatively common, accounts in the colony less than 20 years old About the 6% of all cancers, and account for all cancers in the teenager and young adult (AYA) that the range of age is 15 to 29 years old 3%.Every year about 400 Children and teenagers are have impact in U.S.'s osteosarcoma, be this represent in decades almost without treatment The field of improved little but high demand.Although osteosarcoma (OS) is rare malignant tumour, it is arranged in children's age group For the main cause of cancer-related death.Modern, many medicaments, dose density chemotherapy are combined with operation and are confined in 60-70% 5 years are realized in the nonmetastatic disease of limbs without event survival period.However, main but an open question is metastatic Recurrence or the again poor prognosis of appearance and axis Disease.Additionally, there is no approval for osteosarcomatous product, table in the U.S. The bright novel therapies to treating the disease have tight demand.

The present invention is by providing recombinant listeria bacterium (the Listeria)-HER2/neu generated using LmddA vaccine carriers Vaccine strain meets this demand, and the carrier has clear and definite the attenuation mechanism and without antibiotic selected marker, and it is found that should Carrier is effective in treatment canid osteosarcoma.

The content of the invention

In one aspect, provided herein is invention be related to the immunogenic composition comprising fused polypeptide, wherein described melt Polypeptide is closed comprising being fused to the HER2/neu chimeric antigens of other polypeptide, and wherein fusion protein is applied to is suffered from The experimenter of HER2/neu expression tumours gets around mutation avoidance (mutation avoidance) that tumour causes.In another reality In applying example, it is due to epitope spreading to get around mutation avoidance.In yet another embodiment, it is embedding due to antigen for get around mutation avoiding Close property.

In another embodiment, provided herein is invention be related to the recombinant listeria bacterium vaccine strain comprising nucleic acid molecules, Wherein and in another embodiment, first ORFs of the nucleic acid molecules comprising coded polypeptide, wherein polypeptide is included HER2/neu chimeric antigens, the wherein nucleic acid molecules also the second ORFs comprising encoding metabolic enzyme, and the wherein generation The endogenous gene being mutated in the chromosome for thanking to enzyme complementation recombinant listeria bacterium bacterial strain.

In one embodiment, provided herein is invention be related to treat the HER2/neu expression tumour growths or cancer of experimenter The method of disease, the method includes the step of applying the recombinant attenuated Listeria of the nucleic acid comprising coding fused polypeptide, wherein institute Fused polypeptide is stated comprising the HER2/neu chimeric antigens for being fused to other polypeptide, wherein the nucleic acid molecules are described comprising coding First ORFs of fused polypeptide, wherein the nucleic acid molecules also the second ORFs comprising encoding metabolic enzyme, and And the endogenous gene being wherein mutated in the chromosome of the complementary recombinant listeria bacterium vaccine strain of the metabolic enzyme.At another In embodiment, experimenter is people.In another embodiment, people experimenter can be adult or children.In another embodiment In, experimenter is canid.In another embodiment, chimeric HER2 is the chimeric HER2 of canid.In another enforcement In example, chimeric HER2 is the chimeric HER2 of people.In another embodiment, the fused polypeptide is applied into the experimenter prevents The escape mutant of the intra-tumor.In another embodiment, the people HER2/neu chimeric antigens include at least 5,9,13,14 Or people's MHC I class epi-positions of 17 plottings.

In another embodiment, provided herein is invention be related to prevent the side of HER2/neu expression tumour growths or cancer Method.

In one embodiment, the method for the treatment of HER2/neu expression tumour growths or cancer makes the totality of the experimenter Survival period increases.In another embodiment, treating the method for HER2/neu expression tumour growths or cancer makes turning for experimenter Shifting property disease delays.In another embodiment, treatment strengthens the response of HER2/neu specific T-cells.

In one embodiment, the present invention provides the increasing for expressing HER2/neu tumour growth or cancer for causing experimenter The method of strong immune response, the method includes applying the recombinant attenuated listerial step of the nucleic acid comprising coding fused polypeptide Suddenly, wherein the fused polypeptide includes the HER2/neu chimeric antigens for being fused to other polypeptide, wherein the nucleic acid molecules bag Containing the first ORFs for encoding the fused polypeptide, wherein the nucleic acid molecules also the comprising encoding metabolic enzyme second opening The endogenous base lacked in reading frame, and the chromosome of the complementary recombinant listeria bacterium vaccine strain of wherein described metabolic enzyme Cause.In another embodiment, it is described to cause the method for strengthening immune response to increase the overall survival phase of the experimenter. It is described to cause the method for strengthening immune response to delay the metastatic disease of experimenter in another embodiment.In another reality It is described to cause the method for strengthening immune response to strengthen the response of HER2/neu specific T-cells in applying example.

In one embodiment, the present invention is provided and extends the experimenter's with HER2/neu expression tumour growths or cancer The method of survival period, the method includes the step of applying the recombinant attenuated Listeria of the nucleic acid comprising coding fused polypeptide, its Described in fused polypeptide comprising the HER2/neu chimeric antigens of other polypeptide are fused to, wherein the nucleic acid molecules include coding First ORFs of the fused polypeptide, wherein the nucleic acid molecules also the second open reading comprising encoding metabolic enzyme Frame, and the endogenous gene of the achromasia of the complementary recombinant listeria bacterium vaccine strain of wherein described metabolic enzyme.Another In one embodiment, experimenter is people.In another embodiment, people experimenter can be adult or children.In another reality In applying example, experimenter is canid.In one embodiment, methods described is additionally included in the experimenter and recurs or shift After apply the recombinant attenuated Listeria.

In one embodiment, provided herein is invention be related to delay express tumour growth or cancer with HER2/neu The method of the metastatic disease of experimenter, the method includes applying the recombinant attenuated Liszt of the nucleic acid comprising coding fused polypeptide The step of bacterium, wherein the fused polypeptide includes the HER2/neu chimeric antigens for being fused to other polypeptide, wherein the nucleic acid First ORFs of the molecule comprising the coding fused polypeptide, wherein the nucleic acid molecules also comprising encoding metabolic enzyme the What is lacked in two ORFs, and the chromosome of the complementary recombinant listeria bacterium vaccine strain of wherein described metabolic enzyme is endogenous Property gene.In another embodiment, experimenter is people.In another embodiment, people experimenter can be adult or children. In another embodiment, experimenter is canid.

Description of the drawings

It is considered subject of the present invention and particularly points out and be distinctly claimed in the conclusion part of specification to be protected.However, When being read in conjunction with the accompanying, by reference to detailed description below, the present invention can be best understood (to tissue and operation side All it is such for method) and its objects, features and advantages, in the accompanying drawings：

The structure of Fig. 1 .ADXS31-164.(A) plasmid map of pAdv164, it has and is opened in composing type Listeria p60 Bacillus subtilis (Bacillus subtilis) dal genes under mover control, for the chromosome of complementary LmddA bacterial strains Dal-dat is lacked.It is also comprising the LLO for truncating_(1-441)With the fusion of chimeric people HER2/neu genes, the fusion is by 3 Individual HER2/neu fragments：EC1 (aa 40-170), the direct fusion of EC2 (aa 359-518) and ICI (aa 679-808) and structure Build.The carrier schematic diagram on right side is shown specifically, and express by the LLO for being fused to truncate 2 of people HER2/neu of pAdv164 are extracellular Domain and the chimeric HER2/neu fusion proteins of an Intracellular domain composition.Plasmid by means of the auxotroph of dal genes it is complementary and Maintain restructuring dal/dat/actA^-In Listeria bacterial strain (LmddA) (referring to example).(B) by being printed with anti-LLO antibody The Western blot analysis that the TCA sedimentation cells culture supernatant of mark is carried out have detected Lm-LLO-ChHer2 (Lm-LLO-138) With the expression and secretion of tLLO-ChHer2 in LmddA-LLO-ChHer2 (ADXS31-164).The differential band correspondence of～104KD In tLLO-ChHer2.Endogenous LLO is detected for 58KD bands.Listeria control lacks ChHer2 expression.

The immunogenicity (A) of Fig. 2 .ADXS31-164 is thin in the spleen of immune mouse based on the listerial vaccines of HER2/neu The cytotoxic T cell response caused in born of the same parents is surveyed using NT-2 cells as stimulating factor, 3T3/neu cells as target Examination.Lm- controls are based on LmddA backgrounds that are identical in all fields but expressing uncorrelated antigen (HPV16-E7).(B) immunity FVB/ The IFN-γ that the splenocyte of N mouse is secreted into cell culture medium is stimulated in the NT-2 cells in vitro processed using mitomycin C Determined by ELISA after 24 hours.(C) splenocyte of the HLA-A2 transgenic mices of chimeric immunity is in response to from albumen The external incubation of the peptide of zones of different and secretion of gamma-IFN.Recombinant C hHer2 albumen is as positive control, uncorrelated peptide or without peptide Group constitutes negative control, such as listed in legend.The cell culture supernatant collected after being incubated altogether using 72 hours carries out ELISA Analysis, to determine IFN-γ secretion.Each data point is the +/- standard error of mean value of triplicate data.* P values<0.001.

Fig. 3. the tumor prevention research of Listeria-ChHER2/neu vaccines uses every kind of recombinant listeria bacterium-ChHer2 Or control Listeria vaccine injection HER2/neu transgenic mices six times.Immunity starts in 6 week old, straight per continuity once in three weeks By the 21st week.The outward appearance of tumour is monitored weekly and is represented with the percentage without mice with tumor.*p<0.05, N=9 only per group.

Effect of Fig. 4 .ADXS31-164 immunity to %Treg in spleen.To FVB/N mouse hypodermic inoculations 1 × 10⁶Individual NT- 2 cells, and using every kind of vaccine with one week as Immunity at intervals three times.Collect spleen within 7 days after second immunity.Exempt from separation After epidemic disease cell, it is dyeed, with by AntiCD3 McAb, CD4, CD25 and FoxP3 antibody test Treg.From representative experiment The point of Treg illustrate CD25⁺/FoxP3⁺The frequency of T cell, with total CD3 between different treatment groups⁺Or CD3⁺CD4⁺T cell Percentage is represented.

Effect of Fig. 5 .ADXS31-164 immunity to the tumor-infiltrated Treg of % in NT-2 tumours.Give FVB/N mouse notch grafts Plant 1 × 10⁶Individual NT-2 cells, and using every kind of vaccine with one week as Immunity at intervals three times.Collect within 7 days after second immunity Tumour.After isolating immune cells, it is dyeed, with by AntiCD3 McAb, CD4, CD25 and FoxP3 antibody test Treg. (A). from the point diagram of the Treg of representative experiment.(B).CD25⁺/FoxP3⁺The frequency of T cell, with total between different treatment groups CD3⁺Or CD3⁺CD4⁺The percentage (left illustration) of T cell and intra-tumor CD8/Treg ratio (right illustration) are represented.Data are with 2 Mean value ± the SEM that independent experiment is obtained is represented.

Fig. 6 .ADXS31-164 vaccine inoculations can delay the growth of breast cancer cell line in brain.Balb/c mouse use ADXS31-164 or control Listeria vaccine immunity three times.To anesthetized mice intracranial injection EMT6-Luc cells (5,000). (A) the in vitro imaging of mouse is carried out using Xenogen X-100CCD cameras in specified number of days.(B) image pixel intensities are with photon Number/second/cm2 surface areas are drawn；This is represented with average luminance.(C) EMT6-Luc cells, 4T1-Luc and NT-2 clones HER2/neu is expressed and detected by using the western blot of anti-HER2/neu antibody.Murine macrophages like cell system J774.A2 cells are used as negative control.

Fig. 7. front 18 patients that ADXS31-164 is inoculated with are shown.

Fig. 8. illustrate that ADXS31-164 is applied and be not result in that early stage or advanced cardiac are damaged.A) the ultrasonic cardiography diagram of heart Go out heart outward appearance normal.B the continuous cardiac troponin I level) assessed in the course of the study illustrates that level is normal (another See Figure 26 D).

Fig. 9. A is shown) body temperature and B) and systolic pressure ADXS31-164 associated changes.After baseline and ADXS31-164 are applied Every 2 hour record body temperature and systolic pressure.Parameter during each vaccine inoculation of every dog is shown.Horizontal bar represent each dosage group, The intermediate value of all dogs of each time point.P ＜ 0.05,P ＜ 0.005

Figure 10. the planning chart of ADXS31-164 and Palliative radiotherapy (RT) therapeutic alliance primary disease is shown.

Figure 11. there is the evidence of metastatic disease after the not shown dog proximal humeral fracture of radiogram, and also illustrate that and deposit In poroma, show union.

Figure 12. the timeline of early stage Phase I clinical trial, the monocytosis of the recombinant expressed ADXS31-164 of its assessment Listeria causes the security and effect of the treatment validity antineoplastic immune with the osteosarcomatous dog of limbs.

Therapy-related adverse events and survival curves after Figure 13 .ADXS-31-164 administrations.A) the bad thing of therapy-related Part.B) the equal non-metastatic disease of all dogs when test is selected.The dog of control group receives amputation, be then administered alone carboplatin or Carboplatin adds adriamycin.2 dogs of vaccine group are examined, because they die from incoherent reason, and (1 dog dies from imbedibility lung Inflammation, another dies from nephroblastoma).Vaccine inoculation group：Red line；Control group：Black line.

Figure 14. the radiation image of the primary and metastatic bone sarcoma (OSA) of people (A) and canid (B) patient.Two In individual species, primary focus is characterised by the notable hyperplasia of bone metaphysis (arrow in A) and dissolving region.

The schematic diagram of Figure 15 .I phase 3+3 clinical testings, it assesses ADXS31- in the dog with HER2+ osteosarcoma (OSA) 164 security and effect.Privately owned dog with spontaneous HER2+ limbs OSA receives nursing standard amputation and follow-up carboplatin Chemotherapy.After final carboplatin dose three weeks, to dog intravenous inoculation 2 × 10⁸、5×10⁸、1×10⁹Or 3 × 10⁹CFU's ADXS31-164 (being separated by three weeks carries out three vaccine inoculation).Carried out again by stages until death, to determine vaccine to dog per 2 months Prevent effect of metastatic disease.

Figure 16. the HER2/neu expression in canid primary osteosarcoma.(A) the H＆E dyeing of dog primary OSA is illustrated Pernicious osteoblastic nest and osteoid are deposited.(B) immunohistochemical evaluation of canid primary OSA illustrates HER2/ Expression of the neu in pernicious Gegenbaur's cell.(C) western blot of the primary OSA sample of 5 privately owned dogs illustrates HER2/ Neu expression is variable.Positive control is：MCF-7 MCF-7s and CAMAC2 canid breast cancer cell lines.

Figure 17. the blood indices of 24 hours after baseline and ADXS31-164 administrations.It is every to all dogs in each dosage group Front value and rear value during secondary vaccine inoculation is averaged.P ＜ 0.05,P ＜ 0.005.Illustrate 24 after ADXS31-164 is applied Hour, leucocyte and neutrophil count (A-B) instantaneous but increase statistically significantly, and with blood platelet and Lymphocyte (C-D) is instantaneously reduced.

Leucocyte (WBC), neutrophil leucocyte and the monocyte count that Figure 18 .ADXS31-164 are induced increases and survival period It is related.WBC, neutrophil leucocyte and monocyte count are determined for 24 hours after baseline and vaccine inoculation.In each vaccine inoculation The percentage of increase is calculated afterwards, and every dog is averaged.(A) result shows according to survival (death lives).(B) tie Fruit shows according to the ADXS31-164 dosage for being received.Horizontal bar represents the intermediate value of the group.

Figure 19. the IFN-γ ELISpot assessment results of the Her-2 specific T-cells responses that ADXS31-164 is induced are shown.

Figure 20. illustrate that repetition " reinforcement " vaccine inoculation have stimulated Her-2 specific immunities.(A) illustrate patient 289-003's As a result.(B) result of patient 289-004 is shown.EC1, EC2 and IC1 represent the fragments of peptides of HER2/neu polypeptides.

Figure 21. (A) the Kaplan Meier of transfer time (TTM) and (B) OSA specificity survival periods estimate.

Figure 22. illustrate that ADXS31-164 prevents the development of metastatic disease.(A and B) after Carboplatin in patients 3 weeks (A) and The breast radiation development photo of 3 weeks (B) collection after three ADXS31-164 vaccine inoculations illustrates that the right cranium side lobe of the lung is pre-existing Metastatic tubercle size increases, but metastatic disease will not further develop in remaining lobe of the lung.(C and D) is applied in ICG Afterwards, the thoracoscopy of Lung neoplasm finds to fluoresce under near infrared light (C).The outward appearance removed when MET cuts off is overall just Normal lung tissue illustrates fluorescence (illustration) under near infrared light (D).(E and F) (E) Lung neoplasm and (F) fluoresce normal lung group The H＆E stained tissue pathology knitted illustrate the focal area of encapsulation Lung neoplasm (E) and the overall normal lung tissue inflammation of outward appearance The notable bleeding and necrosis in domain (F).(G and H) immunohistochemistry of Lung neoplasm under low power (G) and high power (H) magnifying power is shown The micro CD3+T cells gone out in the CD3+T cells and tumor tissues of Lung neoplasm.(I and J) is in low power (G) and high power (H) The immunohistochemistry of the normal lung tissue of outward appearance shows the focal accumulation of CD3+T cells under magnifying power.(K) focal pneumonia Magnification at high multiple rate H＆E dyeing big abnormal cell is shown, wherein mitotic figure is surrounded by lymphocyte.(L) pneumonia region Vimentin dyeing illustrates maxicell, and wherein mitotic figure is surrounded by monocyte.

Figure 23 .ADXS31-164 delay/prevent metastatic disease and extend with the spontaneity osteosarcomatous dogs of HER2+ The overall survival phase.Kaplan-Meier survival curves of the vaccine inoculation dog compared with historical control group are shown.Control group is by suffering from The dog processed by the dog of HER2+ limbs OSA, amputation and follow-up chemotherapy but do not receive the dog of ADXS31-164 and constitute.P<0.0001. Vaccine inoculation group：Red line；Control group：Black line.

Figure 24. illustrate that ADXS31-164 destroys the tolerance to HER2/neu.3 weeks after baseline, the 3rd vaccine inoculation (9 weeks) and PBMC is collected within (17 weeks) after 2 months, and the highly conserved IC1 domains by IFN-γ ELISpot analyses to HER2/neu Response.Dog is divided into early stage respondent, late phase responses person and apparent nonresponder by shown result.NA represents the 17 of these dogs All samples are not evaluated.

Figure 25 A-D. illustrate that ADXS31-164 is not adversely affected to cardiac function.Baseline, the vaccine of every dog of assessment During inoculation and thereafter per 2 months cardiac parameters LVID (diastole) (Figure 25 A), LVID (systole phase) (Figure 25 B) and shortening point Number (Figure 25 C).In identical time point assessment cardiac troponin I level (Figure 25 D).

Figure 26. illustrate that ADXS31-164 destroys the immunological tolerance of the highly conserved Intracellular domain to HER2/neu.

It should be appreciated that in order to explanation it is succinct and clear for the sake of, the element illustrated in figure is not necessarily drawn to scale.For example, For clarity, the size of some elements can amplify relative to other elements.In addition, in the appropriate case, drawing reference numeral can be It is recycled and reused for representing corresponding or similar element in figure.

Specific embodiment

In the following specific embodiments, multiple details are set forth, to provide thorough understanding of the present invention.So And, it should be appreciated by those skilled in the art that the present invention can be implemented in the case where not having these details.In other situations In, to avoid making complication of the present invention, well known method, operation and component are not described in detail.

In one embodiment, there is provided herein described for preventing, treating Her2-neu antigen presentations tumour and inoculation The vaccine of tumour, and the immune response of the secondary Dominant Epitopes to Her2-neu antigens is induced, while getting around the group that mutation is avoided Compound and method.In another embodiment, it is due to epitope spreading to get around mutation avoidance.In yet another embodiment, get around Mutation avoidance is the chimeric property due to antigen.

In another embodiment, there is provided herein the immunogenic composition comprising fused polypeptide, wherein the fusion Polypeptide is included and is fused to the HER2/neu chimeric antigens of other polypeptide, and wherein fusion protein is applied to HER2/ The experimenter of neu expression tumours prevents the escape mutant of the intra-tumor.In another embodiment, exempt from there is provided herein including The recombinant listeria bacterium vaccine strain of epidemic disease Immunogenic Compositions.

In one embodiment, experimenter is people experimenter.In another embodiment, people experimenter is adult or youngster It is virgin.In another embodiment, people experimenter is children.In another embodiment, experimenter is canid experimenter. In another embodiment, canid is dog.

In one embodiment, there is provided herein causing the increasing for expressing HER2/neu tumour growth or cancer of experimenter The method of strong immune response, the method includes the step of applying the recombinant listeria bacterium of the nucleic acid comprising coding fused polypeptide, its Described in fused polypeptide comprising being fused to the HER2/neu chimeric antigens of other polypeptide.

In one embodiment, there is provided herein preventing HER2/neu expression tumour growths or the side of cancer of experimenter Method, the method includes the step of applying the recombinant listeria bacterium of the nucleic acid comprising coding fused polypeptide, wherein the fused polypeptide Comprising the HER2/neu chimeric antigens for being fused to other polypeptide.

In another embodiment, there is provided herein the HER2/neu expression tumour growths for the treatment of experimenter or the side of cancer Method, the method includes the step of applying the recombinant listeria bacterium of the nucleic acid comprising coding fused polypeptide, wherein the fused polypeptide Comprising the HER2/neu chimeric antigens for being fused to other polypeptide.

In one embodiment, there is provided herein extending the experimenter's with HER2/neu expression tumour growths or cancer The method of survival period, the method includes the step of applying the recombinant listeria bacterium of the nucleic acid comprising coding fused polypeptide, wherein institute Fused polypeptide is stated comprising the HER2/neu chimeric antigens for being fused to other polypeptide.In one embodiment, experimenter is people. In another embodiment, experimenter is canid.

In one embodiment, there is provided herein delaying the experimenter's with HER2/neu expression tumour growths or cancer The method of metastatic disease, the method includes the step of applying the recombinant listeria bacterium of the nucleic acid comprising coding fused polypeptide, its Described in fused polypeptide comprising being fused to the HER2/neu chimeric antigens of other polypeptide.In one embodiment, experimenter is People.In another embodiment, experimenter is canid.

In one embodiment, there is provided herein the HER2/neu expression tumour growths for the treatment of experimenter or the side of cancer Method, the method includes the step of applying the recombinant attenuated Listeria of the nucleic acid comprising coding fused polypeptide, wherein the fusion Polypeptide includes the HER2/neu chimeric antigens for being fused to other polypeptide, wherein the nucleic acid molecules are more comprising the coding fusion First ORFs of peptide, wherein the nucleic acid molecules also the second ORFs comprising encoding metabolic enzyme, and wherein The endogenous gene being mutated in the chromosome of the complementary recombinant listeria bacterium vaccine strain of the metabolic enzyme.In another embodiment In, experimenter is people.In another embodiment, people experimenter can be adult or children.In another embodiment, it is tested Person is canid.In another embodiment, chimeric HER2 is the chimeric HER2 of canid.In another embodiment, it is embedding It is the chimeric HER2 of people to close HER2.In another embodiment, the fused polypeptide is applied into the experimenter prevents described swollen Escape mutant in knurl.In another embodiment, the people HER2/neu chimeric antigens include at least 5,9,13,14 or 17 People's MHC I class epi-positions of plotting.

In one embodiment, there is provided herein the recombinant listeria bacterium vaccine strain comprising nucleic acid molecules, the wherein nucleic acid First ORFs of the molecule comprising coded polypeptide, the wherein polypeptide include HER2/neu chimeric antigens, the wherein nucleic acid point Son also the second ORFs comprising encoding metabolic enzyme, and the wherein dyeing of the metabolic enzyme complementation recombinant listeria bacterium bacterial strain The endogenous gene lacked in body.In another embodiment, recombinant listeria bacterium vaccine strain also includes encoding metabolic enzyme The 3rd ORFs nucleic acid molecules, and wherein lack in the chromosome of metabolic enzyme complementation recombinant listeria bacterium bacterial strain Endogenous gene.

In one embodiment, nucleic acid molecules are integrated into Listeria genome.In another embodiment, nucleic acid molecules In the plasmid of recombinant listeria bacterium vaccine strain.In yet another embodiment, in the case where there is no antibiotic selection in plasmid In being stably maintained at recombinant listeria bacterium vaccine strain.In another embodiment, plasmid does not give recombinant listeria bacterium antibiotic Resistance.In another embodiment, recombinant listeria bacterium bacterial strain is attenuation.In another embodiment, recombinant listeria bacterium It is attenuation auxotrophic strain.In another embodiment, exogenous antigen expresses a kind of bacterium such as of the invention to bacterium The hypermetabolism burden of applying is also the important mechanisms of attenuation.

In one embodiment, attenuated strain is LmddA.In another embodiment, the bacterial strain applies strong adjuvant effect, The effect is based on the intrinsic property of listerial vaccine.One performance of the adjuvant effect is expression except chimeric HER2/neu Outside antigen Listeria or ADXS-31-164 (expression chimeric HER2/neu) vaccine caused by under intra-tumor Treg quantity 5 times (referring to Fig. 5 of this paper) of drop.In another embodiment, expression not synantigen (HPV16E7) LmddA carriers also with it is swollen The frequency of Treg is remarkably decreased correlation in knurl, it is likely to due to the result of innate immune responses.

In one embodiment, provided herein is attenuation auxotroph Listeria vaccine strain be ADXS-31-164 bacterium Strain.ADXADXS-31-164 is based on Listeria vaccine carrier, and the carrier is attenuated due to the disappearance of virulent gene actA, and The plasmid of the internal and external expression for HER2/neu is maintained due to the complementary of dal genes.In one embodiment, ADXS31-164 expression and secretion are fused to the chimeric HER2/neu eggs of front 441 amino acid of Listeriolysin O (LLO) In vain, in another embodiment, Listeriolysin O is the non-haemolysis LLO for truncating.In another embodiment, ADXS31- 164 play strong and antigentic specificity antitumor response, and the tolerance to HER2/neu can be destroyed in transgenic animals Property (referring to example, Figure 24).In another embodiment, ADXS31-164 bacterial strains are highly attenuated, and with than previous For the higher safety spectrum of Listeria vaccine, because it more quickly can be removed from the spleen of immune mouse.In another enforcement In example, than Lm-LLO-ChHer2, (antibiotic of the vaccine resists the tumor invasion lag phase in ADXS31-164 render transgenic animals The higher form of property and toxicity) longer (referring to Fig. 3).In another embodiment, ADXS31-164 bacterial strains have hyperimmunization Originality, can destroy the tolerance to HER2/neu self-antigen, and prevent the tumour shape in HER2/neu transgenic animals Into.In another embodiment, ADXS31-164 substantially reduces intra-tumor regulatory T cells (Treg).In another enforcement In example, the decline of Treg frequencies in the tumour of LmddA vaccines process raises intra-tumor CD8/Treg ratio, implies in LmddA More favourable tumor microenvironment can be obtained after vaccine immunity.In another embodiment, the use of the chimeric antigen is not produced and escaped Ease mutation, shows that the mutation of tumour will not be away from the treatment effective response to being treated using the novel antigens (referring to example 6).In another embodiment, the periphery immunity of ADXS31-164 has delayed the growth (ginseng of metastatic breast cancer cell line in brain See example 7).In another embodiment, compared with the control subject of ADXS31-164 vaccine inoculations is not received, will including cut The treatment of limb, chemotherapy and ADXS31-164 vaccine inoculations is supplied to and extends survival period with osteosarcoma canid experimenter (referring to example 9 and 10).In another embodiment, compared with the control subject of ADXS31-164 vaccine inoculations is not received, Show being supplied to including the treatment of amputation, chemotherapy and ADXS31-164 vaccine inoculations with osteosarcomatous canid experimenter Go out transfer to reduce (referring to example 10).In another embodiment, with not receive compareing for ADXS31-164 vaccine inoculations tested Person compares, and receives being supplied to including the treatment of amputation, chemotherapy and ADXS31-164 vaccine inoculations with osteosarcomatous canid Examination person shows that induced specific T-cells response strengthens (referring to example 10).

In one embodiment, Lm-LLO-ChHer2 bacterial strains are Lm-LLO-138, and comprising the antibiosis from plasmid expression Plain resistant gene and prfA genes.

In one embodiment, the antibiotic-free Listeria of recombinant attenuated expression chimeric antigen is used to prevent and control Cancer or entity tumor are treated, as illustrated herein.In another embodiment, tumour is HER2/neu positive tumors. In another embodiment, cancer is HER2/neu expression cancers.In another embodiment, cancer is breast cancer, nervous centralis System (CNS) cancer, head and neck cancer, osteosarcoma (OSA), canid osteosarcoma, Ewing's sarcoma (ES) or known in the art Any HER2/neu expresses cancer.In another embodiment, canid osteosarcoma is limbs osteosarcoma.In another enforcement In example, tumour is osteosarcoma, tumor of breast, head and neck neoplasm or any other antigen presentation tumour known in the art.Another In one embodiment, cancer as herein described or entity tumor are the results of recurrence or metastatic disease.

In another embodiment, the recombinant listeria bacterium for expressing chimeric HER2/neu can be used as treating HER2/neu and cross table Up to the therapeutic vaccine of entity tumor.In another embodiment, provided herein is HER2/neu chimeric antigens can be used to treat HER2/neu expresses tumour and prevents the escape mutant of the tumour.In another embodiment, term " escape mutant " is referred to far From the Tumor mutations of the treatment effective response to treatment.

In one embodiment, there is provided herein comprising coding provided herein is recombinant polypeptide first ORFs Nucleic acid molecules, its nucleic acid molecule is resided in recombinant listeria bacterium vaccine strain.In another embodiment, by provided herein is Nucleic acid molecules are used to convert Listeria, to obtain recombinant listeria bacterium.In another embodiment, provided herein is nucleic acid lack Weary virulent gene.In another embodiment, the nucleic acid molecules for being integrated into Listeria genome carry non-functional toxicity base Cause.In another embodiment, virulent gene is mutation in the genome of recombinant listeria bacterium.In another embodiment In, nucleic acid molecules are used to make the inactivation of endogenous gene present in Listeria genome.In yet another embodiment, toxicity base Because being actA genes.In another embodiment, virulent gene is prfA genes.Technical staff will be understood that virulent gene can Being any gene related to toxicity in recombinant listeria bacterium known in the art.

In one embodiment, provided herein is metabolic gene, virulent gene etc. be that the chromosome of Listeria bacterial strain lacks Weary.In another embodiment, provided herein is metabolic gene, virulent gene etc. be the chromosome of Listeria bacterial strain and appoint What what episome gene lacked.Technical staff will be understood that term " episome ", " episome " etc. refer to that unconformity is entered Provided herein is listerial chromosome in plasmid vector or its purposes.In another embodiment, the term refers to whole Close into provided herein is listerial chromosome in plasmid vector.In another embodiment, metabolic gene, virulent gene Lack etc. the genome for being toxic strain.In one embodiment, virulent gene is mutation in chromosome.At another In embodiment, virulent gene is chromosome deficiency.

In another embodiment, provided herein is nucleic acid and plasmid do not give provided herein is recombinant listeria bacterium antibiosis Plain resistance.

In one embodiment, provided herein is nucleic acid molecules include plasmid.In another embodiment, provided herein is Nucleic acid molecules are plasmids.In another embodiment, provided herein is plasmid be integration vector.In another embodiment, matter Grain is non-integrated vector.In another embodiment, plasmid includes integration vector.In another embodiment, integration vector is Site-specific integration carrier.In another embodiment, the nucleic acid molecules of the method for the present invention and composition by this area Any kind of nucleotides known is constituted.

Technical staff will be understood that, term " metabolic enzyme " can be covered and be related in the synthesis of the nutriment needed for host bacteria Enzyme.In another embodiment, the term refers to the enzyme needed for the synthesis of the nutriment needed for host bacteria.At another In embodiment, the term refers to the enzyme being related in the synthesis of the nutriment that host bacteria is utilized.In another embodiment, should Term refers to the enzyme being related in the synthesis of the nutriment needed for the continued propagation of host bacteria.In another embodiment, enzyme Be nutriment synthesis needed for.Every kind of possibility represents the separate embodiments of the present invention.

Technical staff will be understood that term " stable to maintain " can be covered in the feelings that there is no selection (e.g., antibiotic is selected) Nucleic acid molecules or plasmid is set to maintain 10 generations in host cell or bacterium and not produce detectable loss under condition.In another reality In applying example, the cycle was 15 generations.In another embodiment, the cycle was 20 generations.In another embodiment, the cycle is 25 Generation.In another embodiment, the cycle was 30 generations.In another embodiment, the cycle was 40 generations.In another embodiment In, the cycle was 50 generations.In another embodiment, the cycle was 60 generations.In another embodiment, the cycle was 80 generations. In another embodiment, the cycle was 100 generations.In another embodiment, the cycle was 150 generations.In another embodiment In, the cycle was 200 generations.In another embodiment, the cycle was 300 generations.In another embodiment, the cycle is 500 Generation.In another embodiment, the cycle is more generations.In another embodiment, nucleic acid molecules or plasmid be in vitro (e.g., In culture) stable maintenance.In another embodiment, nucleic acid molecules or plasmid are stably maintained in vivo.In another enforcement In example, nucleic acid molecules or plasmid were both stably maintained in vitro or in vivo.

In one embodiment, the present invention provides the recombinant listeria bacterium bacterial strain for expressing the antigen.The present invention also provides bag Recombinant polypeptide containing Listeria hemolysin (LLO) protein fragments for being fused to HER2 chimeric proteins or its fragment is heavy comprising this The vaccine and immunogenic composition of group polypeptide, and induce anti-HER2 immune responses and treatment HER2 expression tumours and inoculation bag The method of the vaccine containing the recombinant polypeptide.

In another embodiment, recombinant listeria bacterium bacterial strain of the invention is passed in animal reservoir.At another In embodiment, this is passed on maximizes effect of the bacterial strain as vaccine carrier.In another embodiment, this passes on stable Lee The immunogenicity of this special bacteria strain.In another embodiment, this passes on the toxicity of stable Listeria bacterial strain.In another reality In applying example, this passes on the immunogenicity for increasing Listeria bacterial strain.In another embodiment, this passes on increase Listeria bacterium The toxicity of strain.In another embodiment, this passes on the unstable sub-strain for removing Listeria bacterial strain.In another embodiment In, this passes on the prevalence of the unstable sub-strain for reducing Listeria bacterial strain.In another embodiment, Listeria bacterial strain is included The genome insertion of gene of the coding containing antigen recombinant peptide.In another embodiment, Listeria bacterial strain carrying package is containing coding The plasmid of the gene containing antigen recombinant peptide.In another embodiment, this is passed on by any other method known in the art Carry out.

In one embodiment, provided herein is recombinant polypeptide include provided herein is fusion protein.In another enforcement In example, recombinant polypeptide is fusion protein.In another embodiment, provided herein is fusion protein comprising chimeric HER2 antigens and It is selected from：A) non-haemolysis LLO albumen or N- terminal fragments, b) PEST sequences or c) the other polypeptide of ActA fragments, in addition wherein The other peptide fusion is to HER2/neu chimeric antigens.In another embodiment, polypeptide in addition is functional. In another embodiment, the fragment of polypeptide in addition has immunogenicity.In another embodiment, polypeptide in addition has and exempts from Epidemic focus.

In another embodiment, provided herein is fusion protein comprising be fused to provided herein is HER2/neu inosculating antibodies The non-haemolysis LLO albumen or N- terminal fragments of original.In another embodiment, the fusion protein of the method for the present invention and composition Comprising the ActA sequences from Listeria organism.The enhancement antigen in the way of similar to LLO of ActA albumen and its fragment is in Pass and immunity.

In another embodiment, the fusion protein of the method for the present invention and composition is included from Listeria organism Truncation ActA sequences.In another embodiment, the ActA of truncation by wild type ActA albumen front 390 amino acid groups Into such as United States Patent (USP) No.7, described in 655,238, the full patent texts are hereby incorporated herein by.In another embodiment In, the ActA of truncation is ActA-N100 or its modification pattern (referred to as ActA-N100*), wherein PEST motifs disappearance, and is wrapped Containing nonconservative QDNKR displacements, as described in U.S. Patent Publication No.2014/0186387.ActA albumen and its fragment are with class The mode enhancement antigen for being similar to LLO is presented and immunity.

In another embodiment of the method for the present invention and composition, provided herein is fusion protein include HER2/neu Antigen and other polypeptide.In one embodiment, polypeptide in addition is non-haemolysis LLO albumen or its fragment (reality of this paper Example).In another embodiment, polypeptide in addition is PEST sequences.In another embodiment, polypeptide in addition is ActA eggs White or its fragment.The enhancement antigen in the way of similar to LLO of ActA albumen and its fragment is presented and immunity.

In another embodiment, the other polypeptide of the method for the present invention and composition is Listeria hemolysin (LLO) peptide.In another embodiment, polypeptide in addition is ActA peptides.In another embodiment, polypeptide in addition is PEST Sequence peptide.In another embodiment, polypeptide in addition is immunogenic any other peptide for being capable of enhancement antigen peptide.It is every kind of Possibility represents the separate embodiments of the present invention.

Fusion protein comprising HER2/neu chimeric antigens can be prepared by any suitable method, including such as clone and The restriction of appropriate sequence or by the direct chemical synthesis of the method being discussed below.Or, subsequence can be cloned and using appropriate Restriction Enzyme cuts appropriate subsequence.Then can junction fragment preparing required DNA sequence dna.In one embodiment, compile Code provided herein is the DNA of antigen can be prepared using DNA cloning method such as PCR (PCR).First, natural The either side of the new end of DNA fragmentation is individually expanded.The 5' ends coding peptide linker of the sequence of one amplification, and another is expanded The 3' ends of sequence also encode peptide linker.5' ends and the 3' termini-complementaries of the second fragment due to the first fragment, two pieces Section (after partial purification, such as on LMP agaroses) can be used as to overlap template in the 3rd PCR reactions.The sequence of amplification will be wrapped Containing the sequence on the fragment (forming amino sequence now) on codon, vent position carboxyl side, joint and vent position amino side Row (forming carboxyl sequences now).In another embodiment, antigen is connected into plasmid.Every kind of method represents the list of the present invention Only embodiment.

The result of the present invention shows that the administration of the composition of the present invention can be used for induction identification and kill the anti-of tumour cell The formation (example of this paper) of former specific T-cells (e.g., cytotoxic T cell).

In one embodiment, the present invention is provided comprising being fused to HER2 chimeric proteins or be fused to the LLO eggs of its fragment The recombinant polypeptide of white N- terminal fragments.In one embodiment, the present invention is provided by being fused to HER2 chimeric proteins or fusion To the recombinant polypeptide of the N- terminal fragments composition of the LLO albumen of its fragment.

In another embodiment, the HER2 chimeric proteins of the method for the present invention and composition are people's HER2 chimeric proteins. In another embodiment, HER2 chimeric proteins are mouse HER2 chimeric proteins.In another embodiment, HER2 chimeric proteins It is rat HER2 chimeric proteins.In another embodiment, HER2 chimeric proteins are primate HER2 chimeric proteins.At another In embodiment, HER2 chimeric proteins are canid HER2 chimeric proteins.In another embodiment, Her-2 albumen is ability People known to domain or the HER2 chimeric proteins or combinations thereof of any other animal species.Every kind of possibility represents the present invention's Separate embodiments.

In another embodiment, Her-2 albumen is referred to as " HER2/neu ", " Erbb2 ", " v-erb-b2 ", " c-erb- The albumen of b2 ", " neu " or " cNeu ".In another embodiment, HER2/neu is referred to herein as " Her-2 ", " Her-2 Albumen ", " HER2 albumen " or " HER2 ".

In one embodiment, provided herein is Her2-neu chimeric proteins there is two extracellular of HER2/neu antigens Section and an intracellular fragment, the antigen illustrates the MHC I class epi-position clusters of oncogene, and in another embodiment, chimeric protein People MHC I class epi-positions (fragment EC1, EC2 and IC1) with 3 H2Dq and at least plotting of 17 HER2/neu antigens (referring to Figure 1A).In another embodiment, chimeric protein has people MHC I class epi-positions (fragment EC2 and IC1) of at least 13 plottings. In another embodiment, chimeric protein has people MHC I class epi-positions (fragment EC1 and IC1) of at least 14 plottings.Another In individual embodiment, chimeric protein has people MHC I class epi-positions (fragment EC1 and IC2) of at least 9 plottings.In another enforcement In example, Her2-neu chimeric proteins are fused to non-haemolysis Listeriolysin O (LLO).In another embodiment, Her2- Neu chimeric proteins are fused to the Listeriolysin O (tLLO) for truncating.In another embodiment, the chimeric eggs of Her2-neu It is fused to front 441 amino acid of listerisa monocytogenes in mjme Listeriolysin O (LLO) albumen in vain, and by Listerisa monocytogenes in mjme attenuation auxotrophic strain LmddA expression and secretion.In another embodiment, from Provided herein is attenuation auxotrophic strain (the chimeric HER2/neu antigens/LLO fusion proteins of expression) fusion protein tLLO- The expression and secretion of ChHer2 and the expression of growth in vitro Lm-LLO-ChHer2 in TCA sedimentation cell culture supernatants after 8 hours With Secretion when (referring to Figure 1B).

In one embodiment, unexposed animal () or uncorrelated Listeria vaccine injection animal CTL activity is not detected by mouse (referring to Fig. 2A).And in another embodiment, provided herein is attenuation auxotroph bacterium Strain (ADXS31-164) can stimulate the secretion (Fig. 2 B) of IFN-γ by the splenocyte of wild type FVB/N mouse.

In another embodiment, provided herein is the metabolic enzyme of method and composition be amino acid metabolism enzyme, and another In one embodiment, metabolic enzyme is alanine racemase.In another embodiment, metabolic enzyme is D- aminotransferases. In another embodiment, the formation of the amino acid of the Cell wall synthesis that metabolism enzymatic is used in recombinant listeria bacterium bacterial strain, and In another embodiment, metabolic enzyme is alanine racemase.

In another embodiment, the gene for encoding the metabolic enzyme is expressed under the control of Listeria p60 promoter. In another embodiment, using inlA (coding internalization element) promoter.In another embodiment, using hly promoters.Another In one embodiment, using ActA promoters.In another embodiment, integrase gene is opened in any other Gram-positive Express under the control of mover.In another embodiment, the gene of encoding metabolic enzyme in Listeria function it is any Express under the control of other promoters.Technical staff will be appreciated that other promoters or polycistronic expression box can be used to drive The expression of gene.Every kind of possibility represents the separate embodiments of the present invention.

In another embodiment, HER2/neu chimeric proteins are by following SEQ ID NO:Nucleic acid sequence encoding shown in 1

gagacccacctggacatgctccgccacctctaccagggctgccaggtggtgcagggaaacctggaactcacctacct gcccaccaatgccagcctgtccttcctgcaggatatccaggaggtgcagggctacgtgctcatcgctcacaaccaag tgaggcaggtcccactgcagaggctgcggattgtgcgaggcacccagctctttgaggacaactatgccctggccgtg ctagacaatggagacccgctgaacaataccacccctgtcacaggggcctccccaggaggcctgcgggagctgcagct tcgaagcctcacagagatcttgaaaggaggggtcttgatccagcggaacccccagctctgctaccaggacacgattt tgtggaagaatatccaggagtttgctggctgcaagaagatctttgggagcctggcatttctgccggagagctttgat ggggacccagcctccaacactgccccgctccagccagagcagctccaagtgtttgagactctggaagagatcacagg ttacctatacatctcagcatggccggacagcctgcctgacctcagcgtcttccagaacctgcaagtaatccggggac gaattctgcacaatggcgcctactcgctgaccctgcaagggctgggcatcagctggctggggctgcgctcactgagg gaactgggcagtggactggccctcatccaccataacacccacctctgcttcgtgcacacggtgccctgggaccagct ctttcggaacccgcaccaagctctgctccacactgccaaccggccagaggacgagtgtgtgggcgagggcctggcct gccaccagctgtgcgcccgagggcagcagaagatccggaagtacacgatgcggagactgctgcaggaaacggagctg gtggagccgctgacacctagcggagcgatgcccaaccaggcgcagatgcggatcctgaaagagacggagctgaggaa ggtgaaggtgcttggatctggcgcttttggcacagtctacaagggcatctggatccctgatggggagaatgtgaaaa ttccagtggccatcaaagtgttgagggaaaacacatcccccaaagccaacaaagaaatcttagacgaagcatacgtg atggctggtgtgggctccccatatgtctcccgccttctgggcatctgcctgacatccacggtgcagctggtgacaca gcttatgccctatggctgcctcttagactaa(SEQ ID NO:1)。

In another embodiment, HER2/neu chimeric proteins include SEQ ID NO:2 sequence：

E T H L D M L R H L Y Q G C Q V V Q G N L E L T Y L P T N A S L S F L Q D I Q E V Q G Y V L I A H N Q V R Q V P L Q R L R I V R G T Q L F E D N Y A L A V L D N G D P L N N T T P V T G A S P G G L R E L Q L R S L T E I L K G G V L I Q R N P Q L C Y Q D T I L W K N I Q E F A G C K K I F G S L A F L P E S F D G D P A S N T AP L Q P E Q L Q V F E T L E E I T G Y L Y I S AW P D S L P D L S V F Q N L Q V I R G R I L H N G A Y S L T L Q G L G I S W L G L R S L R E L G S G L A L I H H N T H L C F V H T V P W D Q L F R N P H Q A L L H T A N R P E D E C V G E G L A C H Q L C A R G Q Q K I R K Y T M R R L L Q E T E L V E P L T P S G A M P N Q A Q M R I L K E T E L R K V K V L G S G A F G T V Y K G I W I P D G E N V K I P V A I K V L R E N T S P K A N K E I L D E A Y V M A G V G S P Y V S R L L G I C L T S T V Q L V T Q L M P Y G C L L D(SEQ ID NO: 2)。

Table 1 below is shown respectively the percentage (%) between the amino acid sequence of people and canid Her-2EC and IC fragment Homogeneity.

Table 1

In another embodiment, the amino acid sequence of encoding human HER2/EC1 fragments is in (SEQ ID NO:69)： SLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLT EILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCK(SEQ ID NO:69) illustrate in.

In another embodiment, the amino acid sequence of canid her2/neu EC1 fragments is encoded in (SEQ ID NO:70)：

SLSFLQDIQEVQGYVLIAHSQVRQIPLQRLRIVRGTQLFEDNYALAVLDNGDPLEGGIPAPGAAPGGLRELQLRSLT EILKGGVLIQRSPQLCHQDTILWKDVFHKNNQLALTLIDTNRSRACPPCSPACK(SEQ ID NO:70) illustrate in.

In another embodiment, the amino acid sequence of encoding human HER2/neu EC2 fragments is in (SEQ ID NO:71)：

TAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGS (SEQ ID NO:71) illustrate in.

In another embodiment, the amino acid sequence of canid her2/neu EC2 fragments is encoded in (SEQ ID NO:72)：

TAPLQPEQLRVFEALEEITGYLYISAWPDSLPNLSVFQNLRVIRGRVLHDGAYSLTLQGLGISWLGLRSLRELGS (SEQ ID NO:72) illustrate in.

In another embodiment, the amino acid sequence of encoding human HER2/neu IC1 fragments is in (SEQ ID NO:73)：

NQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSR LLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLED(SEQ ID NO:73) illustrate in.

In another embodiment, the amino acid sequence of canid her2/neu IC1 fragments is encoded in (SEQ ID NO:74)：

NQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSR LLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLED(SEQ ID NO:74) illustrate in. In one embodiment, amino acid sequence (the SEQ ID NO of people's HER2EC1 fragments:69) with the ammonia of canid HER2EC1 fragments Base acid sequence (SEQ ID NO:70) with 89% homogeneity.In another embodiment, the amino acid of people HER2EC2 fragments Sequence (SEQ ID NO:71) with amino acid sequence (the SEQ ID NO of canid HER2EC2 fragments:72) with 93% it is same One property.In another embodiment, amino acid sequence (the SEQ ID NO of people HER2IC1 fragments:And canid 73) Amino acid sequence (the SEQ ID NO of HER2IC1 fragments:74) with 98% homogeneity.

In one embodiment, provided herein is method and composition HER2 chimeric proteins or its fragment not including its believe Number sequence.In another embodiment, due to the high hydrophobicity of signal sequence, saving for signal sequence enables HER2 fragments to exist Successful expression in Listeria.Every kind of possibility represents the separate embodiments of the present invention.

In another embodiment, the fragment of the HER2 chimeric proteins of the method for the present invention and composition does not include its cross-film Domain (TM).In one embodiment, due to the high hydrophobicity of TM, saving for TM enables the success in Listeria of HER2 fragments Expression.Every kind of possibility represents the separate embodiments of the present invention.

In one embodiment, the nucleotide sequence of encoding rat HER2/neu genes includes SEQ ID NO:45： CCGGAATCGCGGGCACCCAAGTGTGTACCGGCACAGACATGAAGTTGCGGCTCCCTGCCAGTCCTGAGACCCACCTG GACATGCTCCGCCACCTGTACCAGGGCTGTCAGGTAGTGCAGGGCAACTTGGAGCTTACCTACGTGCCTGCCAATGC CAGCCTCTCATTCCTGCAGGACATCCAGGAAGTTCAGGGTTACATGCTCATCGCTCACAACCAGGTGAAGCGCGTCC CACTGCAAAGGCTGCGCATCGTGAGAGGGACCCAGCTCTTTGAGGACAAGTATGCCCTGGCTGTGCTAGACAACCGA GATCCTCAGGACAATGTCGCCGCCTCCACCCCAGGCAGAACCCCAGAGGGGCTGCGGGAGCTGCAGCTTCGAAGTCT CACAGAGATCCTGAAGGGAGGAGTTTTGATCCGTGGGAACCCTCAGCTCTGCTACCAGGACATGGTTTTGTGGAAGG ACGTCTTCCGCAAGAATAACCAACTGGCTCCTGTCGATATAGACACCAATCGTTCCCGGGCCTGTCCACCTTGTGCC CCCGCCTGCAAAGACAATCACTGTTGGGGTGAGAGTCCGGAAGACTGTCAGATCTTGACTGGCACCATCTGTACCAG TGGTTGTGCCCGGTGCAAGGGCCGGCTGCCCACTGACTGCTGCCATGAGCAGTGTGCCGCAGGCTGCACGGGCCCCA AGCATTCTGACTGCCTGGCCTGCCTCCACTTCAATCATAGTGGTATCTGTGAGCTGCACTGCCCAGCCCTCGTCACC TACAACACAGACACCTTTGAGTCCATGCACAACCCTGAGGGTCGCTACACCTTTGGTGCCAGCTGCGTGACCACCTG CCCCTACAACTACCTGTCTACGGAAGTGGGATCCTGCACTCTGGTGTGTCCCCCGAATAACCAAGAGGTCACAGCTG AGGACGGAACACAGCGTTGTGAGAAATGCAGCAAGCCCTGTGCTCGAGTGTGCTATGGTCTGGGCATGGAGCACCTT CGAGGGGCGAGGGCCATCACCAGTGACAATGTCCAGGAGTTTGATGGCTGCAAGAAGATCTTTGGGAGCCTGGCATT TTTGCCGGAGAGCTTTGATGGGGACCCCTCCTCCGGCATTGCTCCGCTGAGGCCTGAGCAGCTCCAAGTGTTCGAAA CCCTGGAGGAGATCACAGGTTACCTGTACATCTCAGCATGGCCAGACAGTCTCCGTGACCTCAGTGTCTTCCAGAAC CTTCGAATCATTCGGGGACGGATTCTCCACGATGGCGCGTACTCATTGACACTGCAAGGCCTGGGGATCCACTCGCT GGGGCTGCGCTCACTGCGGGAGCTGGGCAGTGGATTGGCTCTGATTCACCGCAACGCCCATCTCTGCTTTGTACACA CTGTACCTTGGGACCAGCTCTTCCGGAACCCACATCAGGCCCTGCTCCACAGTGGGAACCGGCCGGAAGAGGATTGT GGTCTCGAGGGCTTGGTCTGTAACTCACTGTGTGCCCACGGGCACTGCTGGGGGCCAGGGCCCACCCAGTGTGTCAA CTGCAGTCATTTCCTTCGGGGCCAGGAGTGTGTGGAGGAGTGCCGAGTATGGAAGGGGCTCCCCCGGGAGTATGTGA GTGACAAGCGCTGTCTGCCGTGTCACCCCGAGTGTCAGCCTCAAAACAGCTCAGAGACCTGCTTTGGATCGGAGGCT GATCAGTGTGCAGCCTGCGCCCACTACAAGGACTCGTCCTCCTGTGTGGCTCGCTGCCCCAGTGGTGTGAAACCGGA CCTCTCCTACATGCCCATCTGGAAGTACCCGGATGAGGAGGGCATATGCCAGCCGTGCCCCATCAACTGCACCCACT CCTGTGTGGATCTGGATGAACGAGGCTGCCCAGCAGAGCAGAGAGCCAGCCCGGTGACATTCATCATTGCAACTGTA GTGGGCGTCCTGCTGTTCCTGATCTTAGTGGTGGTCGTTGGAATCCTAATCAAACGAAGGAGACAGAAGATCCGGAA GTATACGATGCGTAGGCTGCTGCAGGAAACTGAGTTAGTGGAGCCGCTGACGCCCAGCGGAGCAATGCCCAACCAGG CTCAGATGCGGATCCTAAAAGAGACGGAGCTAAGGAAGGTGAAGGTGCTTGGATCAGGAGCTTTTGGCACTGTCTAC AAGGGCATCTGGATCCCAGATGGGGAGAATGTGAAAATCCCCGTGGCTATCAAGGTGTTGAGAGAAAACACATCTCC TAAAGCCAACAAAGAAATTCTAGATGAAGCGTATGTGATGGCTGGTGTGGGTTCTCCGTATGTGTCCCGCCTCCTGG GCATCTGCCTGACATCCACAGTACAGCTGGTGACACAGCTTATGCCCTACGGCTGCCTTCTGGACCATGTCCGAGAA CACCGAGGTCGCCTAGGCTCCCAGGACCTGCTCAACTGGTGTGTTCAGATTGCCAAGGGGATGAGCTACCTGGAGGA CGTGCGGCTTGTACACAGGGACCTGGCTGCCCGGAATGTGCTAGTCAAGAGTCCCAACCACGTCAAGATTACAGATT TCGGGCTGGCTCGGCTGCTGGACATTGATGAGACAGAGTACCATGCAGATGGGGGCAAGGTGCCCATCAAATGGATG GCATTGGAATCTATTCTCAGACGCCGGTTCACCCATCAGAGTGATGTGTGGAGCTATGGAGTGACTGTGTGGGAGCT GATGACTTTTGGGGCCAAACCTTACGATGGAATCCCAGCCCGGGAGATCCCTGATTTGCTGGAGAAGGGAGAACGCC TACCTCAGCCTCCAATCTGCACCATTGATGTCTACATGATTATGGTCAAATGTTGGATGATTGACTCTGAATGTCGC CCGAGATTCCGGGAGTTGGTGTCAGAATTTTCACGTATGGCGAGGGACCCCCAGCGTTTTGTGGTCATCCAGAACGA GGACTTGGGCCCATCCAGCCCCATGGACAGTACCTTCTACCGTTCACTGCTGGAAGATGATGACATGGGTGACCTGG TAGACGCTGAAGAGTATCTGGTGCCCCAGCAGGGATTCTTCTCCCCGGACCCTACCCCAGGCACTGGGAGCACAGCC CATAGAAGGCACCGCAGCTCGTCCACCAGGAGTGGAGGTGGTGAGCTGACACTGGGCCTGGAGCCCTCGGAAGAAGG GCCCCCCAGATCTCCACTGGCTCCCTCGGAAGGGGCTGGCTCCGATGTGTTTGATGGTGACCTGGCAATGGGGGTAA CCAAAGGGCTGCAGAGCCTCTCTCCACATGACCTCAGCCCTCTACAGCGGTACAGCGAGGACCCCACATTACCTCTG CCCCCCGAGACTGATGGCTATGTTGCTCCCCTGGCCTGCAGCCCCCAGCCCGAGTATGTGAACCAATCAGAGGTTCA GCCTCAGCCTCCTTTAACCCCAGAGGGTCCTCTGCCTCCTGTCCGGCCTGCTGGTGCTACTCTAGAAAGACCCAAGA CTCTCTCTCCTGGGAAGAATGGGGTTGTCAAAGACGTTTTTGCCTTCGGGGGTGCTGTGGAGAACCCTGAATACTTA GTACCGAGAGAAGGCACTGCCTCTCCGCCCCACCCTTCTCCTGCCTTCAGCCCAGCCTTTGACAACCTCTATTACTG GGACCAGAACTCATCGGAGCAGGGGCCTCCACCAAGTAACTTTGAAGGGACCCCCACTGCAGAGAACCCTGAGTACC TAGGCCTGGATGTACCTGTA(SEQ ID NO:45)。

In one embodiment, the nucleotide sequence of encoding rat HER2/neu EC1 fragments includes SEQ ID NO:46：

CCCAGGCAGAACCCCAGAGGGGCTGCGGGAGCTGCAGCTTCGAAGTCTCACAGAGATCCTGAAGGGAGGAGTTTTGA TCCGTGGGAACCCTCAGCTCTGCTACCAGGACATGGTTTTGTGGAAGGACGTCTTCCGCAAGAATAACCAACTGGCT CCTGTCGATATAGACACCAATCGTTCCCGGGCCTGTCCACCTTGTGCCCCCGCCTGCAAAGACAATCACTGTTGGGG TGAGAGTCCGGAAGACTGTCAGATCTTGACTGGCACCATCTGTACCAGTGGTTGTGCCCGGTGCAAGGGCCGGCTGC CCACTGACTGCTGCCATGAGCAGTGTGCCGCAGGCTGCACGGGCCCCAAGCA(SEQ ID NO:46)。

In another embodiment, the nucleotide sequence of encoding rat HER2/neu EC2 fragments includes SEQ ID NO:47：

GGTCACAGCTGAGGACGGAACACAGCGTTGTGAGAAATGCAGCAAGCCCTGTGCTCGAGTGTGCTATGGTCTGGGCA TGGAGCACCTTCGAGGGGCGAGGGCCATCACCAGTGACAATGTCCAGGAGTTTGATGGCTGCAAGAAGATCTTTGGG AGCCTGGCATTTTTGCCGGAGAGCTTTGATGGGGACCCCTCCTCCGGCATTGCTCCGCTGAGGCCTGAGCAGCTCCA AGTGTTCGAAACCCTGGAGGAGATCACAGGTTACCTGTACATCTCAGCATGGCCAGACAGTCTCCGTGACCTCAGTG TCTTCCAGAACCTTCGAATCATTCGGGGACGGATTCTCCACGATGGCGCGTACTCATTGACACTGCAAGGCCTGGGG ATCCACTCGCTGGGGCTGCGCTCACTGCGGGAGCTGGGCAGTGGATTGGCTCTGATTCACCGCAACGCCCATCTCTG CTTTGTACACACTGTACCTTGGGACCAGCTCTTCCGGAACCCACATCAGGCCCTGCTCCACAGTGGGAACCGGCCGG AAGAGGATTGTGGTCTCGAGGGCTTGGTCTGTAACTCACTGTGTGCCCACGGGCACTGCTGGGGGCCAGGGCCCACC CA(SEQ ID NO:47)。

In another embodiment, the nucleotide sequence of encoding rat HER2/neu IC1 fragments includes SEQ ID NO:48：

CGCCCAGCGGAGCAATGCCCAACCAGGCTCAGATGCGGATCCTAAAAGAGACGGAGCTAAGGAAGGTGAAGGTGCTT GGATCAGGAGCTTTTGGCACTGTCTACAAGGGCATCTGGATCCCAGATGGGGAGAATGTGAAAATCCCCGTGGCTAT CAAGGTGTTGAGAGAAAACACATCTCCTAAAGCCAACAAAGAAATTCTAGATGAAGCGTATGTGATGGCTGGTGTGG GTTCTCCGTATGTGTCCCGCCTCCTGGGCATCTGCCTGACATCCACAGTACAGCTGGTGACACAGCTTATGCCCTAC GGCTGCCTTCTGGACCATGTCCGAGAACACCGAGGTCGCCTAGGCTCCCAGGACCTGCTCAACTGGTGTGTTCAGAT TGCCAAGGGGATGAGCTACCTGGAGGACGTGCGGCTTGTACACAGGGACCTGGCTGCCCGGAATGTGCTAGTCAAGA GTCCCAACCACGTCAAGATTACAGATTTCGGGCTGGCTCGGCTGCTGGACATTGATGAGACAGAGTACCATGCAGAT GGGGGCAAGGTGCCCATCAAATGGATGGCATTGGAATCTATTCTCAGACGCCGGTTCACCCATCAGAGTGATGTGTG GAGCTATGGAGTGACTGTGTGGGAGCTGATGACTTTTGGGGCCAAACCTTACGATGGAATCCCAGCCCGGGAGATCC CTGATTTGCTGGAGAAGGGAGAACGCCTACCTCAGCCTCCAATCTGCACCATTGATGTCTACATGATTATGGTCAAA TGTTGGATGATTGACTCTGAATGTCGCCCGAGATTCCGGGAGTTGGTGTCAGAATTTTCACGTATGGCGAGGGACCC CCAGCGTTTTGTGGTCATCCAGAACGAGGACTTGGGCCCATCCAGCCCCATGGACAGTACCTTCTACCGTTCACTGC TGGAA(SEQ ID NO:48)。

In one embodiment, the nucleotide sequence of people HER2/neu genes includes SEQ ID NO:49：

ATGGAGCTGGCGGCCTTGTGCCGCTGGGGGCTCCTCCTCGCCCTCTTGCCCCCCGGAGCCGCGAGCACCCAAGTGTG CACCGGCACAGACATGAAGCTGCGGCTCCCTGCCAGTCCCGAGACCCACCTGGACATGCTCCGCCACCTCTACCAGG GCTGCCAGGTGGTGCAGGGAAACCTGGAACTCACCTACCTGCCCACCAATGCCAGCCTGTCCTTCCTGCAGGATATC CAGGAGGTGCAGGGCTACGTGCTCATCGCTCACAACCAAGTGAGGCAGGTCCCACTGCAGAGGCTGCGGATTGTGCG AGGCACCCAGCTCTTTGAGGACAACTATGCCCTGGCCGTGCTAGACAATGGAGACCCGCTGAACAATACCACCCCTG TCACAGGGGCCTCCCCAGGAGGCCTGCGGGAGCTGCAGCTTCGAAGCCTCACAGAGATCTTGAAAGGAGGGGTCTTG ATCCAGCGGAACCCCCAGCTCTGCTACCAGGACACGATTTTGTGGAAGGACATCTTCCACAAGAACAACCAGCTGGC TCTCACACTGATAGACACCAACCGCTCTCGGGCCTGCCACCCCTGTTCTCCGATGTGTAAGGGCTCCCGCTGCTGGG GAGAGAGTTCTGAGGATTGTCAGAGCCTGACGCGCACTGTCTGTGCCGGTGGCTGTGCCCGCTGCAAGGGGCCACTG CCCACTGACTGCTGCCATGAGCAGTGTGCTGCCGGCTGCACGGGCCCCAAGCACTCTGACTGCCTGGCCTGCCTCCA CTTCAACCACAGTGGCATCTGTGAGCTGCACTGCCCAGCCCTGGTCACCTACAACACAGACACGTTTGAGTCCATGC CCAATCCCGAGGGCCGGTATACATTCGGCGCCAGCTGTGTGACTGCCTGTCCCTACAACTACCTTTCTACGGACGTG GGATCCTGCACCCTCGTCTGCCCCCTGCACAACCAAGAGGTGACAGCAGAGGATGGAACACAGCGGTGTGAGAAGTG CAGCAAGCCCTGTGCCCGAGTGTGCTATGGTCTGGGCATGGAGCACTTGCGAGAGGTGAGGGCAGTTACCAGTGCCA ATATCCAGGAGTTTGCTGGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTCTGCCGGAGAGCTTTGATGGGGACCCA GCCTCCAACACTGCCCCGCTCCAGCCAGAGCAGCTCCAAGTGTTTGAGACTCTGGAAGAGATCACAGGTTACCTATA CATCTCAGCATGGCCGGACAGCCTGCCTGACCTCAGCGTCTTCCAGAACCTGCAAGTAATCCGGGGACGAATTCTGC ACAATGGCGCCTACTCGCTGACCCTGCAAGGGCTGGGCATCAGCTGGCTGGGGCTGCGCTCACTGAGGGAACTGGGC AGTGGACTGGCCCTCATCCACCATAACACCCACCTCTGCTTCGTGCACACGGTGCCCTGGGACCAGCTCTTTCGGAA CCCGCACCAAGCTCTGCTCCACACTGCCAACCGGCCAGAGGACGAGTGTGTGGGCGAGGGCCTGGCCTGCCACCAGC TGTGCGCCCGAGGGCACTGCTGGGGTCCAGGGCCCACCCAGTGTGTCAACTGCAGCCAGTTCCTTCGGGGCCAGGAG TGCGTGGAGGAATGCCGAGTACTGCAGGGGCTCCCCAGGGAGTATGTGAATGCCAGGCACTGTTTGCCGTGCCACCC TGAGTGTCAGCCCCAGAATGGCTCAGTGACCTGTTTTGGACCGGAGGCTGACCAGTGTGTGGCCTGTGCCCACTATA AGGACCCTCCCTTCTGCGTGGCCCGCTGCCCCAGCGGTGTGAAACCTGACCTCTCCTACATGCCCATCTGGAAGTTT CCAGATGAGGAGGGCGCATGCCAGCCTTGCCCCATCAACTGCACCCACTCCTGTGTGGACCTGGATGACAAGGGCTG CCCCGCCGAGCAGAGAGCCAGCCCTCTGACGTCCATCGTCTCTGCGGTGGTTGGCATTCTGCTGGTCGTGGTCTTGG GGGTGGTCTTTGGGATCCTCATCAAGCGACGGCAGCAGAAGATCCGGAAGTACACGATGCGGAGACTGCTGCAGGAA ACGGAGCTGGTGGAGCCGCTGACACCTAGCGGAGCGATGCCCAACCAGGCGCAGATGCGGATCCTGAAAGAGACGGA GCTGAGGAAGGTGAAGGTGCTTGGATCTGGCGCTTTTGGCACAGTCTACAAGGGCATCTGGATCCCTGATGGGGAGA ATGTGAAAATTCCAGTGGCCATCAAAGTGTTGAGGGAAAACACATCCCCCAAAGCCAACAAAGAAATCTTAGACGAA GCATACGTGATGGCTGGTGTGGGCTCCCCATATGTCTCCCGCCTTCTGGGCATCTGCCTGACATCCACGGTGCAGCT GGTGACACAGCTTATGCCCTATGGCTGCCTCTTAGACCATGTCCGGGAAAACCGCGGACGCCTGGGCTCCCAGGACC TGCTGAACTGGTGTATGCAGATTGCCAAGGGGATGAGCTACCTGGAGGATGTGCGGCTCGTACACAGGGACTTGGCC GCTCGGAACGTGCTGGTCAAGAGTCCCAACCATGTCAAAATTACAGACTTCGGGCTGGCTCGGCTGCTGGACATTGA CGAGACAGAGTACCATGCAGATGGGGGCAAGGTGCCCATCAAGTGGATGGCGCTGGAGTCCATTCTCCGCCGGCGGT TCACCCACCAGAGTGATGTGTGGAGTTATGGTGTGACTGTGTGGGAGCTGATGACTTTTGGGGCCAAACCTTACGAT GGGATCCCAGCCCGGGAGATCCCTGACCTGCTGGAAAAGGGGGAGCGGCTGCCCCAGCCCCCCATCTGCACCATTGA TGTCTACATGATCATGGTCAAATGTTGGATGATTGACTCTGAATGTCGGCCAAGATTCCGGGAGTTGGTGTCTGAAT TCTCCCGCATGGCCAGGGACCCCCAGCGCTTTGTGGTCATCCAGAATGAGGACTTGGGCCCAGCCAGTCCCTTGGAC AGCACCTTCTACCGCTCACTGCTGGAGGACGATGACATGGGGGACCTGGTGGATGCTGAGGAGTATCTGGTACCCCA GCAGGGCTTCTTCTGTCCAGACCCTGCCCCGGGCGCTGGGGGCATGGTCCACCACAGGCACCGCAGCTCATCTACCA GGAGTGGCGGTGGGGACCTGACACTAGGGCTGGAGCCCTCTGAAGAGGAGGCCCCCAGGTCTCCACTGGCACCCTCC GAAGGGGCTGGCTCCGATGTATTTGATGGTGACCTGGGAATGGGGGCAGCCAAGGGGCTGCAAAGCCTCCCCACACA TGACCCCAGCCCTCTACAGCGGTACAGTGAGGACCCCACAGTACCCCTGCCCTCTGAGACTGATGGCTACGTTGCCC CCCTGACCTGCAGCCCCCAGCCTGAATATGTGAACCAGCCAGATGTTCGGCCCCAGCCCCCTTCGCCCCGAGAGGGC CCTCTGCCTGCTGCCCGACCTGCTGGTGCCACTCTGGAAAGGGCCAAGACTCTCTCCCCAGGGAAGAATGGGGTCGT CAAAGACGTTTTTGCCTTTGGGGGTGCCGTGGAGAACCCCGAGTACTTGACACCCCAGGGAGGAGCTGCCCCTCAGC CCCACCCTCCTCCTGCCTTCAGCCCAGCCTTCGACAACCTCTATTACTGGGACCAGGACCCACCAGAGCGGGGGGCT CCACCCAGCACCTTCAAAGGGACACCTACGGCAGAGAACCCAGAGTACCTGGGTCTGGACGTGCCAGTGTGAACCAG AAGGCCAAGTCCGCAGAAGCCCTGA(SEQ ID NO:49)。

In another embodiment, the nucleotide sequence of the encoding human HER2/neu EC1 fragments implemented with chimera crosses over people The 120-510bp in EC1 regions, and comprising SEQ ID NO:50：

GAGACCCACCTGGACATGCTCCGCCACCTCTACCAGGGCTGCCAGGTGGTGCAGGGAAACCTGGAACTCACCTACCT GCCCACCAATGCCAGCCTGTCCTTCCTGCAGGATATCCAGGAGGTGCAGGGCTACGTGCTCATCGCTCACAACCAAG TGAGGCAGGTCCCACTGCAGAGGCTGCGGATTGTGCGAGGCACCCAGCTCTTTGAGGACAACTATGCCCTGGCCGTG CTAGACAATGGAGACCCGCTGAACAATACCACCCCTGTCACAGGGGCCTCCCCAGGAGGCCTGCGGGAGCTGCAGCT TCGAAGCCTCACAGAGATCTTGAAAGGAGGGGTCTTGATCCAGCGGAACCCCCAGCTCTGCTACCAGGACACGATTT TGTGGAAG(SEQID NO:50)。

In one embodiment, complete EC1 people's HER2/neu fragment spans (58-979bp of people's HER2/neu genes), And by comprising SEQ ID NO:54 nucleic acid sequence encoding：

GCCGCGAGCACCCAAGTGTGCACCGGCACAGACATGAAGCTGCGGCTCCCTGCCAGTCCCGAGACCCACCTGGACAT GCTCCGCCACCTCTACCAGGGCTGCCAGGTGGTGCAGGGAAACCTGGAACTCACCTACCTGCCCACCAATGCCAGCC TGTCCTTCCTGCAGGATATCCAGGAGGTGCAGGGCTACGTGCTCATCGCTCACAACCAAGTGAGGCAGGTCCCACTG CAGAGGCTGCGGATTGTGCGAGGCACCCAGCTCTTTGAGGACAACTATGCCCTGGCCGTGCTAGACAATGGAGACCC GCTGAACAATACCACCCCTGTCACAGGGGCCTCCCCAGGAGGCCTGCGGGAGCTGCAGCTTCGAAGCCTCACAGAGA TCTTGAAAGGAGGGGTCTTGATCCAGCGGAACCCCCAGCTCTGCTACCAGGACACGATTTTGTGGAAGGACATCTTC CACAAGAACAACCAGCTGGCTCTCACACTGATAGACACCAACCGCTCTCGGGCCTGCCACCCCTGTTCTCCGATGTG TAAGGGCTCCCGCTGCTGGGGAGAGAGTTCTGAGGATTGTCAGAGCCTGACGCGCACTGTCTGTGCCGGTGGCTGTG CCCGCTGCAAGGGGCCACTGCCCACTGACTGCTGCCATGAGCAGTGTGCTGCCGGCTGCACGGGCCCCAAGCACTCT GACTGCCTGGCCTGCCTCCACTTCAACCACAGTGGCATCTGTGAGCTGCACTGCCCAGCCCTGGTCACCTACAACAC AGACACGTTTGAGTCCATGCCCAATCCCGAGGGCCGGTATACATTCGGCGCCAGCTGTGTGACTGCCTGTCCCTACA ACTACCTTTCTACGGACGTGGGATCCTGCACCCTCGTCTGCCCCCTGCACAACCAAGAGGTGACAGCAGAGGAT (SEQ ID NO:54)。

In another embodiment, the nucleotide sequence of the encoding human HER2/neu EC2 fragments implemented with chimera crosses over people The 1077-1554bp of HER2/neu EC2 fragments, and extend including 50bp, and comprising SEQ ID NO:51：

AATATCCAGGAGTTTGCTGGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTCTGCCGGAGAGCTTTGATGGGGACCC AGCCTCCAACACTGCCCCGCTCCAGCCAGAGCAGCTCCAAGTGTTTGAGACTCTGGAAGAGATCACAGGTTACCTAT ACATCTCAGCATGGCCGGACAGCCTGCCTGACCTCAGCGTCTTCCAGAACCTGCAAGTAATCCGGGGACGAATTCTG CACAATGGCGCCTACTCGCTGACCCTGCAAGGGCTGGGCATCAGCTGGCTGGGGCTGCGCTCACTGAGGGAACTGGG CAGTGGACTGGCCCTCATCCACCATAACACCCACCTCTGCTTCGTGCACACGGTGCCCTGGGACCAGCTCTTTCGGA ACCCGCACCAAGCTCTGCTCCACACTGCCAACCGGCCAGAGGACGAGTGTGTGGGCGAGGGCCTGGCCTGCCACCAG CTGTGCGCCCGAGGG(SEQ ID NO:51)。

In one embodiment, the 907-1504bp of complete EC2 people's HER2/neu fragment spans people's HER2/neu genes, And by comprising SEQ ID NO:55 nucleic acid sequence encoding：

TACCTTTCTACGGACGTGGGATCCTGCACCCTCGTCTGCCCCCTGCACAACCAAGAGGTGACAGCAGAGGATGGAAC ACAGCGGTGTGAGAAGTGCAGCAAGCCCTGTGCCCGAGTGTGCTATGGTCTGGGCATGGAGCACTTGCGAGAGGTGA GGGCAGTTACCAGTGCCAATATCCAGGAGTTTGCTGGCTGCAAGAAGATCTTTGGGAGCCTGGCATTTCTGCCGGAG AGCTTTGATGGGGACCCAGCCTCCAACACTGCCCCGCTCCAGCCAGAGCAGCTCCAAGTGTTTGAGACTCTGGAAGA GATCACAGGTTACCTATACATCTCAGCATGGCCGGACAGCCTGCCTGACCTCAGCGTCTTCCAGAACCTGCAAGTAA TCCGGGGACGAATTCTGCACAATGGCGCCTACTCGCTGACCCTGCAAGGGCTGGGCATCAGCTGGCTGGGGCTGCGC TCACTGAGGGAACTGGGCAGTGGACTGGCCCTCATCCACCATAACACCCACCTCTGCTTCGTGCACACGGTGCCCTG GGACCAGCTCTTTCGGAACCCGCACCAAGCTCTGCTCCACACTGCCAACCGGCCAGAG(SEQ ID NO:55)。

In another embodiment, the nucleotide sequence of the encoding human HER2/neu IC1 fragments implemented with chimera is included SEQ ID NO:52：

CAGCAGAAGATCCGGAAGTACACGATGCGGAGACTGCTGCAGGAAACGGAGCTGGTGGAGCCGCTGACACCTAGCGG AGCGATGCCCAACCAGGCGCAGATGCGGATCCTGAAAGAGACGGAGCTGAGGAAGGTGAAGGTGCTTGGATCTGGCG CTTTTGGCACAGTCTACAAGGGCATCTGGATCCCTGATGGGGAGAATGTGAAAATTCCAGTGGCCATCAAAGTGTTG AGGGAAAACACATCCCCCAAAGCCAACAAAGAAATCTTAGACGAAGCATACGTGATGGCTGGTGTGGGCTCCCCATA TGTCTCCCGCCTTCTGGGCATCTGCCTGACATCCACGGTGCAGCTGGTGACACAGCTTATGCCCTATGGCTGCCTCT TAGACT(SEQ ID NO:52)。

In another embodiment, the nucleotide sequence for encoding complete people's HER2/neu IC1 fragments crosses over people HER2/neu The 2034-3243 of gene, and comprising SEQ ID NO:56：

CAGCAGAAGATCCGGAAGTACACGATGCGGAGACTGCTGCAGGAAACGGAGCTGGTGGAGCCGCTGACACCTAGCGG AGCGATGCCCAACCAGGCGCAGATGCGGATCCTGAAAGAGACGGAGCTGAGGAAGGTGAAGGTGCTTGGATCTGGCG CTTTTGGCACAGTCTACAAGGGCATCTGGATCCCTGATGGGGAGAATGTGAAAATTCCAGTGGCCATCAAAGTGTTG AGGGAAAACACATCCCCCAAAGCCAACAAAGAAATCTTAGACGAAGCATACGTGATGGCTGGTGTGGGCTCCCCATA TGTCTCCCGCCTTCTGGGCATCTGCCTGACATCCACGGTGCAGCTGGTGACACAGCTTATGCCCTATGGCTGCCTCT TAGACCATGTCCGGGAAAACCGCGGACGCCTGGGCTCCCAGGACCTGCTGAACTGGTGTATGCAGATTGCCAAGGGG ATGAGCTACCTGGAGGATGTGCGGCTCGTACACAGGGACTTGGCCGCTCGGAACGTGCTGGTCAAGAGTCCCAACCA TGTCAAAATTACAGACTTCGGGCTGGCTCGGCTGCTGGACATTGACGAGACAGAGTACCATGCAGATGGGGGCAAGG TGCCCATCAAGTGGATGGCGCTGGAGTCCATTCTCCGCCGGCGGTTCACCCACCAGAGTGATGTGTGGAGTTATGGT GTGACTGTGTGGGAGCTGATGACTTTTGGGGCCAAACCTTACGATGGGATCCCAGCCCGGGAGATCCCTGACCTGCT GGAAAAGGGGGAGCGGCTGCCCCAGCCCCCCATCTGCACCATTGATGTCTACATGATCATGGTCAAATGTTGGATGA TTGACTCTGAATGTCGGCCAAGATTCCGGGAGTTGGTGTCTGAATTCTCCCGCATGGCCAGGGACCCCCAGCGCTTT GTGGTCATCCAGAATGAGGACTTGGGCCCAGCCAGTCCCTTGGACAGCACCTTCTACCGCTCACTGCTGGAGGACGA TGACATGGGGGACCTGGTGGATGCTGAGGAGTATCTGGTACCCCAGCAGGGCTTCTTCTGTCCAGACCCTGCCCCGG GCGCTGGGGGCATGGTCCACCACAGGCACCGCAGCTCATCTACCAGGAGTGGCGGTGGGGACCTGACACTAGGGCTG GAGCCCTCTGAAGAGGAGGCCCCCAGGTCTCCACTGGCACCCTCCGAAGGGGCT(SEQ ID NO:56)。

In one embodiment, for provided herein is the LLO of method and composition be Listeria LLO.In a reality In applying example, LLO from Listeria be listerisa monocytogenes in mjme (LM).In another embodiment, Liszt Bacterium is Vyacheslav Ivanov Listeria (Listeria ivanovii).In another embodiment, Listeria is Wei Erxunlisi Special bacterium (Listeria welshimeri).In another embodiment, Listeria is Xi Er Listeria (Listeria seeligeri).In another embodiment, LLO albumen is non-Listeria LLO albumen.In another embodiment, LLO eggs It is in vain synthesis LLO albumen.In another embodiment, it is restructuring LLO albumen.

In one embodiment, LLO albumen is by comprising SEQ ID NO:3 nucleic acid sequence encoding：

atgaaaaaaataatgctagtttttattacacttatattagttagtctaccaattgcgcaacaaactgaagcaaagga tgcatctgcattcaataaagaaaattcaatttcatccatggcaccaccagcatctccgcctgcaagtcctaagacgc caatcgaaaagaaacacgcggatgaaatcgataagtatatacaaggattggattacaataaaaacaatgtattagta taccacggagatgcagtgacaaatgtgccgccaagaaaaggttacaaagatggaaatgaatatattgttgtggagaa aaagaagaaatccatcaatcaaaataatgcagacattcaagttgtgaatgcaatttcgagcctaacctatccaggtg ctctcgtaaaagcgaattcggaattagtagaaaatcaaccagatgttctccctgtaaaacgtgattcattaacactc agcattgatttgccaggtatgactaatcaagacaataaaatagttgtaaaaaatgccactaaatcaaacgttaacaa cgcagtaaatacattagtggaaagatggaatgaaaaatatgctcaagcttatccaaatgtaagtgcaaaaattgatt atgatgacgaaatggcttacagtgaatcacaattaattgcgaaatttggtacagcatttaaagctgtaaataatagc ttgaatgtaaacttcggcgcaatcagtgaagggaaaatgcaagaagaagtcattagttttaaacaaatttactataa cgtgaatgttaatgaacctacaagaccttccagatttttcggcaaagctgttactaaagagcagttgcaagcgcttg gagtgaatgcagaaaatcctcctgcatatatctcaagtgtggcgtatggccgtcaagtttatttgaaattatcaact aattcccatagtactaaagtaaaagctgcttttgatgctgccgtaagcggaaaatctgtctcaggtgatgtagaact aacaaatatcatcaaaaattcttccttcaaagccgtaatttacggaggttccgcaaaagatgaagttcaaatcatcg acggcaacctcggagacttacgcgatattttgaaaaaaggcgctacttttaatcgagaaacaccaggagttcccatt gcttatacaacaaacttcctaaaagacaatgaattagctgttattaaaaacaactcagaatatattgaaacaacttc aaaagcttatacagatggaaaaattaacatcgatcactctggaggatacgttgctcaattcaacatttcttgggatg aagtaaattatgat(SEQID NO:3)。

In another embodiment, LLO albumen includes sequence SEQ ID NO:4：

M K K I M L V F I T L I L V S L P I A Q Q T E A K D A S A F N K E N S I S S M A P P A S P P A S P K T P I E K K H A D E I D K Y I Q G L D Y N K N N V L V Y H G D A V T N V P P R K G Y K D G N E Y I V V E K K K K S I N Q N N A D I Q V V N A I S S L T Y P G A L V K A N S E L V E N Q P D V L P V K R D S L T L S I D L P G M T N Q D N K I V V K N A T K S N V N N A V N T L V E R W N E K Y A Q A Y P N V S A K I D Y D D E M A Y S E S Q L I A K F G T A F K A V N N S L N V N F G A I S E G K M Q E E V I S F K Q I Y Y N V N V N E P T R P S R F F G K A V T K E Q L Q A L G V N A E N P P A Y I S S V A Y G R Q V Y L K L S T N S H S T K V K A A F D A A V S G K S V S G D V E L T N I I K N S S F K A V I Y G G S A K D E V Q I I D G N L G D L R D I L K K G A T F N R E T P G V P I A Y T T N F L K D N E L A V I K N N S E Y I E T T S K A Y T D G K I N I D H S G G Y V A Q F N I S W D E V N Y D(SEQ ID NO:4)

It is signal sequence corresponding to front 25 amino acid of the front albumen of the sequence, and cuts from LLO in bacterial secretory Cut.Therefore, in this embodiment, the length of total length activity LLO albumen is 504 residues.In another embodiment, LLO eggs The white sequence with shown in GenBank accession number DQ054588, DQ054589, AY878649, U25452 or U25452.Another In individual embodiment, LLO albumen is the variant of LLO albumen.In another embodiment, LLO albumen is the homologue of LLO albumen. Every kind of possibility represents the separate embodiments of the present invention.

In another embodiment, " LLO of truncation " or " tLLO " refer to the LLO fragments comprising PEST domains.At another In embodiment, the term is referred to not comprising aminoterminal activation domain and the LLO fragments not comprising cystine 484.Another In individual embodiment, LLO fragments are made up of PEST sequences.In another embodiment, LLO fragments include PEST sequences.Another In individual embodiment, LLO fragments are made up of 400 to 441 amino acid before the pact of 529 amino acid total length LLO albumen.At another In embodiment, LLO fragments are the non-haemolysis forms of LLO albumen.

In another embodiment of the method for the present invention and composition, the nucleotide sequence of the method for the present invention and composition The recombinant polypeptide of coding is the fusion protein comprising chimeric HER2/neu antigens and other polypeptide, and in another embodiment In, fusion protein especially includes LLO fragments, and the LLO fragments are in one embodiment the non-haemolysis LLO albumen of LM, or another It is the LLO (example of this paper) for truncating in individual embodiment.

In one embodiment, LLO fragments are made up of about residue 1-25.In another embodiment, LLO fragments are by big About residue 1-50 is constituted.In another embodiment, LLO fragments are made up of about residue 1-75.In another embodiment, LLO fragments are made up of about residue 1-100.In another embodiment, LLO fragments are made up of about residue 1-125.Another In individual embodiment, LLO fragments are made up of about residue 1-150.In another embodiment, LLO fragments are by about residue 1175 Composition.In another embodiment, LLO fragments are made up of about residue 1-200.In another embodiment, LLO fragments are by big About residue 1-225 is constituted.In another embodiment, LLO fragments are made up of about residue 1-250.In another embodiment, LLO fragments are made up of about residue 1-275.In another embodiment, LLO fragments are made up of about residue 1-300.Another In individual embodiment, LLO fragments are made up of about residue 1-325.In another embodiment, LLO fragments are by about residue 1-350 Composition.In another embodiment, LLO fragments are made up of about residue 1-375.In another embodiment, LLO fragments are by big About residue 1-400 is constituted.In another embodiment, LLO fragments are made up of about residue 1-425.Every kind of possibility represents this The separate embodiments of invention.

In another embodiment, the fusion protein of the method for the present invention and composition include from LLO albumen or from The PEST sequences of another kind of organism such as prokaryotes body.

In another embodiment, PEST amino acid (AA) sequence is comprising selected from SEQ ID NO:The sequence of 5-9.Another In individual embodiment, PEST sequences are the PEST sequences of listerisa monocytogenes in mjme (Lm) ActA albumen.In another reality In applying example, PEST sequences include KTEEQPSEVNTGPR (SEQ ID NO:5)、KASVTDTSEGDLDSSMQSADESTPQPLK (SEQ ID NO:6)、KNEEVNASDFPPPPTDEELR(SEQ ID NO:7) or RGGIPTSEEFSSLNSGDFTDDENSETTEEEIDR(SEQ ID NO:8).In another embodiment, PEST sequences from The streptococcolysin O protein of streptococcus (Streptococcus sp).In another embodiment, PEST sequences are from wine Streptococcus pyrogenes (Streptococcus pyogenes) streptolysin O, the such as KQNTASTETTTTNEQPK of AA 35-51 (SEQ ID NO:9).In another embodiment, PEST sequences are from streptococcus equisimilis (Streptococcus KQNTANTETTTTNEQPK (the SEQ ID NO of equisimilis) streptolysin O, such as AA38-54:10).At another In embodiment, PEST sample sequences are derived from another PEST AA sequence of prokaryotes body.In another embodiment, PEST sequences are any other PEST sequences known in the art.Every kind of possibility represents the separate embodiments of the present invention.

The PEST sequences of Antigen Fusion to Lm enhance the cell-mediated immunity and antineoplastic immune of antigen.Therefore, antigen It is fused to derive from other PEST sequences of other prokaryotes bodies also by the immunogenicity of enhancement antigen.Can be according to such as by example Such as Rechsteiner and Rogers (1996, Trends Biochem.Sci.21:267-271) for the method identification described in Lm The PEST sequences of other prokaryotes bodies.Or, the PEST AA sequences from other prokaryotes bodies can reflect also according to the method It is fixed.Other prokaryotes bodies that wherein expection has PEST AA sequences include but is not limited to other Listeria species.Another In one embodiment, in the embedded antigen protein of PEST sequences.Therefore, in another embodiment, " fusion " refer to both comprising anti- The former antigen protein for also including the PEST amino acid sequences in being connected to one end of antigen or embedded antigen.

In another embodiment, there is provided herein the vaccine of the recombinant polypeptide comprising the present invention.In another embodiment In, there is provided herein the composition of the recombinant polypeptide comprising the present invention.In another embodiment, there is provided herein by the present invention Recombinant polypeptide composition vaccine.In another embodiment, there is provided herein the combination being made up of the recombinant polypeptide of the present invention Thing.In another embodiment, composition is immunogenic composition.

In another embodiment, there is provided herein the nucleic acid molecule of the recombinant polypeptide of the coding present invention.At another In embodiment, there is provided herein the vaccine comprising nucleic acid molecule.In another embodiment, there is provided herein including nucleotides The composition of molecule.

In another embodiment, there is provided herein the nucleic acid molecule of the recombinant polypeptide of the coding present invention.

In another embodiment, there is provided herein the recombinant polypeptide of the nucleic acid molecule coding of the present invention.

In another embodiment, there is provided herein the vaccine of the nucleic acid molecule comprising the present invention or recombinant polypeptide.

In another embodiment, there is provided herein the immunogenicity of the nucleic acid molecule comprising the present invention or recombinant polypeptide Composition.

In another embodiment, there is provided herein the carrier of the nucleic acid molecule comprising the present invention or recombinant polypeptide.

In another embodiment, there is provided herein the listerial restructuring shape of the nucleic acid molecule comprising the present invention Formula.

In another embodiment, there is provided herein the vaccine of the listerial recombinant forms comprising the present invention.

In another embodiment, there is provided herein the immunogenicity group of the listerial recombinant forms comprising the present invention Compound.

In another embodiment, there is provided herein the culture of the listerial recombinant forms of the present invention.

In one embodiment, for the method for the present invention vaccine or composition comprising any form as herein described or The restructuring listerisa monocytogenes in mjme of embodiment.In one embodiment, for the present invention vaccine or composition by The restructuring listerisa monocytogenes in mjme composition of the invention of any form as herein described or embodiment.In another reality In applying example, for the method for the present invention vaccine or composition substantially by any form as herein described or embodiment this Bright restructuring listerisa monocytogenes in mjme composition.

In one embodiment, term "comprising" is referred in vaccine or composition comprising restructuring monocytosis Li Si Special bacterium, and including other vaccines, composition or process can be known in the art.In another embodiment, term is " basic On by ... constitute " refer to that function ingredients are the vaccine of listerisa monocytogenes in mjme of recombinating, but may include directly not relate to And other vaccines or composition component of the therapeutic effect of vaccine, and can for example refer to be conducive to recombinate monocytosis The component of listerial effect (e.g., stable, maintenance etc.).In another embodiment, term " composition " is referred to comprising restructuring The vaccine of listerisa monocytogenes in mjme.

In another embodiment, the method for the present invention includes applying the restructuring of any form as herein described or embodiment The step of listerisa monocytogenes in mjme.In one embodiment, the method for the present invention is as herein described any by applying The step of restructuring listerisa monocytogenes in mjme of the invention of form or embodiment, constitutes.In another embodiment, The method of the present invention is substantially by the restructuring monocytosis of the invention for applying any form as herein described or embodiment Property listerial step composition.In one embodiment, term " including " refers to that method increases including administered recombinant monocyte The step of many property Listerias, and including additive method or process can be known in the art.In another embodiment, art Language " substantially by ... constitute " refers to the method that functional component is administered recombinant listerisa monocytogenes in mjme, so And the additive method step of the therapeutic effect that may include to be not directed to method, and can for example refer to be conducive to recombinate monokaryon it is thin The step of increasing property of born of the same parents listerial application effect.In one embodiment, term " composition " is referred to without other step The method of administered recombinant listerisa monocytogenes in mjme.

In another embodiment, the Listeria of the method for the present invention and composition is monocytosis Liszt Bacterium.In another embodiment, Listeria is Vyacheslav Ivanov Listeria (Listeria ivanovii).In another reality In applying example, Listeria is Wei Erxun Listerias.In another embodiment, Listeria is Xi Er Listerias.It is every kind of The Listeria of type represents the separate embodiments of the present invention.

In one embodiment, the Listeria bacterial strain of the method for the present invention and composition is ADXS31-164 bacterial strains. In another embodiment, ADXS31-164 stimulates the splenocyte secretion of gamma-IFN of wild type FVB/N mouse.In addition, provided herein is Data display, ADXS31-164 can cause the anti-HER2/neu specificity of people's epi-position of the not same area for being pointed to target antigen to exempt from Epidemic disease response.

In another embodiment, the present invention provides the nucleic acid molecule comprising coding HER2 chimeric proteins or its fragment Listerial recombinant forms.

In one embodiment, the method that the present invention provides the anti-HER2 immune responses of induction experimenter, the method includes The recombinant polypeptide of the N- terminal fragments for including the LLO albumen for being fused to HER2 chimeric proteins or being fused to its fragment is applied to and is received Examination person, so as to induce the anti-HER2 immune responses of experimenter.

In one embodiment, two molecules (LLO, ActA fragment or PEST sequences and antigen) of fusion protein directly connect Connect.In another embodiment, two molecules are connected by the short spacer peptide being made up of one or more amino acid.In a reality In applying example, without specific in addition to other spatial relationships of spacer peptide except the certain minimum range of connection albumen or maintenance or between them BA.In another embodiment, select spacer peptide constitutes amino acid to affect the properties of molecule, such as rolls over Folded, net charge or hydrophobicity.In another embodiment, two protein moleculars (LLO fragments and antigen) be it is separately synthesized or Do not merge.In another embodiment, two protein moleculars are separately synthesized from identical nucleic acid.In yet another embodiment, Two molecules each synthesize from single nucleic acid.Every kind of possibility represents the separate embodiments of the present invention.

In one embodiment, coding provided herein is recombinant polypeptide nucleic acid also encoded signal peptide or sequence.Another In individual embodiment, the fusion protein of the method for the present invention and composition is comprising the LLO signal sequences from LLO.In an enforcement In example, heterologous antigen can be believed by using signal sequence such as Listeria signal sequence such as hemolysin signal sequence or actA Number sequence table reaches.Or, for example, foreign gene can be expressed in listerisa monocytogenes in mjme promoter downstream, and not shape Into fusion protein.In another embodiment, signal peptide is bacterium (listerial or non-listerial).In a reality In applying example, signal peptide is that bacterium is natural.In another embodiment, signal peptide is bacterium external source.In another embodiment In, signal peptide is the signal peptide of listerisa monocytogenes in mjme, such as secA1 signal peptides.In another embodiment, believe Number peptide is the Usp45 signal peptide or Bacillus anthracis (Bacillus of Lactococcus lactis (Lactococcus lactis) Anthracis protection antigen signals peptide).In another embodiment, signal peptide is secA2 signal peptides, such as monocyte The listerial p60 signal peptides of increasing property.Additionally, recombinant nucleic acid molecules optionally include coding p60 or more than the 3 of its fragment Nucleotide sequence.In another embodiment, signal peptide is Tat signal peptides, and such as bacillus subtilis Tat signal peptides are (e.g., PhoD).In one embodiment, signal peptide is in the identical translation reading frame of encoding recombinant polypeptide.

In another embodiment, there is provided herein the method for the anti-HER2 immune responses of induction experimenter, the method bag Include the recombinant polypeptide of the N- terminal fragments that coding is included the LLO albumen for being fused to HER2 chimeric proteins or being fused to its fragment Recombinant nucleotide is applied to experimenter, so as to induce the anti-HER2 immune responses of experimenter.

In one embodiment, there is provided herein causing the enhancing immune response for expressing HER2/neu tumour of experimenter Method, and in another embodiment, the method include including provided herein is recombinant listeria bacterium vaccine strain combination Thing is applied to experimenter.In another embodiment, the suboptimum to HER2 albumen is included to the immune response that HER2 expresses tumour The immune response of gesture epi-position.In another embodiment, the immune response that HER2 expresses tumour is included to many of HER2 albumen The immune response of individual Dominant Epitopes.In another embodiment, the immune response that HER2 expresses tumour is included to HER2 eggs The immune response of at least 1-5 white time Dominant Epitopes.In another embodiment, the immune response bag of tumour is expressed HER2 Include the immune response of at least 1-10 to HER2 albumen time Dominant Epitopes.In another embodiment, tumour is expressed to HER2 Immune response include the immune response of at least 1-17 to HER2 albumen Dominant Epitopes.In another embodiment, it is right The immune response of HER2 expression tumours includes the immune response of at least 17 to HER2 albumen Dominant Epitopes.

It was reported that, when based on the listerial vaccine of small fragment or the trastuzumab (born of the same parents for being located at HER2/neu antigens The monoclonal antibody of the epi-position of foreign lands) when targetting these tumours, the point mutation of carcinogenic protein HER2/neu or amino acid deletions are situated between The treatment of cells of resistant tumors is led.This document describes the composition based on chimeric HER2/neu, said composition has HER2/ Two extracellular segments and an intracellular fragment of neu antigens, the antigen illustrates the MHC I class epi-position clusters of oncogene.The chimeric egg There are in vain 3 H2Dq and at least people's MHC I class epi-positions of the plotting of 17 HER2/neu antigens, and be fused to monocyte increasing Front 441 amino acid of many property Listeria Listeriolysin O albumen, by listerisa monocytogenes in mjme bacterium is attenuated Strain LmddA expression and secretion.

Preceding report is illustrated, when the small fragment using expression respectively and secretion HER2/neu antigens is based on listerial During vaccine (every kind of vaccine only H2Dq epi-position with HER2/neu oncogenes) immunity HER2/neu transgenic mices, due to The mutation of those epi-positions of the HER2/neu antigens of every kind of vaccine targeting, HER2/neu overexpression tumour can escape (referring to Singh R,Paterson Y.Immunoediting sculpts tumor epitopes during immunotherapy.Cancer Res 2007；67:1887-92).Unpredictable consequence is shown herein, when three or more tables of HER2/neu albumen When position is incorporated to chimeric, the selection and escape of these tumours can be eliminated by escape mutant.It is chimeric using new HER2/neu Listeria vaccine immunity is not result in any escape that may be related to the point mutation of HER2/neu antigens or amino acid deletions Mutation is (referring to the example 4 of this paper).

In one embodiment, there is provided herein Listeria vaccine strain is engineered to express HER2 chimeric proteins or table Up to the method for the recombinant polypeptide of chimeric protein, the method includes converting Listeria bacterial strain with nucleic acid molecules.In another enforcement In example, first ORFs of the nucleic acid molecules comprising coded polypeptide, the wherein polypeptide include HER2/neu chimeric antigens.Another In one embodiment, nucleic acid molecules also the second ORFs comprising encoding metabolic enzyme, and wherein described metabolic enzyme is complementary The endogenous gene lacked in the chromosome of recombinant listeria bacterium bacterial strain, so as to Listeria vaccine strain is engineered into expression HER2 chimeric proteins.

In one embodiment, provided herein is method and composition also include adjuvant, and in another embodiment, assistant Agent includes granulocyte/macrophage colony stimulatory factor (GM-CSF) albumen, nucleic acid molecule, the Saponaria officinalis of coding GM-CSF albumen Glycosides QS21, monophosphoryl lipid A, without methylated containing CpG ODN or any adjuvant known in the art.

In one embodiment, the present invention uses attenuation Listeria bacterial strain, such as LM δ-actA mutant (Brundage et al,1993,Proc.Natl.Acad.Sci.,USA,90:11890-11894), listerisa monocytogenes in mjme δ- plcA(Camilli et al,1991,J.Exp.Med.,173:751-754) or δ-ActA, δ INL-b (Brockstedt et 5al,2004,PNAS,101:13832–13837).In another embodiment, Listeria bacterial strain is attenuated by introducing one Or multiple Attenuating mutations build, understand when understanding this disclosure such as one of ordinary skill in the art.Such bacterial strain Example include but is not limited to aromatic amino acid auxotroph Listeria bacterial strain (Alexander et al, 1993, Infection and Immunity 1061:2245-2248) and formed lipoteichoicacid mutant (Abachin et al, 2002,Mol.Microbiol.43:Those bacterial strains 1-14) and due to lacking virulent gene being attenuated are (referring to the reality of this paper Example).

In another embodiment, the nucleic acid molecules of the method for the present invention and composition may be operably coupled to promoter/ Regulating and controlling sequence.In another embodiment, the first ORFs of the method for the present invention and composition may be operably coupled to Promoter/regulating and controlling sequence.In another embodiment, nucleic acid molecules are included and may be operably coupled to promoter/regulating and controlling sequence Second ORFs.In another embodiment, each ORFs may be operably coupled to promoter/regulating and controlling sequence. Every kind of possibility represents the separate embodiments of the present invention.

Technical staff understand provided herein is present disclosure and during method, will be apparent from different transcripting startings Son, terminator, carrier or specific gene sequence (e.g., those in commercially available cloning vector) can be successfully used to the side of the present invention Method and composition.As contemplated by the present invention, these functions are provided in the commercial vector of pUC series is for example referred to as.At another In embodiment, nonessential DNA sequence dna (e.g., antibiotics resistance gene) is removed.Every kind of possibility represents the independent enforcement of the present invention Example.In another embodiment, the present invention uses commercially available plasmid.Such plasmid can be obtained from multiple sources, for example Invitrogen (La Jolla, CA), Stratagene (La Jolla, CA), Clontech (Palo Alto, CA), or can make Built with method well known in the art.

Another embodiment is the plasmid of such as pCR2.1 (Invitrogen, La Jolla, CA), and the plasmid is that have original Core replication orgin and promoter/controlling element are so as to the prokaryotic expression carrier for being conducive to being expressed in prokaryotes body.Another In individual embodiment, the size and increase for removing exogenous nucleotide sequence to reduce plasmid is placed in the size of box therein.

Such method is well known in the art, and in such as 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 it is described in).

Antibiotics resistance gene is used for into conventional selection and the clone's technique that molecular biology and vaccine preparation are usually used. It is contemplated by the invention that antibiotics resistance gene including but not limited to give ampicillin, penicillin, methicillin, streptomysin, Erythromycin, kanamycins, tetracycline, chloramphenicol (CAT), neomycin, hygromycin, gentamicin and it is well known in the art other The gene outcome of antibiotic resistance.The separate embodiments of each gene representation present invention.

The method of transform bacteria is well known in the art, and is worn including the method for calcium chloride competent cell, electricity is based on Kong Fa, bacteriophage mediation transduction, chemically and physically transformation technology (de Boer et al, 1989, Cell 56:641-649； 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,Washington,DC；Miller,1992,A Short Course in Bacterial Genetics,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.).Another In individual embodiment, the Listeria vaccine strain of the present invention passes through Electroporation Transformation.Every kind of method represents the independent enforcement of the present invention Example.

In another embodiment, engagement is used to for inhereditary material and/or plasmid to introduce bacterium.The method of engagement is this Known to field, and for example in Nikodinovic J et al. (A second generation snp-derived Escherichia coli-Streptomyces shuttle expression vector that is generally transferable by conjugation.Plasmid.2006Nov；56(3):223-7) with Auchtung JM et al (Regulation of a Bacillus subtilis mobile genetic element by intercellular signaling and the global DNA damage response.Proc Natl Acad Sci U S A.2005Aug 30；102(35):It is described in 12554-9).Every kind of method represents the separate embodiments of the present invention.

In one embodiment, " conversion " be equal to use with term " transfection ", refer to by bacterial cell be engineered to absorb Plasmid or other heterologous DNA moleculars.In another embodiment, " conversion " refer to and bacterial cell be engineered into expression plasmid Gene or other heterologous DNA moleculars.Every kind of possibility represents the separate embodiments of the present invention.

The available plasmid of the present invention and other expression vectors are described in this paper other places, and may include for example to start The replication orgin of son/regulating and controlling sequence, Gram-negative bacteria and gram-positive bacteria, the detached nucleic acid of encoding fusion protein and The features such as the detached nucleic acid of coded amino acid metabolic gene.In addition, the separation of encoding fusion protein and amino acid metabolising gene Nucleic acid will have the promoter for being applied to the expression for driving such detached nucleic acid.For driving what is expressed in bacterial system Promoter is well known in the art, and including phageλ, the bla promoters of the beta-lactam enzyme gene of pBR322 and The CAT promoters of the chloramphenicol acetyl transferasegene of pBR325.The other example of prokaryotic promoter includes the master of 5 phageλs Want right and left promoter (PL and PR), colibacillary trp, recA, lacZ, lad and gal promoter, AMS (Ulmanen et al,1985.J.Bacteriol.162:176-182) with the S28 specificity promoters of bacillus subtilis (Gilman et al,1984Gene 32:11-20), the bacteriophage of bacillus (Bacillus) promoter (Gryczan, 1982,In:The Molecular Biology of the Bacilli, Academic Press, Inc., New York) with And Streptomyces promoter (Ward et al, 1986, Mol.Gen.Genet.203:468-478).It is contemplated by the invention that it is other Prokaryotic promoter is in such as Glick (1987, J.Ind.Microbiol.1:277-282)；Cenatiempo,(1986, Biochimie,68:505-516) and Gottesman, (1984, Ann.Rev.Genet.18:It is reviewed in 415-442. It is contemplated by the invention that the other example of promoter/controlling element include but is not limited to Listeria prfA promoter, Listeria Hly promoters, Listeria p60 promoter and Listeria actA promoter (GenBank accession number NC_003210) or its piece Section.

In another embodiment, gene of the plasmid of the method for the present invention and composition comprising encoding fusion protein. In another embodiment, clone subsequence and cut appropriate subsequence using appropriate Restriction Enzyme.In another embodiment In, the DNA sequence dna needed for then junction fragment is generated.In another embodiment, the DNA of coding for antigens uses DNA cloning method It is prepared by such as PCR (PCR).First, the either side in the new end of n DNA fragment is individually expanded.One expansion The 5' ends coding peptide linker of the sequence of increasing, and the 3' ends of the sequence of another amplification also encode peptide linker.Due to first piece The 3' termini-complementaries of 5' ends and second fragment of section, two fragments (after partial purification, such as on LMP agaroses) can be the It is used as to overlap template in three PCR reactions.The sequence of amplification is by comprising fragment (the present shape on codon, vent position carboxyl side Into amino sequence), the sequence (forming carboxyl sequences now) on joint and vent position amino side.Antigen is connected into plasmid. Every kind of method represents the separate embodiments of the present invention.

In another embodiment, present invention additionally comprises for the chromosomal integration system based on bacteriophage of clinical practice System.Using indispensable enzyme (including but not limited to D-alanine racemase) auxotroph host strain, such as Lmdal (-) dat (-).In another embodiment, to avoid " bacteriophage cures step " (phage curing step), using based on PSA's Bacteriophage integration system (Lauer, et al., 2002J Bacteriol, 184:4177-4186).In another embodiment, This needs the continuous selection of antibiotic to maintain integrator gene.Therefore, in another embodiment, the present invention allows foundation to be not required to The chromosomal integration system based on bacteriophage for wanting antibiotic to select.Conversely, auxotroph host strain will be complementary.

In another embodiment, the recombinant protein of the present invention is synthesized using recombinant DNA method.In one embodiment, This is related to form the DNA sequence dna of encoding fusion protein, the expression cassette that DNA is placed under the control of concrete promoter/controlling element In (plasmid such as of the invention), and expressing protein.In another embodiment, by any suitable method, including example As appropriate sequence clone and limit or by such as Narang et al. (1979, Meth.Enzymol.68:Phosphorus 90-99) Sour triester method, Brown et al. (1979, Meth.Enzymol 68:Approach 109-151), Beaucage et al.(1981,Tetra.Lett.,22:15 1859-1862) diethyl phosphoramidite method and United States Patent (USP) No.4,458,066 Solid phase vector the direct chemical synthesis of method preparing the fusion protein (e.g., non-haemolysis LLO/ antigens) of the coding present invention DNA。

In another embodiment, single stranded oligonucleotide is prepared using chemical synthesis.In various embodiments, the single-stranded widow Nucleotides by with complementary sequence hybridization or using it is single-stranded be polymerized by archaeal dna polymerase as template be converted to double-stranded DNA.Ability Domain it will be recognized that although the chemical synthesis of DNA is only limitted to the sequence of about 100 bases, longer sequence can Obtained by the connection of shorter sequence.In another embodiment, clone subsequence and cut using appropriate Restriction Enzyme Appropriate subsequence.Then junction fragment is with the DNA sequence dna needed for preparing.

In another embodiment, using DNA cloning method such as PCR (PCR) the clones coding present invention's The DNA of fusion protein or recombinant protein.Therefore, using the sense primer comprising suitable restriction site and comprising another limit The antisense primer PCR in property site processed (restriction site for e.g., differing to be conducive to cloning) expands the gene of non-haemolysis LLO. The detached nucleic acid of the identical step amplification coding antigen of repetition.Connect non-haemolysis LLO and antigen sequence and insert plasmid or load Body, to generate the carrier that coding is connected to the non-haemolysis LLO of antigen end.Two molecules are introduced directly or by restriction site Short spacer peptide connection.

In another embodiment, molecule is generally spaced by the spacer peptide isolation being made up of one or more amino acid Without particular biological activity in addition to other spatial relationships of peptide except the certain minimum range of connection albumen or maintenance or between them. In another embodiment, the composition AA of spacer peptide is selected to affect the properties of molecule, such as folding, net charge or hydrophobic Property.In another embodiment, the nucleotide sequence of coding fusion or recombinant protein is converted to various host cells, including large intestine Bacillus, other bacterial hosts such as Listeria, yeast and various higher eucaryotic cells such as COS, CHO and HeLa clone with And myeloma cell line.Recombination fusion protein gene will may be operably coupled to the appropriate expression control sequenc of each host.Open Mover/regulating and controlling sequence is described in detail in this paper other places.In another embodiment, plasmid is also comprising other promoter regulation unit Part, and ribosome bind site and transcription stop signals.For eukaryotic, regulating and controlling sequence including promoter and will be derived from The such as enhancer of immunoglobulin gene, SV40, cytomegalovirus, and polyadenylation se-quence.In another enforcement In example, sequence includes donor splicing site and receptor sequence.

In one embodiment, term " being operably connected " refers to that the part of wherein such description is in and allows it The juxtaposition of the relation of function in a desired manner.The connected mode of the control sequence of " being operably connected " to coded sequence makes The expression for obtaining coded sequence is realized under conditions of compatible with control sequence.

In another embodiment, in order to select the auxotrophic bacterium comprising plasmid, the auxotroph of conversion is made Bacterium grows on the culture medium that will select amino acid metabolising gene expression.In another embodiment, using comprising for D- The plasmid conversion D-Glu synthesis auxotrophic bacterium of the gene of glutamic acid synthesis, and the auxotrophic bacterium is not Grow in the case of there is D-Glu, and the plasmid conversion is not used or the albumen encoded for D-Glu synthesis is not expressed The auxotrophic bacterium of plasmid then can not grow.In another embodiment, when the plasmid of conversion and the expression present invention, If the detached nucleic acid of the amino acid metabolism enzyme that plasmid synthesizes comprising coding for D-alanine, D-alanine synthesis nutrition Deficiency bacterium will grow in the case where there is no D-alanine.It is such for prepare include or lack necessary growth factor, The method of the appropriate culture medium of replenishers, amino acid, vitamin, antibiotic etc. is well known in the art, and commercially available is obtained Obtain (Becton-Dickinson, Franklin Lakes, NJ).Every kind of method represents the separate embodiments of the present invention.

In another embodiment, once selecting the auxotroph of the plasmid comprising the present invention thin in appropriate culture medium After bacterium, bacterium can breed in the case where there is selection pressure.Such propagation includes making bacterium without the auxotroph factor Culture medium in grow.There is the plasmid of express amino acid metabolic enzyme in auxotrophic bacterium guarantees that plasmid will be together with bacterium Replicate, so as to continuously select the bacterium with plasmid.Technical staff is readily able to when the present disclosure and method of this paper is understood Listeria vaccine carrier is expanded by the amount of the residing culture medium of the adjustment growth of the auxotrophic bacterium comprising plasmid Prepare.

Technical staff will be appreciated that, in another embodiment, using other auxotrophic strains and complementary system with The present invention is used together.

In one embodiment, there is provided herein hindering the method that the HER2 of experimenter expresses tumour growth, wherein and In another embodiment, the method includes being applied to the composition comprising recombinant listeria bacterium vaccine strain as herein described and receives The step of examination person.

In another embodiment, there is provided herein hindering or delaying to express the transfer of tumour from the HER2 of experimenter Property disease method, wherein and in another embodiment, the method includes will be comprising recombinant listeria bacterium as herein described The step of composition of vaccine strain is applied to experimenter.

In another embodiment, there is provided herein the enhancing immunity for expressing HER2/neu tumour for causing experimenter is answered The method answered, wherein and in another embodiment, the method is included comprising recombinant listeria bacterium vaccine as herein described The step of composition of strain is applied to experimenter.In yet another embodiment, the immune response bag of tumour is expressed HER2/neu Include the immune response of at least one Dominant Epitopes to HER2/neu albumen.

In one embodiment, there is provided herein preventing the method that HER2/neu expresses the escape mutant in oncotherapy, Wherein and in another embodiment, the method include will include provided herein is recombinant listeria bacterium vaccine strain composition The step of being applied to the experimenter.

In another embodiment, there is provided herein preventing the side of the HER2/neu antigen presentation tumor invasions of experimenter Method, wherein and in another embodiment, the method include will include provided herein is recombinant listeria bacterium vaccine strain group The step of compound is applied to experimenter.

In one embodiment, there is provided herein reduce intra-tumor regulatory T cells frequency method, wherein and In another embodiment, the method include will include provided herein is recombinant listeria bacterium vaccine strain composition be applied to it is tested The step of person.

In another embodiment, there is provided herein reduce intra-tumor regulatory T cells frequency method, wherein and In another embodiment, the method include including provided herein is the composition of recombinant listeria bacterium vaccine strain be applied to and receive The step of examination person.

In one embodiment, there is provided herein reduce intra-tumor derived from bone marrow SC frequency method, Wherein and in another embodiment, the method include will include provided herein is recombinant listeria bacterium vaccine strain composition The step of being applied to experimenter.

In another embodiment, there is provided herein reduce derived from bone marrow SC frequency method, wherein And in another embodiment, the method include will include provided herein is recombinant listeria bacterium vaccine strain composition apply The step of experimenter.

In one embodiment, there is provided herein preventing from forming the method that HER2/neu expresses tumour in experimenter, wherein And in another embodiment, the method include will include provided herein is recombinant listeria bacterium vaccine strain composition apply The step of experimenter.In another embodiment, there is provided herein preventing from being formed in experimenter from HER2/neu expression The method of the metastatic disease of tumour, wherein and in another embodiment, the method include will include provided herein is weight The step of composition of group Listeria vaccine strain is applied to experimenter.In one embodiment, there is provided herein treatment is tested The method that the HER2/neu of person expresses tumour, wherein and in another embodiment, the method include including provided herein is The composition of recombinant listeria bacterium vaccine strain the step of be applied to experimenter.In another embodiment, there is provided herein controlling The method for treating the metastatic disease that tumour is expressed from HER2/neu of experimenter, wherein and in another embodiment, The method include including provided herein is the composition of recombinant listeria bacterium vaccine strain the step of be applied to experimenter.At one In embodiment, there is provided herein the method for applying the composition of the present invention.In another embodiment, there is provided herein applying this The method of the vaccine of invention.In another embodiment, there is provided herein applying the recombinant polypeptide or recombinant nucleotide of the present invention Method.In another embodiment, the step of applying composition, vaccine, recombinant polypeptide or the recombinant nucleotide of the present invention is logical Cross each sheet is carried out as the listerial attenuation recombinant forms of discrete embodiment, and the Listeria includes composition, epidemic disease Seedling, recombinant nucleotide or expression recombinant polypeptide.In another embodiment, apply is carried out by different attenuation bacteria carriers. In another embodiment, apply is carried out by DNA vaccination (e.g., naked DNA vaccine).In another embodiment, it is of the invention The administration of recombinant polypeptide generates albumen by restructuring, then is applied to experimenter to carry out by recombinant protein.Every kind of possibility table Show the separate embodiments of the present invention.

In one embodiment, the repetitive administration (booster) of composition of the invention can be after first course for the treatment of of treatment Carry out immediately or carry out behind the interval of a few days, a few weeks or months, to realize tumor regression.In another embodiment, weight Multiple dosage can immediately be carried out after first course for the treatment of of treatment or carried out behind the interval of a few days, a few weeks or months, to realize swelling The suppression of knurl growth.Assessment can be determined by any technology known in the art, including diagnostic method such as imaging technique, serum Tumor markers analysis, biopsy, the presence of tumor-related symptoms, do not exist or improve.

In another embodiment, the immune response that the method for the present invention and composition cause includes CD8⁺T cell is mediated Response.In another embodiment, immune response is mainly by CD8⁺The response composition of T cell mediation.In another embodiment In, the only detectable part of immune response is CD8⁺The response of T cell mediation.

In another embodiment, provided herein is the immune response that causes of method and composition include CD4⁺T cell is situated between The response led.In another embodiment, immune response is mainly by CD4⁺The response composition of T cell mediation.In another enforcement In example, the only detectable part of immune response is CD4⁺The response of T cell mediation.In another embodiment, CD4⁺T Cell-mediated response is with the determined antibody response to antigen.In another embodiment, CD4⁺The response of T cell mediation Without the determined antibody response to antigen.

In another embodiment, the present invention provides secondary advantage CD8 to antigen of induction experimenter⁺T cell epitope CD8⁺The method of the immune response of T cell mediation, the method is comprised the following steps：(a) make coding Her2-neu chimeric antigens or The nucleic acid molecule of its fragment is fused to the nucleic acid molecule of the N- terminal fragments for encoding LLO albumen, so as to form coding LLO- The recombinant nucleotide of antigen coalescence protein；And recombinant nucleotide or LLO- Antigen Fusion bodies are applied to experimenter by (b)；So as to Secondary advantage CD8 of the induction to antigen⁺The CD8 of t cell epitope⁺The immune response of T cell mediation.

In one embodiment, there is provided herein improve intra-tumor CD8+/regulatory T cells ratio method, wherein simultaneously And in another embodiment, the method is included the recombinant polypeptide comprising the present invention, recombinant listeria bacterium or recombinant vector The step of composition is applied to experimenter.

In another embodiment, there is provided herein improve intra-tumor CD8+/regulatory T cells ratio method, wherein And in another embodiment, the method is included the recombinant polypeptide comprising the present invention, recombinant listeria bacterium or recombinant vector Composition the step of be applied to experimenter.

In another embodiment, provided herein is the immune response that causes of method and composition include to antigen at least The immune response of one Dominant Epitopes.In another embodiment, immune response does not include that the immunity to secondary Dominant Epitopes should Answer.In another embodiment, immune response is mainly made up of the immune response at least one Dominant Epitopes.At another In embodiment, the part that only can determine of immune response is the immune response at least one Dominant Epitopes.Per species The immune response of type represents the separate embodiments of the present invention.

In one embodiment, the method for the present invention destroys experimenter and expresses tumour or the experimenter to HER2/ The tolerance of cancer, wherein and in another embodiment, the method include will include provided herein is recombinant listeria bacterium The step of composition of vaccine strain is applied to experimenter.

The method for determining immune response is well known in the art, and thin including the such as suppression of measure tumour growth, streaming Born of the same parents' art, target cell lysis analysis (e.g., chromium release analysis), tetramer are used etc..Every kind of method represents that the present invention's is independent Embodiment.

In another embodiment, the present invention provides and delays or suppress experimenter's to express turning for tumour from Her-2 Move property disease method, wherein and in another embodiment, the method include will comprising be fused to HER2 chimeric proteins or The recombinant polypeptide of N- terminal fragments or the recombinant nucleotide of encoding recombinant polypeptide of the LLO albumen of its fragment is applied to experimenter, Wherein the experimenter produces the immune response for expressing HER2 tumour, so as to delay or suppress expressing from HER2 for experimenter The metastatic disease of tumour.

In another embodiment, the present invention provides the method for enhancing antigenicity for improving HER2 chimeric proteins, wherein and In another embodiment, the method includes that the nucleotides of the N- terminal fragments for making coding LLO albumen is fused to encode Her-2 albumen Or the nucleotides of its fragment, the step of to form recombinant polypeptide, so as to improve the antigenicity of HER2 chimeric proteins.

In another embodiment, there is provided herein improving the method for enhancing antigenicity of HER2 chimeric proteins, wherein and In another embodiment, the method includes being engineered to Listeria bacterial strain to express recombinant nucleotide.In another embodiment In, using different bacteria carrier expression recombinant nucleotides.In another embodiment, bacteria carrier is attenuation.Another In individual embodiment, using DNA vaccination (e.g., naked DNA vaccine) recombinant nucleotide is expressed.In another embodiment, nucleotides is compiled The administration of the LLO-HER2 chimera fusogenic peptides of code generates albumen by restructuring, then is applied to experimenter to enter by recombinant protein OK.Every kind of possibility represents the separate embodiments of the present invention.

In one embodiment, the present invention is provided to the method for " epitope spreading " of tumour.In another embodiment, Using provided herein is composition and the immune induction of method there is antigen in addition to the antigen that the vaccine of the present invention is carried Epitope spreading in other tumours.

In another embodiment, Dominant Epitopes or secondary Dominant Epitopes are respectively advantage in the experimenter for being treated Or it is co-dominant.In another embodiment, Dominant Epitopes or secondary Dominant Epitopes be in the colony treated advantage or It is co-dominant.

In one embodiment, there is provided herein by epitope spreading treatment, checking or suppressing the cancer of experimenter or swollen The method of knurl growth, wherein and in another embodiment, the cancer and the antigen that includes in the composition of the present invention or The expression of its fragment is related.In another embodiment, method is included recombinant polypeptide, recombinant listeria bacterium comprising the present invention Or the composition of recombinant vector is applied to the experimenter.In yet another embodiment, experimenter creates antagonism former expression cancer Or the immune response of antigen presentation tumour, so as to treat, check or suppress the cancer or tumour growth of experimenter.

In one embodiment, " advantage CD8⁺T cell epitope " is finger protein or wraps protein-contg pathogen or cancer cell Vaccine inoculation, infection or antigentic specificity CD8 more than 30% that causes of malignancy⁺The epi-position of T cell identification.Another In individual embodiment, the term refers to antigentic specificity CD8 more than 35% for thus causing⁺The epi-position of T cell identification.Another In individual embodiment, the term refers to more than 40% antigentic specificity CD8⁺The epi-position of T cell identification.In another embodiment In, the term refers to more than 45% antigentic specificity CD8⁺The epi-position of T cell identification.In another embodiment, the term Refer to more than 50% antigentic specificity CD8⁺The epi-position of T cell identification.In another embodiment, the term is referred to more than 55% antigentic specificity CD8⁺The epi-position of T cell identification.In another embodiment, the term refers to more than 60% antigen Specific C D8⁺The epi-position of T cell identification.In another embodiment, the term refers to more than 65% antigentic specificity CD8⁺T The epi-position of cell recognition.In another embodiment, the term refers to more than 70% antigentic specificity CD8⁺T cell identification Epi-position.In another embodiment, the term refers to more than 75% antigentic specificity CD8⁺The epi-position of T cell identification.Another In one embodiment, the term refers to more than 80% antigentic specificity CD8⁺The epi-position of T cell identification.In another embodiment In, the term refers to more than 85% antigentic specificity CD8⁺The epi-position of T cell identification.In another embodiment, the term Refer to more than 90% antigentic specificity CD8⁺The epi-position of T cell identification.In another embodiment, the term is referred to more than 95% antigentic specificity CD8⁺The epi-position of T cell identification.In another embodiment, the term refers to more than 96% antigen Specific C D8⁺The epi-position of T cell identification.In another embodiment, the term refers to more than 97% antigentic specificity CD8⁺T The epi-position of cell recognition.In another embodiment, the term refers to more than 98% antigentic specificity CD8⁺T cell identification Epi-position.

In one embodiment, " secondary advantage CD8⁺T cell epitope " is that finger protein or the protein-contg pathogen of bag or cancer are thin Antigentic specificity CD8 less than 30% that the vaccine inoculation of born of the same parents, infection or malignancy cause⁺The epi-position of T cell identification.Another In one embodiment, the term refers to antigentic specificity CD8 less than 28%⁺The epi-position of T cell identification.In another embodiment In, the term refers to more than 26% antigentic specificity CD8⁺The epi-position of T cell identification.In another embodiment, the term Refer to antigentic specificity CD8 less than 24%⁺The epi-position of T cell identification.In another embodiment, the term is referred to more than 22% antigentic specificity CD8⁺The epi-position of T cell identification.In another embodiment, the term refers to the antigen less than 20% Specific C D8⁺The epi-position of T cell identification.In another embodiment, the term refers to more than 18% antigentic specificity CD8⁺T The epi-position of cell recognition.In another embodiment, the term refers to antigentic specificity CD8 less than 16%⁺T cell identification Epi-position.In another embodiment, the term refers to more than 14% antigentic specificity CD8⁺The epi-position of T cell identification.Another In one embodiment, the term refers to more than 12% antigentic specificity CD8⁺The epi-position of T cell identification.In another embodiment In, the term refers to antigentic specificity CD8 less than 10%⁺The epi-position of T cell identification.In another embodiment, the term Refer to more than 8% antigentic specificity CD8⁺The epi-position of T cell identification.In another embodiment, the term is referred to less than 6% Antigentic specificity CD8⁺The epi-position of T cell identification.In another embodiment, the term refers to the antigen-specific less than 5% Property CD8⁺The epi-position of T cell identification.In another embodiment, the term refers to more than 4% antigentic specificity CD8⁺T cell The epi-position of identification.In another embodiment, the term refers to antigentic specificity CD8 less than 3%⁺The epi-position of T cell identification. In another embodiment, the term refers to antigentic specificity CD8 less than 2%⁺The epi-position of T cell identification.In another reality In applying example, the term refers to antigentic specificity CD8 less than 1%⁺The epi-position of T cell identification.In another embodiment, the art Language refers to antigentic specificity CD8 less than 0.5%⁺The epi-position of T cell identification.

Each type of Dominant Epitopes and time Dominant Epitopes represent the separate embodiments of the present invention.

In one embodiment, the antigen in the method for the present invention and composition with detectable level in the non-tumour of experimenter Express on cell.In another embodiment, antigen with detectable level at least a certain percentage (e.g., 0.01%, 0.03%th, 0.1%, 0.3%, 1%, 2%, 3% or experimenter's non-tumor cell 5%) on express.In one embodiment, " non-tumor cell " refers to the cell of Tumor in Vitro.In another embodiment, " non-tumor cell " refers to non-malignant cell. In another embodiment, " non-tumor cell " refers to unconverted cell.In another embodiment, non-tumor cell is that body is thin Born of the same parents.In another embodiment, non-tumor cell is reproduction cell.Every kind of possibility represents the separate embodiments of the present invention.

In one embodiment, " detectable level " refers to the level that can use standard analysis detection.In one embodiment In, analysis is immunoassay.In one embodiment, analysis is ELISA (ELISA).In another embodiment, Analysis is western blot.In another embodiment, analysis is FACS.Technical staff will be appreciated that, provided herein is method Can be using available any other analysis in this area.In another embodiment, the back of the body of the detectable level relative to concrete analysis Scape level determines.Method for performing every kind of these technologies is well known to those skilled in the art, and every kind of technology represents The separate embodiments of the present invention.

In one embodiment, using recombinant antigen expression LM vaccine inoculation is carried out induction of epitope spreading.At another In embodiment, even if outside the scope of Her2, using the vaccine inoculation of LLO- Antigen Fusion bodies also induction of epitope spreading.It is every kind of Possibility represents the separate embodiments of the present invention.

In another embodiment, the present invention provides the method for hindering the growth of HER2 expression tumour in experimenter, the party Method includes for the recombinant polypeptide of the N- terminal fragments comprising the LLO albumen for being fused to HER2 chimeric antigens being applied to experimenter, its Middle antigen has one or more advantage CD8⁺T cell epitope, wherein experimenter create antagonism the former immunity for expressing tumour should Answer, so as to hinder experimenter in HER2 express tumour growth.In another embodiment, antigen does not include any advantage CD8⁺ T cell epitope.In another embodiment, there is provided herein hindering the method that HER2 expresses tumour growth in experimenter, the party Method include will include coding provided herein is recombinant polypeptide recombinant nucleotide listerial recombinant forms be applied to it is tested Person.

In another embodiment, the present invention provides induction and cytotoxic T cell is formed in the host with cancer Method, the method includes for the composition of the present invention being applied to host, and cell is formed in the host with cancer so as to induce Cytotoxic T cell.

In another embodiment, the present invention provides the method that cancer occurs that reduces, and the method includes applying the present invention's Composition.In another embodiment, the present invention provides the method for improving cancer, and the method includes applying the combination of the present invention Thing.Every kind of possibility represents the separate embodiments of the present invention.

In one embodiment, composition is applied in vitro the cell of experimenter；In another embodiment, will combine Thing is applied in vitro the cell of donor；In another embodiment, the cell of donor will be applied in composition body, be then shifted To experimenter.Every kind of possibility represents the separate embodiments of the present invention.

In one embodiment, the cancer of method of the present invention treatment is breast cancer.In another embodiment, cancer is Cancer containing Her2.In another embodiment, cancer is melanoma.In another embodiment, cancer is cancer of pancreas.Another In individual embodiment, cancer is oophoroma.In another embodiment, cancer is cancer of the stomach.In another embodiment, cancer is pancreas The carcinous stove of gland.In another embodiment, cancer is adenocarcinoma of lung.In another embodiment, cancer is colorectal adenocarcinoma. In another embodiment, cancer is lung squamous gland cancer.In another embodiment, cancer is sdenocarcinoma of stomach.In another enforcement In example, cancer is Ovarian surface epithelium knurl (e.g., their benign, Hypertrophic or malignant form).In another embodiment, cancer Disease is oral squamous cell carcinoma.In another embodiment, cancer is non-small cell lung cancer.In another embodiment, Cancer is CNS cancers.In another embodiment, cancer is carcinoma of endometrium.In another embodiment, cancer is bladder Cancer.In another embodiment, cancer is celiothelioma.In another embodiment, cancer is malignant mesothelioma (MM).Another In individual embodiment, cancer is head and neck cancer.In another embodiment, cancer is prostate cancer.In another embodiment, cancer It is osteosarcoma.In another embodiment, cancer is HER2/neu expression osteosarcoma.In another embodiment, osteosarcoma is Canid osteosarcoma.In another embodiment, osteosarcoma is localized osteosarcoma.In another embodiment, osteosarcoma is Metastatic bone sarcoma.In another embodiment, osteosarcoma is height osteosarcoma.In another embodiment, osteosarcoma is dog Section's animal limb osteosarcoma.

In another embodiment of the method for the present invention, experimenter creates antagonism the immunity of former expression tumour or target antigen Response, so as to mediating antitumor effect.

In another embodiment, the present invention is provided to the immunogenic composition for the treatment of cancer, said composition is included The fusion of the LLO and HER2 chimeric protein of truncation.In another embodiment, also comprising expression, this melts immunogenic composition Fit Listeria bacterial strain.

In another embodiment, the present invention is provided to the immunogenic composition for the treatment of cancer, said composition is included The Listeria bacterial strain of expression HER2 chimeric proteins.

In one embodiment, therapeutic scheme of the invention is curative.In another embodiment, scheme is prevention Property.In another embodiment, vaccine of the invention or composition are used for protection due to familial inheritance or make it tend to suffer from There are other situations of these disease types and there is the people of cancer (such as breast cancer or other kinds of tumour containing HER2) risk, As it will be understood by the skilled person.In another embodiment, vaccine is after the growth of operation, conventional chemotherapy or radiotherapy destroyed tumor As immunotherapy for cancer.After such treatment, the administration of the vaccine of the present invention causes the CTL responses of the tumour antigen to vaccine Destroy remaining alleviation shifted and extend cancer.In another embodiment, vaccine and operation, conventional chemotherapy or put Treat joint and be used as immunotherapy for cancer.In another embodiment, such therapeutic alliance is used to not be amenable to the experimenter of amputation. In another embodiment, such therapeutic alliance is used to suffer from the osteosarcomatous experimenter for not being amenable to amputation of primary.Another In one embodiment, the vaccine of the present invention is used to affect the growth of the tumour set up before this and kills existing tumour cell.

In one embodiment, " tumour antigen or its fragment ", " tumor associated antigen or its fragment ", " heterogenetic antigen Or its fragment " or " Antigenic Peptide or its fragment " be used interchangeably herein, and including any antigen known in the art, bag Include tumour antigen, angiogenesis antigen or infectious diseases antigen.In another embodiment, antigen is self-antigen.

In one embodiment, provided herein is antigenic source in tumor associated antigen, in one embodiment, it be with One of lower tumour antigen：Survivin, MAGE (melanoma associated antigen E) albumen such as MAGE1, MAGE 2, MAGE 3, MAGE 4th, tyrosinase；Mutated ras protein；Mutant p 53 Protein；The related ras peptides of p97 melanoma antigens, TCA or p53 peptides； (cancer embryo resists the related CEA of the related KLH antigens of the related antigens of HPV 16/18 of cervix cancer, breast cancer, colorectal cancer It is former), the related PSA antigens of the related MART1 antigens of gp100, melanoma or prostate cancer.In another embodiment, it is used for Provided herein is composition and the antigen of method be melanoma associated antigen, in one embodiment, it be TRP-2, MAGE-1, MAGE-3, gp-100, tyrosinase, HSP-70, β-HCG or combinations thereof.Present invention further contemplates it is known in the art its His tumor associated antigen.

In another embodiment, antigen or its fragment from selected from HPV-E7 (from HPV16 or HPV18 bacterial strains), HPV-E6 (from HPV16 or HPV18 bacterial strains), Her-2/neu, NY-ESO-1, Telomerase (TERT), SCCE, CEA, LMP-1, P53, carbonic anhydrase IX (CAIX), PSMA, prostate stem cell antigen (PSCA), HMW-MAA, WT-1, HIV-1Gag, protease 3rd, tyrosinase related protein1, PSA (PSA), EGFR-III, survivin, apoptosis repetitive sequence include rod Shape virus inhibitory factor 5 (BIRC5), LMP-1, p53, PSMA, PSCA, Muc1, PSA (PSA) or they The antigen of combination.

In another embodiment, the compositions and methods of the invention are used for the vaccine inoculation for tumour or cancer.

In one embodiment, therapeutic scheme of the invention is curative.In another embodiment, scheme is prevention Property.In another embodiment, vaccine of the invention or composition are used for protection due to familial inheritance or make it tend to suffer from There are other situations of these disease types and there is the people of cancer (such as breast cancer or other kinds of tumour containing HER2) risk, As it will be understood by the skilled person.In another embodiment, vaccine is after the growth of operation, conventional chemotherapy or radiotherapy destroyed tumor As immunotherapy for cancer.After such treatment, the administration of the vaccine of the present invention causes the CTL responses of the tumour antigen to vaccine Destroy remaining alleviation shifted and extend cancer.In another embodiment, vaccine and operation or conventional chemotherapy connection Share and make immunotherapy for cancer.In another embodiment, such therapeutic alliance is used to not be amenable to the experimenter of amputation.Another In one embodiment, such therapeutic alliance is used to suffer from the osteosarcomatous experimenter for not being amenable to amputation of primary.At another In embodiment, the vaccine of the present invention is used to affect the growth of the tumour set up before this and kills existing tumour cell.

In another embodiment, the vaccine and immunogenic composition used by any of the above described method has the epidemic disease of the present invention Any characteristic of seedling and immunogenic composition.The separate embodiments of every kind of personality presentation present invention.

Present invention contemplates the various embodiments of dosage range.In one embodiment, for vaccine carrier, dosage exists 0.4LD₅₀In the range of/agent.In another embodiment, dosage is for about 0.4-4.9LD₅₀/ agent.In another embodiment, agent Amount is for about 0.5-0.59LD₅₀/ agent.In another embodiment, dosage is for about 0.6-0.69LD₅₀/ agent.In another embodiment In, dosage is for about 0.7-0.79LD₅₀/ agent.In another embodiment, dosage is for about 0.8LD₅₀/ agent.In another embodiment In, dosage is 0.4LD₅₀/ agent is to 0.8LD₅₀/ agent.

In another embodiment, dosage is 10⁷Individual bacterium/agent.In another embodiment, dosage is 1.5 × 10⁷It is individual Bacterium/agent.In another embodiment, dosage is 2 × 10⁷Individual bacterium/agent.In another embodiment, dosage is 3 × 10⁷It is individual Bacterium/agent.In another embodiment, dosage is 4 × 10⁷Individual bacterium/agent.In another embodiment, dosage is 6 × 10⁷It is individual Bacterium/agent.In another embodiment, dosage is 8 × 10⁷Individual bacterium/agent.In another embodiment, dosage is 1 × 10⁸It is individual Bacterium/agent.In another embodiment, dosage is 1.5 × 10⁸Individual bacterium/agent.In another embodiment, dosage is 2 × 10⁸ Individual bacterium/agent.In another embodiment, dosage is 3 × 10⁸Individual bacterium/agent.In another embodiment, dosage is 4 × 10⁸ Individual bacterium/agent.In another embodiment, dosage is 6 × 10⁸Individual bacterium/agent.In another embodiment, dosage is 8 × 10⁸ Individual bacterium/agent.In another embodiment, dosage is 1 × 10⁹Individual bacterium/agent.In another embodiment, dosage be 1.5 × 10⁹Individual bacterium/agent.In another embodiment, dosage is 2 × 10⁹Individual bacterium/agent.In another embodiment, dosage be 3 × 10⁹Individual bacterium/agent.In another embodiment, dosage is 5 × 10⁹Individual bacterium/agent.In another embodiment, dosage be 6 × 10⁹Individual bacterium/agent.In another embodiment, dosage is 8 × 10⁹Individual bacterium/agent.In another embodiment, dosage be 1 × 10¹⁰Individual bacterium/agent.In another embodiment, dosage is 1.5 × 10¹⁰Individual bacterium/agent.In another embodiment, dosage For 2 × 10¹⁰Individual bacterium/agent.In another embodiment, dosage is 3 × 10¹⁰Individual bacterium/agent.In another embodiment, agent Measure as 5 × 10¹⁰Individual bacterium/agent.In another embodiment, dosage is 6 × 10¹⁰Individual bacterium/agent.In another embodiment, Dosage is 8 × 10¹⁰Individual bacterium/agent.In another embodiment, dosage is 8 × 10⁹Individual bacterium/agent.In another embodiment In, dosage is 1 × 10¹¹Individual bacterium/agent.In another embodiment, dosage is 1.5 × 10¹¹Individual bacterium/agent.In another reality In applying example, dosage is 2 × 10¹¹Individual bacterium/agent.In another embodiment, dosage is 3 × 10¹¹Individual bacterium/agent.At another In embodiment, dosage is 5 × 10¹¹Individual bacterium/agent.In another embodiment, dosage is 6 × 10¹¹Individual bacterium/agent.Another In individual embodiment, dosage is 8 × 10¹¹Individual bacterium/agent.In another embodiment, dosage is 5.0 × 10⁸Individual bacterium/agent. In another embodiment, dosage is 3.3 × 10⁹Individual bacterium/agent.In another embodiment, for provided herein is method Composition includes 3.3 × 10⁹Individual Listeria/agent.Every kind of possibility represents the separate embodiments of the present invention.

In one embodiment, the vaccine or immunogenic composition of the present invention are separately administered into experimenter.Another In individual embodiment, vaccine or immunogenic composition are applied together with another kind of cancer therapy.Every kind of possibility represents the present invention Separate embodiments.

In one embodiment, the recombinant listeria bacterium of the method for the present invention and composition with coding HER2 chimeric antigens or The construct of LLO-HER2 Chimeric Antigen Fusion bodies stable conversion together.In one embodiment, construct comprising polylinker with Be conducive to other subclone.The listerial multiple technologies of Prepare restructuring are known.

In one embodiment, construct or nucleic acid molecules are integrated into Listeria chromosome using homologous recombination.Together Source recombinant technique is well known in the art, and for example in Baloglu S, Boyle SM, et al. (Immune responses of mice to vaccinia virus recombinants expressing either Listeriamonocytogenes partial listeriolysin or Brucella abortus ribosomal L7/ L12protein.Vet Microbiol 2005,109(1-2):11-7) with Jiang LL, Song HH, et al., (Characterization of a mutant Listeriamonocytogenes strain expressing green fluorescent protein.Acta Biochim Biophys Sin(Shanghai)2005,37(1):In 19-24) Description.In another embodiment, homologous recombination such as United States Patent (USP) No.6, is carried out described in 855,320.In this case, The restructuring LM bacterial strains of expression E7 are by E7 genes (under the control of hly promoters, and including hly signal sequences, to guarantee gene The secretion of product) chromosomal integration preparing, so as to obtain the referred to as recombinant of Lm-AZ/E7.In another embodiment, Recombinant is selected using temperature sensitive type plasmid.Every kind of technology represents the separate embodiments of the present invention.

In another embodiment, construct or nucleic acid molecules are integrated into Listeria dyeing using transposons insertion Body.Transposons insertion technology is well known in the art, and particularly by Sun et al. (Infection and Immunity 1990,58:3770-3778) it is described in the structure of DP-L967.In another embodiment, transposon mutagenesis have The advantage of stable genome insertion mutation body can be formed, but also has the shortcomings that foreign gene inserts the Location-Unknown of genome.

In another embodiment, construct or nucleic acid molecules are integrated into Listeria dye using bacteriophage integration site Colour solid (Lauer P, Chow MY et al, Construction, characterization, and use of two Listeriamonocytogenes site-specific phage integration vectors.J Bacteriol 2002；184(15):4177-86).In some embodiments of the method, using the integrase gene and connection site of bacteriophage Heterologous gene is inserted corresponding connection site by (e.g., U153 or PSA Listerias bacteriophage), and the connection site can be base Any appropriate site (e.g., 3 ' ends of comK or arg tRNA genes) in because of group.In another embodiment, in construct Or cure endogenous prophage from connection site used before the integration of heterologous gene.In another embodiment, this Method forms single copy integron.Every kind of possibility represents the separate embodiments of the present invention.

In another embodiment, the fusion egg using one of various promoters expression antigen or comprising the antigen In vain.In one embodiment, using Lm promoters, the such as promoter of gene hly, actA, plca, plcB and mpl, these genes It is separately encoded Listeria albumen hemolysin, actA, phosphatidylinositols specific phospholipase, phospholipase C and metalloproteinases.Often Plant the separate embodiments that possibility represents the present invention.

In another embodiment, the method for the present invention and composition adopt the homologue or the present invention of HER2 chimeric proteins LLO sequences.In another embodiment, the method for the present invention and composition are fitted together to egg using the HER2 of non-human mammal In vain.In one embodiment, when term " homology ", " homologous " etc. are related to any albumen or peptide, refer in aligned sequence With introducing room (if necessary), to realize maximum homology percentage, and a part for sequence iden is not considered as Any conservative substitution after, with the percentage of the residue identical amino acid residue of corresponding natural polypeptides in candidate sequence.With In compare method and computer program be well known in the art.

In another embodiment, similarly, when term " homology " is related to any nucleotide sequence, candidate sequence is represented In percentage with the nucleotides identical nucleotides of corresponding native sequence nucleic acid.

In another embodiment, the present invention provides encoded signal peptide or the recombinant polypeptide of the present invention or dividing for fusion protein From nucleic acid.In one embodiment, detached nucleic acid includes the recombinant polypeptide or fusion egg with encoded signal peptide or the present invention The sequence of total at least 65% homology of white nucleic acid.In another embodiment, detached nucleic acid is included and encoded signal peptide Or the sequence of the recombinant polypeptide of the present invention or total at least 75% homology of the nucleic acid of fusion protein.In another embodiment, It is homologous that detached nucleic acid includes total with the nucleic acid of the recombinant polypeptide or fusion protein of encoded signal peptide or the present invention at least 85% The sequence of property.In another embodiment, detached nucleic acid includes the recombinant polypeptide or fusion with encoded signal peptide or the present invention The sequence of total at least 90% homology of the nucleic acid of albumen.In another embodiment, detached nucleic acid is included and encoded signal The sequence of total at least 95% homology of the nucleic acid of the recombinant polypeptide or fusion protein of peptide or the present invention.In another embodiment In, it is same that detached nucleic acid includes total with the nucleic acid of the recombinant polypeptide or fusion protein of encoded signal peptide or the present invention at least 97% The sequence of source property.In another embodiment, detached nucleic acid comprising with the recombinant polypeptide of encoded signal peptide or the present invention or melt The sequence of total at least 99% homology of the nucleic acid of hop protein.

In one embodiment, homology is true by the computerized algorithm of sequence alignment by method that this area describes in detail It is fixed.For example, the computerized algorithm analysis of nucleic acid sequence homology may include to adopt and be available from such as BLAST, DOMAIN, BEAUTY (BLAST Enhanced Alignment Utility), multiple software kits of GENPEPT and TREMBL software kits.

In another embodiment, " homology " refer to selected from provided herein is sequence (nucleic acid or amino acid sequence) The homogeneity of sequence is more than 65%.In another embodiment, " homology " refer to selected from provided herein is sequence sequence Homogeneity be more than 70%.In another embodiment, homogeneity is more than 75%.In another embodiment, homogeneity is more than 78%.In another embodiment, homogeneity is more than 80%.In another embodiment, homogeneity is more than 82%.At another In embodiment, homogeneity is more than 83%.In another embodiment, homogeneity is more than 85%.In another embodiment, it is same Property be more than 87%.In another embodiment, homogeneity is more than 88%.In another embodiment, homogeneity is more than 90%. In another embodiment, homogeneity is more than 92%.In another embodiment, homogeneity is more than 93%.In another embodiment In, homogeneity is more than 95%.In another embodiment, homogeneity is more than 96%.In another embodiment, homogeneity is more than 97%.In another embodiment, homogeneity is more than 98%.In another embodiment, homogeneity is more than 99%.At another In embodiment, homogeneity is 100%.Every kind of possibility represents the separate embodiments of the present invention.

In another embodiment, homology determined by candidate sequence hybridization assays, its method be it is well known that (see, for example, " Nucleic Acid Hybridization " Hames, B.D., and Higgins S.J., Eds. (1985)；Sambrook et al.,2001,Molecular Cloning,ALaboratory 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, with coding day So the method for the complementary sequence hybridization of the DNA of caspase peptide can be carried out under moderate to stringent condition.As a example by hybridization conditions As being incubated overnight at 42 DEG C in the solution, the solution is included：10-20% formamides, 5 × SSC (150mM NaCl, 15mM lemons Sour trisodium), 50mM sodium phosphates (pH 7.6), 5 × Denhardt solution, 10% dextran sulfate and 20 μ g/ml denaturation shearing salmon Smart DNA.

In one embodiment of the invention, " nucleic acid " refers to a string at least two base-sugar-phosphates combinations.At one In embodiment, the term includes DNA and RNA.In one embodiment, " nucleotides " refers to the monomeric unit of nucleic acid polymers. In one embodiment, RNA can be tRNA (transfer RNA), snRNA (small nuclear rna), rRNA (rRNA), mRNA (couriers RNA), the form of antisense RNA, siRNA (siRNA), microRNA (miRNA) and ribozyme.The use of siRNA and miRNA is It is described (Caudy AA et al, Genes＆Devel 16:2491-96 and references cited therein).DNA can be The form of DNA, viral DNA, linear DNA or chromosomal DNA or the derivative of these packets.Additionally, the DNA of these forms Can be single-stranded, double-strand, three chains or four chains with RNA.In another embodiment, the term also includes that other types can be included Main chain but with the artificial nucleic acid of identical base.In one embodiment, artificial nucleic acid is PNA (peptide nucleic acid).PNA includes peptide Main chain and nucleotide base, and in one embodiment can be with reference to two kinds of molecules of DNA and RNA.In another embodiment, Nucleotides is oxetanes modification.In another embodiment, nucleotides is by replacing one or many with D2EHDTPA key Individual phosphodiester bond is modifying.In another embodiment, phosphoric acid master of the artificial nucleic acid comprising natural acid known in the art Any other variant of chain.The use of phosphorothioate nucleic acids and PNA is known to those skilled in the art, and for example Neilsen PE,Curr Opin Struct Biol 9:353-57 and Raz NK et al Biochem Biophys Res Commun.297:It is described in 1075-84.The preparation of nucleic acid and using being known to those skilled in the art, and Such as Molecular Cloning, (2001), Sambrook and Russell, eds. and Methods in Enzymology:Methods for molecular cloning in eukaryotic cells(2003)Purchio and It is described in G.C.Fareed.Every kind of nucleic acid derivative represents the separate embodiments of the present invention.

In one embodiment, by methods known in the art, including immunoblotting assay, or using it is any it is various can Software kit is analyzed by the computerized algorithm of amino acid sequence, and by the method set up any ammonia listed herein is determined The albumen and/or peptide homology of base acid sequence.Some in these software kits may include FASTA, BLAST, MPsrch or Scanps software kits the, and it can be deployed in the overall situation/local of such as Smith and Waterman algorithms and/or analysis or BLOCKS ratios It is right.Every kind of method for determining homology represents the separate embodiments of the present invention.

In another embodiment, the present invention provides the kit included for performing the reagent of the method for the present invention. In another embodiment, the present invention provides the kit of the composition, instrument or instrument that include the present invention.

In one embodiment, term " contact " or " administration " be instigate the present invention composition directly contact cancer cell or Tumour.In another embodiment, the term is the composition mediate contact cancer cell or tumour for instigating the present invention.At another In embodiment, the method for the present invention includes the composition of wherein experimenter's contact present invention, and then composition passes through to spread or this Any other active transport known to field or passive transportation (compound is circulated in vivo by the process) contact cancer are thin Born of the same parents or the method for tumour.In another embodiment, the method for the present invention may include the composition of at least single administration present invention, Wherein in another embodiment, the method for the present invention may include the composition for repeatedly applying the present invention.Every kind of possibility is represented The separate embodiments of the present invention.

In another embodiment, term " gene " and " recombination " refer to the opening of the polypeptide comprising the coding present invention The nucleic acid molecules of reading frame.Such natural allelic variation can generally cause the 1-5% of the nucleotide sequence of given gene to become Change.Allele can be substituted by the sequencing identification of gene of interest in multiple Different Individuals or organism.This is easy to pass through Identify in multiple individual or organisms identical genetic loci using hybridization probe to carry out.As natural allelic variation Result and do not change any and all such nucleotide diversity and amino acid polymorphism or the variation of gained of functional activity It is intended within the scope of the present invention.

Pharmaceutical composition

In another embodiment, by any method known to those skilled in the art, by such as parenteral, cancer Side, transmucosal, intranasal, intramuscular, intravenous, intracutaneous, interior subcutaneous, intraperitoneal, ventricle, encephalic, intravaginal or intra-tumor will be included The vaccine of the present invention and the pharmaceutical composition of composition are applied to experimenter.

Provided herein is method and composition another embodiment in, vaccine or composition oral are applied, therefore are matched somebody with somebody It is made as being applied to Orally administered form, i.e. solid or liquid preparation.Suitable solid orally ingestible include tablet, capsule, Pill, granule, pilule etc..Suitable liquid oral medicine includes solution, suspension, dispersion, emulsion, finish etc. Deng.In another embodiment of the present invention, active component is formulated as capsule.According to the embodiment, except reactive compound and Outside inert carrier or diluent, the composition of the present invention also includes hard gelatin capsule.

In another embodiment, vaccine or composition are by the injection of the intravenous of liquid preparation, intra-arterial or intramuscular Apply.Suitable liquid preparation includes solution, suspension, dispersion, emulsion, finish etc..In one embodiment, medicine group The intravenous administration of compound, therefore be formulated as being applied to the form of intravenous administration.In another embodiment, pharmaceutical composition is moved Apply in arteries and veins, therefore be formulated as being applied to the form that intra-arterial is applied.In another embodiment, pharmaceutical composition intramuscular is applied With, therefore be formulated as being applied to the form that intramuscular is applied.

In one embodiment, term " treatment " refers to cure diseases.In another embodiment, " treatment " refer to and prevent Disease.In another embodiment, " treatment " refer to reduce disease generation.In another embodiment, " treatment " refer to and change The symptom of kind disease.In another embodiment, " treatment " refer to improve patient the progresson free survival phase or the overall survival phase. In another embodiment, " treatment " refers to the progress of stable disease.In another embodiment, " treatment " refer to inducer remission. In another embodiment, " treatment " refer to the progress for slowing down disease.In another embodiment, term " reduction ", " checking " " suppression " refers to mitigation or reduces.Every kind of possibility represents the separate embodiments of the present invention.

As used herein, term " about " is quantity term, it is intended that add deduct 5%, or in another embodiment, is added deduct 10%, or in another embodiment, add deduct 15%, or in another embodiment, add deduct 20%.

Technical staff understands that term " experimenter " can cover mammal, including need to treat illness or its sequelae or The adult humanses being easily affected by or children, juvenile or teenager, and may also include non-human mammal, such as dog, cat, Pig, cow, sheep, goat, horse, rat and mouse., it will also be appreciated that the term can cover livestock.Anyway, term " is received Examination person " is not excluded for normal individual.

In one embodiment, term " experimenter " is also contemplated by pet dog and cat, including the dog and cat that are not amenable to amputation. In another embodiment, term " experimenter " is also contemplated by the people for not being amenable to perform the operation.In another embodiment, term " is received Examination person " is also contemplated by not being amenable to the people of amputation.In another embodiment, term " experimenter " is also contemplated by human child.

Technical staff will be appreciated that for therapeutic purposes, term " mammal " is referred to and is classified as any of mammal Animal, including but not limited to people, domestic animal and farming animals and zoo, match animal or pet, such as including the canid of dog With horse, cat, ox, pig, sheep etc..

" therapeutically effective amount " for being related to oncotherapy is the amount for referring to produce less than one or more effect：(1) one Determine to suppress tumour growth in degree, including slowing down and complete growth retardation；(2) quantity of tumour cell is reduced；(3) tumour is reduced Size；(4) tumor cell invasion peripheral organ is suppressed (that is, reduce, alleviate or prevent completely)；(5) suppress (that is, reduce, delay Solution is prevented completely) transfer；(6) anti-tumor immune response is strengthened, this can cause tumor regression or repulsion with (but not necessarily)； And/or (7) mitigate to a certain extent one or more symptoms related to imbalance.For the purpose of oncotherapy, carry herein For vaccine " therapeutically effective amount " can by rule of thumb and with customary manner determination.

There is provided following instance the preferred embodiments of the present invention are more completely shown.However, they should not anyway It is interpreted the broad range for limiting the present invention.

Example

Material and method

Oligonucleotides is synthesized by Invitrogen (Carlsbad, CA), and DNA sequencing is by Genewiz Inc, South Plainfield, NJ are carried out.Flow cytometry reagent is purchased from Becton Dickinson Biosciences (BD, San Diego,CA).Except as otherwise noted, otherwise cell culture medium, replenishers and every other reagent are purchased from Sigma (St.Louise,MO).HER2/neu HLA-A2 peptides are synthesized by EZbiolabs (Westfield, IN).RPMI 1640 completely (C-RPMI) culture medium includes 2mM glutamine, 0.1mM nonessential amino acid and 1mM Sodium Pyruvates, 10% hyclone, green grass or young crops Mycin/streptomysin, Hepes (25mM).As described above, and anti-HER2/neu antibody is purchased from Anti-TNF-α LLO antibody Sigma。

Mouse and clone

All zooperies are all in accordance with University of Pennsylvania or Rutgers University's The scheme of IACUC approvals is carried out.FVB/N mouse are purchased from Jackson laboratories (Bar Harbor, ME).Overexpression is big The FVB/N HER2/neu transgenic mices of mouse HER2/neu cancer proteins are closed in the dynamic of University of Pennsylvania Thing is raised center (animal core facility) and is bred.Express the NT-2 tumours of high-caliber rat HER2/neu albumen Clone derives from the spontaneous gland tumor of these mouse, and grows as described above.DHFR-G8 (3T3/neu) cell Derive from ATCC and grown according to ATCC suggested designs.EMT6-Luc clones are by John doctor Ohlfest (University Of Minnesota, MN) generosity is given, and grow in complete C-RPMI culture mediums.Bioluminescence is operated in University Under the guidance of Small Animal Imaging Facility (SAIF) of of Pennsylvania (Philadelphia, PA) Carry out.

Listeria construct and antigen presentation

HER2/neu-pGEM7Z is provided by the Mark doctors Greene friendship of University of Pennsylvania, And comprising being cloned into total length people HER2/neu (hHer2) gene of pGEM7Z plasmids (Promega, Madison WI).The matter Grain is used as the template of three hHER2/neu fragments of amplification, i.e. EC1, EC2 and IC1, and the amplification uses pfx archaeal dna polymerases (Invitrogen) oligonucleotides and shown in table 1 is carried out by PCR.

Table 1：The chimeric cloning primers of people HER2-

HER2/neu chimeric constructs directly merge generation by SOEing PCR methods, each single hHER2/neu piece Duan Zuowei templates.Primer is illustrated in table 2.

For expanding the primer sequence in different fragments people Her2 regions

Cut ChHer2 genes from pAdv138 using XhoI and SpeI Restriction Enzymes, and with Lmdd shuttle vector pAdv134 The non-haemolysis fragments of LLO of middle truncation are in same reading inframe clone.Insetion sequence LLO and hly promoter is analyzed by DNA sequencing Confirm.The plasmid electroporation is converted to Electrocompetent actA, dal, dat mutant Listeria monocytogenes Listeria bacterial strain, LmddA and positive colony are selected on brain heart infusion (BHI) agar plate comprising streptomysin (250 μ g/ml).In some experiments In, express the similar Listeria bacterial strain of hHER2/neu (Lm-hHER2) fragment for comparative purposes.These contents have above It is described.In all researchs, including uncorrelated Listeria construct (Lm- controls), to consider Listeria to immunity The antigen-independent effect of system.Lm- controls are based on and ADXS31-164 identical Listeria platforms, but express different Antigen, such as HPV16-E7 or NY-ESO-1.Test the expression and secretion of listerial fusion protein.In each construct body Pass on twice.

Cytotoxicity analysis

With 3-5 FVB/N mouse as one group, using 1 × 10⁸The Lm-LLO-ChHer2 of individual colony forming unit (CFU), ADXS31-164, Lm-hHer2ICI or Lm- control (the uncorrelated antigen of expression) is with one week as Immunity at intervals three times or keeps not sudden and violent Dew.NT-2 cell in-vitro growths are made, by the de- wall of trypsase, and (250 μ g/ml, are dissolved in serum-free C- with mitomycin C RPMI culture mediums) process 45 minutes at 37 ° DEG C.After washing 5 times, by itself and the spleen collected from immune or unexposed animal Cell is with 1:5 (stimulating factors:Respondent) ratio at 37 ° DEG C and 5%CO₂Incubate 5 days altogether down.Standard cytotoxicity assay makes 3T3/neu (DHFR-G8) cells marked with europium are carried out as target according to preceding method.Light splitting is used after incubating at 4 hours Photometer (Perkin Elmer, Victor²) europium discharged from the target cell for killing is determined at 590 nm.Specific lytic Percentage is defined as (cracking of experimental group-spontaneity cracking)/(maximum cracking-spontaneous cracking).

The interferon-γ of the splenocyte secretion of immune mouse

With 3-5 FVB/N or HLA-A2 transgenic mice as one group, using 1 × 10⁸The negative Listeria pair of individual CFU According to ADXS31-164 (the uncorrelated antigen of expression) is with one week as Immunity at intervals three times or keeps unexposed.It is latter in last immunity Week separates the splenocyte of FVB/N mouse, and with 5 × 10⁶There is silk in C-RPMI culture mediums in 24 orifice plates in individual cells/well Co-culture in the case of the NT-2 cells of rimocidin C process.There is 1 μM of HLA-A2 in the splenocyte of HLA-A2 transgenic mices Specific peptide or 1 μ g/ml restructuring His mark ChHer2 albumen (are prepared, by the affinity chromatography system based on nickel in Escherichia coli System purifying) in the case of incubate.Sample was obtained from supernatant after 24 or 72 hours, using mouse IFN-γ Enzyme-linked Immunosorbent Assay Analysis (ELISA) kit tests the presence of interferon-γ (IFN-γ) according to the suggested design of manufacturer.

INF- γ ELISpot are analyzed

In the PBMC of each time point defrosting refrigeration specified, in 37 DEG C of left overnights, then count.2.5 μ of cell M overlap people's HER2/Neu peptides storehouse (11 units overlap 5 amino acid) (EC1 of HER2/Neu present in expression chimeric, EC2 and IC1 domains) and recombinant human il-2 (Invitrogen, Fredrick, MD) stimulate 5 days.Cell is collected, is washed in 1 × PBS Wash twice and count.IFN-γ ELISpot analyses use commercialization canid IFN-γ ELISpot assay kit (R＆D Systems, Minneapolis, MN) carried out according to the scheme of manufacturer.In brief, by the thin of 0.8-2 × 105 Jing stimulations Born of the same parents incubate together with adding IL-2 or single IL-2 (to determine background count) with 2.5 μM of EC1, EC2 or IC1 peptide storehouses.All points Analysis is carried out in duplicate.Flat board is developed the color according to the explanation of manufacturer.Using CTL-Immunospot analyzers (C.T.L, Shaker Heights, OH) spot is counted.The quantity of spot passes through the twice for deducting the amount of speckle not stimulated in hole It is normalized.

Tumor research in Her2 transgenic animals

Six week old FVB/N rat HER2/neu transgenic mices (9-14/group) use 5 × 10⁸The Lm-LLO- of individual CFU ChHer2, ADXS31-164 or Lm- control immunity 6 times.The appearance of spontaneous gland tumor is observed weekly twice, using electronic card Chi measurement tumour, most 52 weeks.When average diameter size reaches 1cm²When cut the tumour of escape, be stored at -20 DEG C In RNAlater °.Impact of the mutation in determine HER2/neu albumen to these tumor escapes, is separated using genomic DNA Kit extracts genomic DNA, and is sequenced.

Effects of the ADXS31-164 to regulatory T cells in spleen and tumour

To mouse subcutaneous (s.c.) implantation 1 × 10⁶Individual NT-2 cells.At the 7th, 14 and 21 days, using 1 × 10⁸Individual CFU's ADXS31-164, LmddA- control carries out immunity or keeps unexposed to them.Tumour and spleen were extracted at the 28th day and is led to Cross facs analysis test CD3⁺/CD4⁺/FoxP3⁺The presence of Treg.In brief, by homogenizing two in C-RPMI culture mediums Spleen between slide and separating Morr. cell.Tumour is shredded using sterile razor blade, and DNA enzymatic (12U/ml) and is dissolved in including The buffer solution digestion of the clostridiopetidase A (2mg/ml) of PBS.After incubation 60min is stirred at room temperature, separated thin by fierce piping and druming Born of the same parents.With RBC lysis buffer splitting erythrocytes, then washed repeatedly with the complete RPMI-1640 culture mediums comprising 10%FBS. After nylon net filter, tumour cell and splenocyte are resuspended in FACS buffer solution (2%FBS/PBS), with AntiCD3 McAb- PerCP-Cy5.5, CD4-FITC, CD25-APC antibody staining, is then changed and anti-Foxp3-PE dyeing thoroughly.Using 4 colors FACS calibur (BD) carry out flow cytometry, using cell quest softwares (BD) analyze datas.

Statistical analysis

Logarithm order Chi-square Test is used for into survival period data, and Student t-test is used for into CTL and elisa assay, it is described Analysis is carried out in triplicate.In these analyses, it is aobvious that p- values less than 0.05 (with * marks) are considered to have statistically Work property.All statistical analysis using Prism softwares V.4.0a (2006) or SPSS softwares V.15.0 (2006) are carried out.For All FVB/N rats HER2/neu transgenic researches, we used 8-14 mouse/group, for all wild types FVB/N are ground Study carefully, we used at least 8 mouse/groups, except as otherwise noted.Long-term tumour in except HER2/neu transgene mouse models Research is outer, and all researchs are repeated at least once more.

Example 1

Produce the listerisa monocytogenes in mjme bacterial strain that secretion is fused to the LLO fragments of Her-2 fragments：ADXS31- 164 structure。

The structure of chimeric HER2/neu genes (ChHer2) is as described above.In brief, ChHer2 genes pass through SOEing PCR methods directly merge two extracellular segments (aa 40-170 and aa359-433) of HER2/neu albumen and a born of the same parents Interior fragment (aa 678-808) is preparing.Chimeric protein has the known people's MHC I class epi-positions of the major part of albumen.From plasmid PAdv138 (it is used to build Lm-LLO-ChHer2) cuts ChHer2 genes and is cloned into LmddA shuttle plasmids, obtains matter Grain pAdv164 (Figure 1A).There are two Main Differences between the two plasmid backbones.1) pAdv138 uses chloramphenicol resistance marker (cat) the external selection of recombinant bacteria is carried out, and pAdv164 has the D-alanine racemase gene of bacillus subtilis (dal), it uses the complementary way of metabolism that the external selection in the LmddA bacterial strains for lacking dal-dat genes and internal plasmid keep Footpath.This vaccine platform is designed and develops into the concern of the antibiotic resistance for solving FDA to being engineered Listeria vaccine strain.2) Different from pAdv138, pAdv164 does not have the prfA gene copies (referring to following sequence and Figure 1A) in plasmid, because this is not It is the internal complementary required of Lmdd bacterial strains.LmddA vaccine strains also lack actA genes (be responsible for the movement of listerial intracellular and Cell-to-cell spread), thus from this skeleton recombinant vaccine strain toxicity ratio from its parental strain Lmdd those are little 100 times.Vaccine based on LmddA is also fast than the vaccine based on Lmdd (less than 48 hours from the removing of immune mouse spleen It is interior).TCA sedimentation cells after carrying out the expression and secretion of the fusion protein tLLO-ChHer2 of bacterial strain since then and growing 8 hours in vitro Lm-LLO-ChHer2 in culture supernatant is suitable (Figure 1B), because being examined by anti-LLO antibody using Western blot analysis Measure～the band of 104KD.The Listeria skeleton bacterial strain for only expressing tLLO is used as negative control.

PAdv164 sequences (7075 base-pairs) (referring to Fig. 1)：

cggagtgtatactggcttactatgttggcactgatgagggtgtcagtgaagtgcttcatgtggcaggagaaaaaagg ctgcaccggtgcgtcagcagaatatgtgatacaggatatattccgcttcctcgctcactgactcgctacgctcggtc gttcgactgcggcgagcggaaatggcttacgaacggggcggagatttcctggaagatgccaggaagatacttaacag ggaagtgagagggccgcggcaaagccgtttttccataggctccgcccccctgacaagcatcacgaaatctgacgctc aaatcagtggtggcgaaacccgacaggactataaagataccaggcgtttccccctggcggctccctcgtgcgctctc ctgttcctgcctttcggtttaccggtgtcattccgctgttatggccgcgtttgtctcattccacgcctgacactcag ttccgggtaggcagttcgctccaagctggactgtatgcacgaaccccccgttcagtccgaccgctgcgccttatccg gtaactatcgtcttgagtccaacccggaaagacatgcaaaagcaccactggcagcagccactggtaattgatttaga ggagttagtcttgaagtcatgcgccggttaaggctaaactgaaaggacaagttttggtgactgcgctcctccaagcc agttacctcggttcaaagagttggtagctcagagaaccttcgaaaaaccgccctgcaaggcggttttttcgttttca gagcaagagattacgcgcagaccaaaacgatctcaagaagatcatcttattaatcagataaaatatttctagccctc ctttgattagtatattcctatcttaaagttacttttatgtggaggcattaacatttgttaatgacgtcaaaaggata gcaagactagaataaagctataaagcaagcatataatattgcgtttcatctttagaagcgaatttcgccaatattat aattatcaaaagagaggggtggcaaacggtatttggcattattaggttaaaaaatgtagaaggagagtgaaacccat gaaaaaaataatgctagtttttattacacttatattagttagtctaccaattgcgcaacaaactgaagcaaaggatg catctgcattcaataaagaaaattcaatttcatccatggcaccaccagcatctccgcctgcaagtcctaagacgcca atcgaaaagaaacacgcggatgaaatcgataagtatatacaaggattggattacaataaaaacaatgtattagtata ccacggagatgcagtgacaaatgtgccgccaagaaaaggttacaaagatggaaatgaatatattgttgtggagaaaa agaagaaatccatcaatcaaaataatgcagacattcaagttgtgaatgcaatttcgagcctaacctatccaggtgct ctcgtaaaagcgaattcggaattagtagaaaatcaaccagatgttctccctgtaaaacgtgattcattaacactcag cattgatttgccaggtatgactaatcaagacaataaaatagttgtaaaaaatgccactaaatcaaacgttaacaacg cagtaaatacattagtggaaagatggaatgaaaaatatgctcaagcttatccaaatgtaagtgcaaaaattgattat gatgacgaaatggcttacagtgaatcacaattaattgcgaaatttggtacagcatttaaagctgtaaataatagctt gaatgtaaacttcggcgcaatcagtgaagggaaaatgcaagaagaagtcattagttttaaacaaatttactataacg tgaatgttaatgaacctacaagaccttccagatttttcggcaaagctgttactaaagagcagttgcaagcgcttgga gtgaatgcagaaaatcctcctgcatatatctcaagtgtggcgtatggccgtcaagtttatttgaaattatcaactaa ttcccatagtactaaagtaaaagctgcttttgatgctgccgtaagcggaaaatctgtctcaggtgatgtagaactaa caaatatcatcaaaaattcttccttcaaagccgtaatttacggaggttccgcaaaagatgaagttcaaatcatcgac ggcaacctcggagacttacgcgatattttgaaaaaaggcgctacttttaatcgagaaacaccaggagttcccattgc ttatacaacaaacttcctaaaagacaatgaattagctgttattaaaaacaactcagaatatattgaaacaacttcaa aagcttatacagatggaaaaattaacatcgatcactctggaggatacgttgctcaattcaacatttcttgggatgaa gtaaattatgatctcgagacccacctggacatgctccgccacctctaccagggctgccaggtggtgcagggaaacct ggaactcacctacctgcccaccaatgccagcctgtccttcctgcaggatatccaggaggtgcagggctacgtgctca tcgctcacaaccaagtgaggcaggtcccactgcagaggctgcggattgtgcgaggcacccagctctttgaggacaac tatgccctggccgtgctagacaatggagacccgctgaacaataccacccctgtcacaggggcctccccaggaggcct gcgggagctgcagcttcgaagcctcacagagatcttgaaaggaggggtcttgatccagcggaacccccagctctgct accaggacacgattttgtggaagaatatccaggagtttgctggctgcaagaagatctttgggagcctggcatttctg ccggagagctttgatggggacccagcctccaacactgccccgctccagccagagcagctccaagtgtttgagactct ggaagagatcacaggttacctatacatctcagcatggccggacagcctgcctgacctcagcgtcttccagaacctgc aagtaatccggggacgaattctgcacaatggcgcctactcgctgaccctgcaagggctgggcatcagctggctgggg ctgcgctcactgagggaactgggcagtggactggccctcatccaccataacacccacctctgcttcgtgcacacggt gccctgggaccagctctttcggaacccgcaccaagctctgctccacactgccaaccggccagaggacgagtgtgtgg gcgagggcctggcctgccaccagctgtgcgcccgagggcagcagaagatccggaagtacacgatgcggagactgctg caggaaacggagctggtggagccgctgacacctagcggagcgatgcccaaccaggcgcagatgcggatcctgaaaga gacggagctgaggaaggtgaaggtgcttggatctggcgcttttggcacagtctacaagggcatctggatccctgatg gggagaatgtgaaaattccagtggccatcaaagtgttgagggaaaacacatcccccaaagccaacaaagaaatctta gacgaagcatacgtgatggctggtgtgggctccccatatgtctcccgccttctgggcatctgcctgacatccacggt gcagctggtgacacagcttatgccctatggctgcctcttagactaatctagacccgggccactaactcaacgctagt agtggatttaatcccaaatgagccaacagaaccagaaccagaaacagaacaagtaacattggagttagaaatggaag aagaaaaaagcaatgatttcgtgtgaataatgcacgaaatcattgcttatttttttaaaaagcgatatactagatat aacgaaacaacgaactgaataaagaatacaaaaaaagagccacgaccagttaaagcctgagaaactttaactgcgag ccttaattgattaccaccaatcaattaaagaagtcgagacccaaaatttggtaaagtatttaattactttattaatc agatacttaaatatctgtaaacccattatatcgggtttttgaggggatttcaagtctttaagaagataccaggcaat caattaagaaaaacttagttgattgccttttttgttgtgattcaactttgatcgtagcttctaactaattaattttc gtaagaaaggagaacagctgaatgaatatcccttttgttgtagaaactgtgcttcatgacggcttgttaaagtacaa atttaaaaatagtaaaattcgctcaatcactaccaagccaggtaaaagtaaaggggctatttttgcgtatcgctcaa aaaaaagcatgattggcggacgtggcgttgttctgacttccgaagaagcgattcacgaaaatcaagatacatttacg cattggacaccaaacgtttatcgttatggtacgtatgcagacgaaaaccgttcatacactaaaggacattctgaaaa caatttaagacaaatcaataccttctttattgattttgatattcacacggaaaaagaaactatttcagcaagcgata ttttaacaacagctattgatttaggttttatgcctacgttaattatcaaatctgataaaggttatcaagcatatttt gttttagaaacgccagtctatgtgacttcaaaatcagaatttaaatctgtcaaagcagccaaaataatctcgcaaaa tatccgagaatattttggaaagtctttgccagttgatctaacgtgcaatcattttgggattgctcgtataccaagaa cggacaatgtagaattttttgatcccaattaccgttattctttcaaagaatggcaagattggtctttcaaacaaaca gataataagggctttactcgttcaagtctaacggttttaagcggtacagaaggcaaaaaacaagtagatgaaccctg gtttaatctcttattgcacgaaacgaaattttcaggagaaaagggtttagtagggcgcaatagcgttatgtttaccc tctctttagcctactttagttcaggctattcaatcgaaacgtgcgaatataatatgtttgagtttaataatcgatta gatcaacccttagaagaaaaagaagtaatcaaaattgttagaagtgcctattcagaaaactatcaaggggctaatag ggaatacattaccattctttgcaaagcttgggtatcaagtgatttaaccagtaaagatttatttgtccgtcaagggt ggtttaaattcaagaaaaaaagaagcgaacgtcaacgtgttcatttgtcagaatggaaagaagatttaatggcttat attagcgaaaaaagcgatgtatacaagccttatttagcgacgaccaaaaaagagattagagaagtgctaggcattcc tgaacggacattagataaattgctgaaggtactgaaggcgaatcaggaaattttctttaagattaaaccaggaagaa atggtggcattcaacttgctagtgttaaatcattgttgctatcgatcattaaattaaaaaaagaagaacgagaaagc tatataaaggcgctgacagcttcgtttaatttagaacgtacatttattcaagaaactctaaacaaattggcagaacg ccccaaaacggacccacaactcgatttgtttagctacgatacaggctgaaaataaaacccgcactatgccattacat ttatatctatgatacgtgtttgtttttctttgctggctagcttaattgcttatatttacctgcaataaaggatttct tacttccattatactcccattttccaaaaacatacggggaacacgggaacttattgtacaggccacctcatagttaa tggtttcgagccttcctgcaatctcatccatggaaatatattcatccccctgccggcctattaatgtgacttttgtg cccggcggatattcctgatccagctccaccataaattggtccatgcaaattcggccggcaattttcaggcgttttcc cttcacaaggatgtcggtccctttcaattttcggagccagccgtccgcatagcctacaggcaccgtcccgatccatg tgtctttttccgctgtgtactcggctccgtagctgacgctctcgccttttctgatcagtttgacatgtgacagtgtc gaatgcagggtaaatgccggacgcagctgaaacggtatctcgtccgacatgtcagcagacgggcgaaggccatacat gccgatgccgaatctgactgcattaaaaaagccttttttcagccggagtccagcggcgctgttcgcgcagtggacca ttagattctttaacggcagcggagcaatcagctctttaaagcgctcaaactgcattaagaaatagcctctttctttt tcatccgctgtcgcaaaatgggtaaatacccctttgcactttaaacgagggttgcggtcaagaattgccatcacgtt ctgaacttcttcctctgtttttacaccaagtctgttcatccccgtatcgaccttcagatgaaaatgaagagaacctt ttttcgtgtggcgggctgcctcctgaagccattcaacagaataacctgttaaggtcacgtcatactcagcagcgatt gccacatactccgggggaaccgcgccaagcaccaatataggcgccttcaatccctttttgcgcagtgaaatcgcttc atccaaaatggccacggccaagcatgaagcacctgcgtcaagagcagcctttgctgtttctgcatcaccatgcccgt aggcgtttgctttcacaactgccatcaagtggacatgttcaccgatatgttttttcatattgctgacattttccttt atcgcggacaagtcaatttccgcccacgtatctctgtaaaaaggttttgtgctcatggaaaactcctctcttttttc agaaaatcccagtacgtaattaagtatttgagaattaattttatattgattaatactaagtttacccagttttcacc taaaaaacaaatgatgagataatagctccaaaggctaaagaggactataccaactatttgttaattaa(SED ID NO:53)

Example 2：The immunogenicity of ADXS31-164 is as LM-LLO-ChHER2.

ADXS31-164 is produced the immunogene of anti-HER2/neu specific cytotoxic t lymphocytes in the analysis of standard CTL Property is compared with Lm-LLO-ChHer2 vaccines.Two kinds of vaccines cause the HER2/neu antigens expressed 3T3/neu target cells Strong but similar cytotoxic T cell response.Therefore, the Li Si of the Her2 intracellular fragments of LLO is fused to using only expression The mouse of special bacterial immunity shows the lytic activity lower than the chimera comprising multiple MHC I class epi-positions.Unexposed dynamic CTL activity (Fig. 2A) is not detected by the mouse of thing or uncorrelated Listeria vaccine injection.ADXS31-164 also can stimulate The splenocyte secretion of gamma-IFN (Fig. 2 B) of wild type FVB/N mouse.In the NT-2 cells processed with mitomycin C, (expression is high for this The HER2/neu antigens of level) co-culture these cells culture supernatant in detect (Fig. 5 C).

Proper treatment and the presentation of ADXS31-164 immunity descendant's MHC I class epi-positions are tested in HLA-A2 mouse.Jing The splenocyte of the HLA-A2 transgenic animals of immunity and extracellular (the HLYQGCQVV SEQ ID NO for being located at HER2/neu molecules:11 Or KIFGSLAFL SEQ ID NO:Or intracellular (RLLQETELV SEQ ID NO 12):13) HLA-A2 of the plotting in domain limits table The corresponding peptide in position is incubated altogether 72 hours (Fig. 2 C).Recombinant C hHer2 albumen is as positive control, uncorrelated peptide or without peptide as the moon Property control.The data display of the experiment, ADXS31-164 can cause the anti-of people's epi-position of the not same area for being pointed to target antigen HER2/neu specific immune responses.

Example 3：ADXS31-164 suppresses the morbidity of spontaneous gland tumor more more effective than LM-LLO-ChHER2.

The GVT of ADXS31-164 is compared with the Lm-LLO-ChHer2 in HER2/neu transgenic animals, should HER2/neu transgenic animals suffer from the spontaneous gland tumor of slow growth in 20-25 week old.It is all using uncorrelated Li Si The animal of special bacterium control vaccine immunity suffers from tumor of breast in 21-25 is all and put to death before the 33rd week.By contrast, Li Si Special bacterium-HER2/neu recombinant vaccines make the formation of tumor of breast significantly delay.ADXS31-164 epidemic diseases at the 45th week, more than 50% The mouse (9 5 for merely hitting) of seedling inoculation still without tumour, by contrast, is only had using the mouse of Lm-LLO-ChHer2 immunity 25%.At the 52nd week, using 2 in 8 mouse of ADXS31-164 immunity still without tumour, and other experimental groups was all little Mouse has died from disease (Fig. 3).These results indicate that it is higher despite attenuation degree, but ADXS31-164 prevents HER2/neu The morbidity of the spontaneous gland tumor of transgenic animals is more more effective than Lm-LLO-ChHer2.

Example 4：The mutation of HER2/NEU genes when ADXS31-164 is immune.

The mutation of the MHC I class epi-positions of HER2/neu has been considered in small fragment vaccine or trastuzumab (Herceptin) It is responsible for tumor escape during (a kind of monoclonal antibody of the epi-position in extracellular domain of targeting HER2/neu) immunity.To assess the work With, the escape tumour from transgenic animals extracts genomic material, and to neu bases in the tumour of chimeric or control vaccine immunity The homologous segment of cause is sequenced.Mutation is not observed in the HER2/neu genes of the tumor sample of any vaccine inoculation, secretly Show that presence substitutes escape mechanism (data are not shown).

Example 5：ADXS31-164 substantially reduces intra-tumor regulatory T cells.

To illustrate impacts of the ADXS31-164 to regulatory T cells frequency in spleen and tumour, NT-2 tumour cells are planted Enter mouse.Separating Morr. cell and tumour endolymph cell and Treg is dyeed after three immunity, Treg is defined as CD3⁺/CD4⁺/ CD25⁺/FoxP3⁺Cell, but when individually analysis, FoxP3 or CD25 marks obtain similar result.As a result show, with Uncorrelated Listeria vaccine or unexposed animal are compared, and ADXS31-164 immunity is on the frequency of Treg in spleen without impact (referring to Fig. 4).By contrast, presence of the Listeria vaccine immunity to Treg in tumour has a huge impact (Fig. 5 A).And All CD3 on average in untreated tumour⁺The 19.0% of T cell is Treg, and for uncorrelated vaccine, the frequency is reduced to 4.2%, for ADXS31-164 is reduced to 3.4%, the frequency of intra-tumor Treg reduces by 5 times (Fig. 5 B).Arbitrary LmddA vaccines are controlled The frequency of intra-tumor Treg reduces that the difference of tumor size can not be attributed in the mouse for the treatment of.In representative experiment, ADXS31- The tumour of the mouse of 164 immunity is significantly less than [average diameter (mm) ± SD, 6.71 ± 0.43, n=5] untreated mouse (8.69 ± 0.98, n=5, p<0.01) or uncorrelated vaccine therapy mouse (8.41 ± 1.47, n=5, p=0.04) it is swollen Knurl, and last two groups of the significant difference (p=0.73) statistically for not showing tumor size.At LmddA vaccines The decline of Treg frequencies in the tumour of reason raises intra-tumor CD8/Treg ratio, and hint can be obtained after LmddA vaccine immunities More favourable tumor microenvironment.However, the vaccine (ADXS31-164) of only expression target antigen HER2/neu can slow down tumour Growth, shows that the reduction of Treg has effect in the case of only there is antigentic specificity response in tumour.

Example 6：The chimeric Listeria vaccines of expression HER-2 are not introduced into escape mutant

Collect the mouse of difference vaccine such as Lm-LLO-138, LmddA164 and uncorrelated vaccine Lm-LLO-NY immunity Tumor sample.From these Sample Purification on Single DNA, the corresponding DNA fragmentation of amplification HER2/neu regions IC1, EC1 and EC2, and be sequenced with Determine whether there is any immunologic escape mutation.The sequence alignment of each DNA is carried out using CLUSTALW.Analysis result shows, Without mutation in the DNA sequence dna collected from tumour.The labor of these sequences is as follows.

The comparison (975-1029bp of HER2/neu) of EC2

With 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

With 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

With 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

With 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

With reference to

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

With 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

With 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

With 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

With 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

With 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

With 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-------------------------------------

The comparison (2114-3042bp of HER2/neu) of IC1

With 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

With 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

With 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

With reference to CTCCTAAAGCCAACAAAGAAATTCTAGATGAAGCGTATGTGATGGCTGGTGTGGGT TCTC (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

With 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

With 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

With 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

With 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

With 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

With 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

With 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

With reference to

CCAAACCTTACGATGGAATCCCAGCCCGGGAGATCCCTGATTTGCTGGAGAAGGGAGAA(SEQ ID NO:36)

Lm-LLO-NY-1

CCAAACCTTACGATGGAATCCCAGCCCGGGAGATCCCTGATTTGCTGGAGAAGGGAGAA

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

With 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

With 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

With 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

The comparison (399-758bp of HER2/neu) of EC1

With 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

With 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

With 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

With 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

With 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：The periphery immunity of ADXS31-164 can delay the growth of metastatic breast cancer cell line in brain.

Using ADXS31-164 or uncorrelated Lm- control vaccine IP immune mouses, then 5,000 expression of encephalic implantation is glimmering The EMT6-Luc tumour cells (Fig. 6 C) of light element enzyme and low-level HER2/neu.Different time after inoculation passes through anesthetized mice In vitro Imaging: Monitoring tumour.The 8th day after tumor inoculation, the tumour in all control-animals is detected, but in ADXS31-164 Mouse do not show any detectable tumour (Fig. 6 A and B).ADXS31-164 can substantially delay the morbidity of these tumours, Because it is all that all mouse after tumor inoculation in the 11st day negative control group have died from tumour, but ADXS31-164 groups Mouse still survives, and only shows the sign of a small amount of tumour growth.These result strong hints, the periphery of ADXS31-164 The immune response that administration is obtained may reach central nervous system, and the vaccine based on LmddA can have treatment cns tumor Potential use.

Example 8：By ADXS31-164 immunization therapy canid osteosarcoma.

Canid osteosarcoma is the cancer of long (leg) bone, is the primary killers of towser of the age more than 10 years old.Standard is controlled Treatment is amputation immediately after diagnosis, then chemotherapy.However, invariably cancer metastasis is to lung.Deposited with untreated 6-12 month Current is compared, and by chemotherapy dog can be made to survive about 18 months.It is believed that HER2 antigens are present in up to 50% osteosarcoma. ADXS31-164 forms immune attack to the cell for expressing this antigen, and is developed for treating human breast carcinoma.

Histodiagnosis expresses the dog of the evidence of HER2/neu for osteosarcoma and with malignant cell and meets eligibility.

Canid osteosarcoma is tested

In first team, amputation is carried out, then receive a few wheel chemotherapeutic treatments.3 doses of Her-2 vaccines are subsequently applied, between 6 months Strengthen after the phase or do not strengthen.

All dogs receive the Carboplatin in patients of 4 weeks.The surrounding after the administration of last carboplatin, dog received an ADXS- per three weeks HER2, is administered 3 times altogether.1st group (3 dogs) receives 1 × 10⁸Individual CFU/ agent, the 2nd group (3 dogs) is per only acceptance 5 × 10⁸It is individual CFU/ agent, the 3rd group (3 dogs) receives 1 × 10⁹Individual CFU/ agent.If it is observed that potential dose limiting toxicity, then add in group Enter other dog to collect more data.Therefore, 9-18 dog can be treated in Primary Study.

In the second team, repetitive therapy identical with first team except for the difference that only applies single dose epidemic disease before chemotherapy (before 1 month) Seedling, is administered 4 times altogether.

In addition, applying single dose to two teams within latter month in chemotherapy.

Example 9：Assessment ADXS-cHER2 is in the osteosarcomatous companion dog of canid with HER2/NEU overexpression 1 phase dose escalation study of security

Carrying out early stage I phase dose escalation study can safely and effectively stimulate the tumour with osteosarcomatous dog special to determine The dosage of the listerisa monocytogenes in mjme of expression people's HER2/neu recombinant vaccines of specific immunological.Routine is collected due to bosom Doubt or confirm to be supplied to PennVet to carry out the tumour of all dogs of amputation with OSA, and carry out histopathological evaluation with true Recognize the diagnosis of OSA.Additionally, assess the tumor biopsy of all dogs by IHC and Western blot analysis, to determine that tumour is No expression HER2/neu.Only histodiagnosis is OSA and the dog of the evidence with malignant cell expression HER2/neu just accords with Close eligibility.The single cell suspension of the tumor tissues gathered in operation is refrigerated, and is used as in chromium release analysis autologous swollen Knurl target, to determine antineoplastic immune.

It has been selected in most 18 with limbs OSA and confirmation form has reached the privately owned dog (Fig. 7) of Her2-neu.When selected (most Afterwards 3 weeks after Carboplatin in patients), all dogs receive basic clinical labororatory's test, including CBC (CBC), chemistry sieve Select (CS) and urinalysis (UA) and the baseline estimate of cardiac function is carried out by ECHOCARDIOGRAPH and specific heart is determined Property Troponin I (cTnI) level.Lung metastases are determined whether there is by breast radiation development.Only without the dog of Lung metastases evidence Just meet research eligibility.When selected, collect PMBC (PBMC) to assess the baseline water of antineoplastic immune Put down (referring to the assessment of antineoplastic immune).Additionally, blood sampling assessment baseline immunologic function, to guarantee them no longer by carboplatin immunity suppression System.Only there is fully functional immune dog just to meet the qualification for receiving Listeria vaccine.

Lm restructuring administration and data acquisition

It is inoculated with to all dogs using single ADXS31-164ADXS31-164 recombinant vaccines.Three weeks after the administration of last carboplatin First time Lm-huHer2-neu vaccine is given, was hereafter given once per three weeks, 3 times (Fig. 7) is administered altogether.

1st group (3 dogs) receives 1 × 10⁸ADXS31-164 (Lm- people the is fitted together to HER2/neu) vaccine of individual CFU/ agent, the 2nd Group (3 dogs) is per only acceptance 5 × 10⁸Individual CFU/ agent, the 3rd group (3 dogs) receives 1 × 10⁹Individual CFU/ agent and 3.3 × 10⁹Individual CFU/ Agent (1 dog).Restructuring Lm Jing are applied for 30 minutes with slow intravenous infusion.Dosage selected by 1st group is to be fitted together in mouse The determination safe dose of ADXS31-164 recombinants.In people, the non-toxic of Lovaxin C is only than the dosage of determination in mouse High an order of magnitude, and the dosage is the dosage of the 3rd group of assessment in the pre-stage test.

When Lm is applied, the evidence of the general adverse effect of dog is monitored.During being transfused, heart rate is monitored by ECG With the rhythm of the heart and recording respiration rate.In addition, monitoring heart injury (Fig. 8) using ultrasonic wave and by determining troponin I levels. After infusion, dog is monitored 48 hours closely.Using the Vital Sense of MiniMitterRespironics manufactures after infusion The continuous monitoring core body temperature of continuous body temperature monitoring system (veterinary clinic test center (VCIC) common instrument)<12 hours.Front 6 Hour per hour and thereafter pulse frequency, pulse rhythm and pulse condition, respiratory rate and respiratory effort were monitored and recorded per 4 hours, And blood pressure and body temperature (Fig. 9).Record it is all meet immunostimulating symptom, as needed using fluid, anodyne, antemetic Severe reaction is controlled with antihistaminic.Observation six times daily of all dogs, record the sign of any poisonous effect of recombinant, including Uncomfortable, drowsiness, Nausea and vomiting and diarrhoea.24,48 and 72 hours collection blood samples enter after the vaccine inoculation of first time ADXS31-164 Row culture, to assess systemic administration after Lm removing.

The assessment of antineoplastic immune

Last carboplatin administration after three weeks, dog receive routine clinical inspection and baseline blood work, including CBC, CS, UA and CTnI levels.The baseline estimate of antineoplastic immune is carried out in time collection PBMC.In each vaccine inoculation and final vaccine The Immunological evaluation that three weeks are repeated after inoculation.(ELISpot and qRT-PCR) and CTL are generated by CFSE propagation, cell factor Analytic approach analyzing HER2/neu specific T-cells responses of the PBMC to autologous tumor target, summarize (Figure 12) by following article.

As a result

So far, we have carried out 41 ADXS31-164 infusions altogether in 16 dogs.

The scope of ADXS31-164 dosage is 1 × 10⁸、5×10⁸、1×10⁹With 3.3 × 10⁹Individual CFU.

The standard practice instructions of vaccine administration

The standard practice instructions that exploitation ADXS31-164 is applied.In the previous hour of vaccine inoculation, patient passes through intramuscular injection Receive 2mg/kg diphenhydramines, and slow intravenous injection 0.2mg/kg Ondansetrons.Vaccine is maintained at -80 DEG C, to trouble Thaw when person applies.It is dissolved in Jing in 200ml 0.9%NaCl and applies for 30 minutes.Then rinse defeated with 30ml Plasmalyte Note pipeline.Send dog back to cage, a course for the treatment of applies Amoxicillin (72 hours after vaccine inoculation start) within three days, treatment in 7 days Journey applies liver replenishers (S- Adenosyl-Methionines), to help cell growth and reparation.

The Primary Endpoint of research is to determine the maximum tolerated dose of ADXS31-164.

Dog of the body weight in the range of 25kg to 67kg can well tolerate up to 3.3 × 10⁹Dosage.The pair of all reports Effect is I level toxicity, and maximum tolerated dose not yet reaches.Generally there is side effect in the 2-4 hours of vaccine administration.Hyperpyrexia Generally disappeared with maintaining the isotonic fluid 2-4 hours of speed (4ml/kg/ hours) intravenous delivery.Heating wherein reaches In the case of two kinds of 104.7 and Geng Gao, single subcutaneous injection Carprofen makes temperature recovery normal in 1-2 hours.Nausea and vomit Tell typically self limiting, but in the case of recorded repeatedly outbreak wherein, apply 1mg/kg Cerenia, this to prevent into The nausea and vomiting of one step is highly effective.In 48 hours of vaccine administration, altogether there are slight, I levels and rises in the liver enzyme of 5 dogs Height, this disappears after vaccine inoculation one week.

Listerial removing

After blood culture is carried out to all 16 dogs vaccinated so far, 24 hours any one after vaccine inoculation Listeria is not detected by the peripheral circulation of dog.Listerial stream in the urine and excrement of vaccine inoculation dog is not assessed Go out.

The secondary endpoints of research are progresson free survival phase and overall survival phase.ADXS31- is applied after amputation and 4 doses of carboplatins Have observed that when 164 with overall survival phase advantage of the osteosarcomatous dog with conspicuousness statistically.Front two doses of groups (6 Dog) earlier results show, compared with its owner selects to be not involved in 6 dogs for testing but tracking survival period, receive ADXS31- 164 dog has significant survival advantage (p=0.003) (Figure 13).The mean survival time of not vaccinated dog is 239.5 days.The mean survival time of vaccinated dog is not reached.When analysis includes all dogs in treatment of purpose group, this Also set up.

Therefore, not evidence show and significant short-term or long-term secondary work are caused to angiocarpy, hematopoiesis, liver or renal system With.Additionally, in the case where there is minimal residual disease, the administration of ADXS31-164 can delay/prevent metastatic disease, and extend The overall survival phase with the positive osteosarcomatous dogs of HER2/neu.

Example 10：

1 clinical trial phase of ADXS31-164 is assessed in the spontaneous canid model of osteosarcoma (OSA)

Material and method

Vaccine is manufactured

The design and generation of ADXS31-164.In brief, with the pADV plasmids for carrying chimeric people's HER2/neu constructs The dal dat actA mutants which hads of transfection listerisa monocytogenes in mjme (Lm).The construct includes people HER2/neu 2 extracellular domains (EC1 and EC2) of molecule and an Intracellular domain (IC1), people HER2/neu molecules are fused to truncate comprising major part Listeriolysin O construct HLA-A2 limit immunodominant epitope.Transferring plasmid also includes bacillus p60dal Gene, and maintained in mutant Lm by auxotroph complementation.There is no bacterial resistance box.Vaccine by Vibalogics GmbH (Cuxhaven, Germany) are manufactured, and -80 DEG C are stored in before the use.

Histopathology, by stages and immunohistochemistry

The histopathological evaluation of all primary limbs osteosarcoma tumors by committee's certification veterinary pathologist (J.E.) carry out.Tumour is described as into Gegenbaur's cell tumour, chondroblast tumour, fibroblast according to histologic characteristics to swell Knurl and distensibility of blood vessel tumour.It is swollen to primary according to mitotic index, core polymorphy and matrix and the amount of necrosis Knurl is scored.Histological score is converted into rank (I, II or III).

For HER2/neu dyeing, 5 microns thick serial section of formalin fix, decalcification, paraffin-embedded tissue are pacified It is attached on electronegative slide.Section is heated 20 minutes at 80 DEG C, in immersion Pro Par (clearant), in ethanol Rehydration.Antigen retrieval is carried out by boiling section in sodium citrate buffer solution (pH～9.0).Sealed using 3% hydrogen peroxide Close endogenous peroxydase.Using rabbit-anti people's HER2/neu antibody (Neu (c-18):sc-284,Santa Cruz ) or rabbit igg isotype (general negative control sera (Universal Negative Control Biotecnology Serum), NC498, Biocare Medical) dyeed.Using the system (Universal of universal chain Avidin-Biotin 2 Streptavadin-Biotin2System) the antibody that (DAKO/LSAB2, HRP) detection is combined.Tissue is joined with 3,3'- diaminourea Aniline solution (DAKO) is dyeed, and uses haematoxylin redyeing color.Using the calibrated upright microscopes of Nikon E600infinity Observation slide.Bright vision image is obtained using Nikon Digital Sight DS-Fi1 color cameras, and uses NIS- Element BR3.0 carry out graphical analysis.By the virologist (J.E.) of committee's certification according to the HER2/ of neoplastic cell Neu dyeing percentages (<10%=1,10%-50%=2,>50%=3) and HER2/neu staining powers (it is weak=1, in=2, By force=3) the HER2/neu positives of histotomy are estimated and are scored.The score of each histotomy is based in 10hpf points The cell of analysis.The tumour cell percentage and HER2/neu dyeing that the HER2/neu scores of merging pass through HER2/neu stained positives Two single scores that intensity is given are multiplied and obtain.The dog ability that only tumour cell of HER2/neu stained positives is more than 10% Pass Test eligibility.

Criterion of acceptability and clinical trial design

Histopathology and immunohistochemical diagnosis are received by amputation for the dog of HER2/neu positive OSA or protect limb The primary tumor that operation is carried out is extractd, and receives 4 doses of 300mg/m²Carboplatin as adjuvant chemotherapy, carboplatin was given per 3 weeks (or if there is bone marrow suppression, then per 4 weeks once) is given once, such dog meets screening qualification.Three after last Carboplatin in patients Week screens to dog.Carry out comprehensive health check-up, CBC (CBC), Chemical Screening (CS) and urinalysis (UA) to determine General health status.Respectively using the analysis of fluidic cell neutrophil leucocyte oxidative burst and the lymphocyte of mitogen induction Proliferation assay is testing substantially congenital and adaptive immunity function.By electrocardiography, ECHOCARDIOGRAPH and serum Cardiac troponin I proficiency assessment baseline heart state.Carry out breast radiation to develop to determine the presence (ginseng of Lung metastases disease See Figure 14 B).Only it is found that whole body health, congenital and adaptive immunity it is fully functional, without potential heart disease evidence and Those dogs of apneumia metastatic disease evidence just meet eligibility.In the course of the study dead dog receives postmortem.Record The presence of metastatic disease and position, carry out histopathology and immunohistochemical analysis, to assess metastasis (metastases) in HER2/neu is expressed.

Immunoassay

Neutrophil leucocyte oxidative burst is analyzed.Using 0.83%NH₄Red blood cell in Cl cracking liquaemin anticoagulations, remaining Leucocyte wash twice in 1 × PBS.With 15 μ g/ml dihydrorbodamine 123 (DHR-123；Molecular Probes, Grand Island, NY) mark cell, and with 3nM phorbol -12- myristoyl -13- acetic acid esters (PMA, Sigma, St.Louis, MO) activate 30 minutes at 37 DEG C.Cell is placed in 15 minutes on ice, flow cytometry is then carried out. Cell is obtained on FACS Canto cell counters (BD Biosciences, San Jose, CA), and using FloJo softwares (Treestar, San Carlos, CA) is analyzed.

Lymphocyte proliferation assays.By density centrifugation from liquaemin anticoagulated whole blood separating periphery blood monocytic cell (PBMC).PBMC is washed twice and counted in 1 × PBS.Cells are marked with 5 μM of CFSE, and with 1.25 μM of concanavalin A stimulates 5 days at 37 DEG C.Cell is collected, is washed twice in FACS buffer solution, the rat anti-canidae animal CD4 being conjugated with APC The rat anti-canidae animal CD8 antibody Serotec, Raleigh, NC being conjugated with PE) mark, and by flow cytometry. For Analysis of Immunological Function, use from the peripheral blood of healthy colony dog (IACUC#804197) collection as positive control.

T cell subgroup is analyzed.Before baseline, each vaccine inoculation, again by stages when and thereafter per 2 months gather PBMC, carries out CD4 and the analysis of cd8 t cell subgroup.In brief, thaw refrigeration cell, FACS buffer solution (1 × PBS, 0.2%BSA components V and 4mM sodium azide) in wash twice, then with mouse anti-canidae animal CD3, PE mark rat resist The anti-dog CD4 (Serotec, Raleigh, NC) of rat of dog CD8 or Alexa mark carries out padding.In Row cytometric acquisition Use vital stain 7-ADD Incubate cells immediately before.By the fluidic cell determined using Cell Dyn 3700CS blood analysers Percentage and total lymphocyte count are calculating CD4⁺And CD8⁺T cell sum.

Vaccine administration

Before vaccine inoculation, dog receives intravenous administration 5HT3 antagonist Ondansetrons (0.2mg/kg) and intramuscular administration H1 receptor blocking pharmacon diphenhydramines (2mg/kg), is respectively used to prevention nausea and allergy.Using standard 3+3 clinical trial design. ADXS31-164 is applied by following dosage；1st group (2 × 10⁸Individual CFU), the 2nd group (5 × 10⁸Individual CFU), the 3rd group (1 × 10⁹It is individual ) and the 4th group (3.3 × 10 CFU⁹Individual CFU).In 100ml 0.9%NaCl (the 1st and 2 group) and 200ml 0.9%NaCl the (the 3rd With 4 groups) middle dilution ADXS31-164, and Jing intravenous administrations in 30 minutes.Monitor temperature pulse respiration per hour after infusion Rate, heart rate and the rhythm of the heart (by EKG) and blood pressure.In the case that wherein body temperature is more than 103 ℉, give dog intravenous administration 4ml/ Kg/h Plasmalyte, until body temperature is down under 103 ℉.Drowsiness, the n or V sign of dog are monitored per hour.24 Hour and vaccine inoculation one week after gather blood sample, to assess any change of hematology or biochemical parameters, and in vaccine Carry out blood culture within 24 hours after inoculation, to determine blood flow in bacterium living lasting existence.72 hours after vaccine inoculation, institute There is dog to receive Amoxicillin and the S-adenosylmethionine (SAMe) of Low doses, to kill the Listeria of any residual, and Antioxidant is provided for liver to support.

The owner of the dog of at least 5 months non-metastatic diseases is optionally connected after final vaccine in initial series are received receives 1×10⁹The booster vaccine of the standard dose of individual CFU.Booster vaccine is applied as described above, and as described above after infusion Dog is monitored.

Toxicity

According to Veterinary Co-operative Oncology Group-Common Terminology Criteria for Adverse Events (VCOG-CTCAE) (the Essential Terms marks of animal doctor's cooperation oncology group-adverse events It is accurate) toxicity is classified.3 weeks and passed through per 2 months thereafter in baseline, each vaccine inoculation, after final vaccine inoculation Continuous ECG, echocardiogram and serum cardiac troponin I level carry out the assessment of cardiac toxic, until death.Commented The parameter estimated includes left room Fractional shortening (LVFS) and LVED (LVIDd) and left room end systolic diameter (LVIDs).LVIDd and LVIDs are normalized into body weight, to consider dog in wide scope body size.

ELISpot is analyzed

In the PBMC of each time point defrosting refrigeration specified, in 37 DEG C of left overnights, then count.2.5 μ of cell M overlap people's HER2/Neu peptides storehouse (11 units overlap 5 amino acid) (EC1 of HER2/Neu present in expression chimeric, EC2 and IC1 domains) and recombinant human il-2 (Invitrogen, Fredrick, MD) stimulate 5 days.Cell is collected, is washed in 1 × PBS Wash twice and count.IFN-γ ELISpot analyses use commercialization canid IFN-γ ELISpot assay kit (R＆D Systems, Minneapolis, MN) carried out according to the scheme of manufacturer.In brief, by 0.8-2 × 10⁵It is thin that individual Jing stimulates Born of the same parents incubate together with adding IL-2 or single IL-2 (to determine background count) with 2.5 μM of EC1, EC2 or IC1 peptide storehouses.All points Analysis is carried out in duplicate.Flat board is developed the color according to the explanation of manufacturer.Using CTL-Immunospot analyzers (C.T.L, ShakerHeights, OH) spot is counted.

Main and Minor consequence is determined

Transfer time (TTM) is with amputation and the Time Calculation between metastatic disease occurs.OSA specificity survival period with cut Time Calculation between limb and death.Patient to dying from uncorrelated reason examines in death.

As a result

18 dogs for reaching criterion of acceptability are selected in the Phase I clinical trial.Record the age, kind, sex, knub position, Subclass, grade and HER2/neu states (table 4).Using standard 3+3 clinical trial design.ADXS31-164 is applied by following dosage With；1st group：2×10⁸Individual CFU (n=3), the 2nd group：5×10⁸Individual CFU (n=3), the 3rd group：1×10⁹Individual CFU (n=9) and 4th group：3×10⁹Individual CFU (n=3).For caring in nursing, five other to be accredited as with pre-existing lung in screening The dog of metastatic disease also receives ADXS31-164 (table 4).Four in these dogs exist>50% going to live in the household of one's in-laws on getting married from primary tumor Dye with strong HER2/neu in natural disposition cell.Screening when, three in these dogs have multiple Lung metastases tubercles, two Dog has single metastatic tubercle.Dog with multiple Lung neoplasms is exited and is ground per only receiving before progression of disease a vaccinating agent Studying carefully carries out replacement therapy.With single tubercle two dogs are per only receiving whole three vaccinating agent.With pre-existing metastatic The dog of disease receives 1 × 10⁹Individual CFU (n=3) or 3 × 10⁹Individual CFU (n=2) ADXS31-164 (table 5).

Figure 15 shows the timeline schematic diagram of 1 clinical trial phase, wherein three vaccine inoculation is in amputation and follow-up chemotherapy After apply.

Table 4：The feature interpretation and tumoral character of selected dog

Table 5：The feature interpretation and tumour of the dog with pre-existing metastatic disease treated for caring in nursing Feature

As a result

Security and the security of all 23 vaccine inoculation dogs of toxicity=assessment.All dogs can tolerate well ADXS31-164 is applied, and is only observing instantaneous low level toxicity (table 6) vaccine inoculation day.In all groups, ADXS31-164 Occur having within 4 hours after administration the body temperature of conspicuousness statistically to raise, (Fig. 9 A) unrelated with dosage.At any time point or Low blood pressure (Fig. 9 B) is not observed under any dosage.8/18 dog (being not suffering from pre-existing metastatic disease) and 3/5 dog (suffering from pre-existing metastatic disease) occurs in 4 hours of vaccine inoculation>The heating of 103 ℉, now gives Intravenous Supplement Liquid.Three dogs receive the NSAIDs of single dose, to reduce body temperature.In all cases, the regression of heating is without the need in addition Intervene.Occur not needing the instantaneous drowsiness of Results, nausea and vomiting in 4 hours of vaccine inoculation, it is unrelated with dosage. After vaccine inoculation soon, instantaneous list or bigeminy VPB are identified in two dogs.In 2 hours of vaccine inoculation, one There is Ventricular Tachycardia in dog with pre-existing metastatic disease.However, lidocaine, procainamide, Suo Taluo You do not have effect with the treatment of corticosteroid, and arrhythmia cordis disappeared in 72 hours.After ADXS31-164 is applied 24 hours The instantaneous but leucocyte with conspicuousness statistically and neutrophil count occur increases, and with blood platelet and Lymphocyte is instantaneously reduced (Figure 17).Although non-correlation between ADXS31-164 dosage and hematological change amplitude, survival Dog and the dog of death between leucocyte, neutrophil leucocyte and monocyte response amplitude there is significant difference (Figure 18 A- F).There is slight, instantaneous rising in the liver enzyme serum-concentration of approximately half of dog, with the microbial mild inflammation of close liver Liszt Unanimously (table 6).The all changes identified in peripheral blood are asymptomatic, and apply in ADXS31-164 one week is interior disappears Move back.The significant changes of renal function are not recorded in any dog.19/23 dog is carried out for 24 hours after ADXS31-164 administrations Blood culture, and feminine gender is, it is with the quick removing of highly attenuated LmddA bacterial strains consistent.

In view of HER2/neu targetings monoclonal antibody can cause cardiac toxic, we are before baseline, each vaccine inoculation And have evaluated the biomarker of heart injury and the echocardiography measurement amount of dysfunction per 2 months thereafter, including Cardiac troponin I, Fractional shortening (%), LVIDd and LVIDs.Heart flesh calcium is not identified in any vaccine inoculation dog Notable, the persistently change (Figure 26 A-D) of protein I, Fractional shortening, LVIDd or LVIDs.However, the 3rd group of a dog is each The step that serum cardiac troponin I is shown during vaccine inoculation is raised, without the dysfunction sign of echocardiography. Value returns baseline after final vaccine inoculation, and does not raise in repeat assessment.

In whole clinical testing, cardiac troponin I level, and Fractional shortening, left room end systolic diameter are determined (LVIDs) and LVED (LVIDd), as shown in Figure 25 (A-D), after the administration of ADXS31-164, do not exist The evidence of long-term or short-term cardiac toxic.

Table 6 below provide data illustrate, few therapy-related adverse events are reported during clinical testing.

Table 6：The therapy-related adverse events occurred in ADXS31-164 vaccine inoculations or in 48 hours.

Conclusion：ADSX31-164 toxicity is low-level and is instantaneous.

Immune response to ADXS31-164

Figure 18 provide as a result, it was confirmed that in the dog for receiving vaccine, the early immune response to ADXS31-164 is predicted The survival period of dog.Figure 18 shows that ADXS31-164 is induction of the WBC related to survival period, neutrophil leucocyte and monocyte meter Number increases, and with the instantaneous reduction (Figure 17) of blood platelet and lymphocyte.

ADXS31-164 inductions are have evaluated during clinical testing and immune response is maintained, HER2/neu is specifically induced The ability of Specific T cell immunity.In order to assess immune response and determine whether ADXS31-164 is special induction of HER2/neu Specific T cell response, by IFN-γ ELISpot HER2/neu specific T-cells quantity is have evaluated.In baseline, (carboplatin is applied 3 weeks afterwards), each vaccine inoculation when and thereafter per 2 months gather sample.Figure 19 shows the result that ELISpot is analyzed.

HER2/neu specific immune responses.In baseline 4/18,6/18 and 1/18 dog is detected to people HER2/ respectively The immunity in EC1, EC2 and IC1 domain (having 89%, 93% and 98% homogeneity with canid HER2/neu respectively) of neu should Answer.Induction to one or more HER2/neu domains was detected to 7 dogs in 3 weeks after the vaccine inoculation of third time ADXS31-164 IFN-γ response (table 7).There is the immune response to highly conserved IC1 domains in five in these dogs.After 2 months five it is another There is the IFN-γ response to IC1 domains in outer dog.In recurrence, three other dogs occur to single EC2, EC2 and IC1 or The IFN-γ response (dog 001,002 and 017) of EC1, EC2 and EC3.Assessed in initial vaccination by IFN-γ ELISpot Occur 3 dogs of the immune response to HER2/neu during series 15 to 17 months.However, not maintaining HER2/neu specific IFN-γ response, during this period dog still non-metastatic disease.10 dogs receive other booster vaccine inoculation, wherein 6 only comment Estimate, 2 months after booster vaccine inoculation, 2 dogs detect HER2/neu specificity IFN-γ responses to be increased.In ADXS31-164 3 weeks after administration, in 8 dogs of recurrence, the HER2/neu specificity IFN-γ responses of 5 do not increase.

Table 7

Booster vaccine is inoculated with.When 18 selected in the dog of non-metastatic disease, ten 5 and 10 after initial vaccine series Single dose booster vaccine is applied between individual month.In these dogs four only accept and are given between 4 and 15 months after booster vaccine first Other booster vaccine.It is the same with initial vaccination series when booster vaccine is inoculated with, recorded be similar to low level, Instantaneous side effect.

Figure 20 (A and B) is illustrated, is repeated booster vaccine inoculation and also have stimulated HER2 specific immunities.6 and 10 months to dynamic Thing 289-003 was applied and is repeated booster vaccine inoculation, and animal 289-004 is applied at 8 months.Clinical Outcome.In vaccine inoculation group 8/18 dog recurrence, 4 suffer from Lung metastases disease, and 4 Bone tumour occur.The dog of two Bone tumours proceeds to Lung metastases.One There is the dog of osteopathy stove to die from aspiration pneumonia for rumpbone, and a dog with solitary pulmonary nodules dies from nephroblastoma, however, It is unsuitable for the osteosarcomatous histopathology confirmation of metastatic respectively from the postmortem sample of bone and tuberculosis stove.It is specific by OSA Survival period is analyzed, and this two dogs are examined.The dog of recurrence receives different redemption chemotherapy and radiations when first visit judges.4 The dog of Bone tumour only with anodyne treatment (1 dog), single Palliative radiotherapy (1 dog) or with chemotherapy combined (2 dogs). Two dogs receive adriamycin, and 1 dog receives Palladia, to treat Lung metastases disease.The not up to intermediate value of vaccine inoculation dog OSA specificity survival periods.The Kaplan-Meier survival curves of TTM and OSA specificity survival periods are as shown in figure 21.Vaccine connects 1 year of kind of dog and 2 years overall survival rates are respectively 71.4% and 57%.Occurs HER2/neu in 2 months of vaccine inoculation special In 12 dogs of different in nature IFN-γ response, 9 (3 dogs that still survive>900 days, 1 dog>700 days, 3 dogs>400 days, 2 Dog>300 days, and 7 at present still without tumour (table 7)).The result that Figure 24 is provided shows that ADXS31-164 is destroyed to HER2/ The tolerance of Neu.This treatment to OSA and other HER2/neu tumours and/or cancer is important.

Autopsy findings.6/18 dog is dead during studying, and 4 in these dogs are performed an autopsy on sb..Three dogs are found With multifocal II and III level metastatic bone sarcoma, it is related to lung (3 dogs), bone (2 dogs), mediastinum (1 dog) and (1, kidney Dog).One dog is carried out euthanasia due to large-scale progressive kidney lump, is found with nephroblastoma.This dog is also With solitary pulmonary nodules, but histopathological evaluation is not regrettably passed through.

Survival period, long survival period, tumour progression after ADXS31-164 administrations

Three dogs with multiple metastatic Lung neoplasms in screening, for caring in nursing, per only connecing before disease is carried out By one vaccine therapy, and remove from research.In screening, two dogs for only existing isolatism metastatic Lung neoplasm receive all Three vaccinating agents (referring to the feature interpretation and tumoral character of table 5).Although having carried out vaccine inoculation, in these dogs still occurs Progressive Lung metastases disease.In second dog, although pre-existing Lung neoplasm was per 3 weeks size doubles, do not occur another Outer tuberculosis stove (Figure 22 A and B).One week after is inoculated with final vaccine, CT scan confirms there is no other metastasis (metastases), and And dog receives MET excision.Before surgery, the intravenous dyestuff indoles cyanines applied for detecting borderline tumor and areas of inflammation Green (ICG), in operation, observes that multiple other outward appearances in Lung neoplasm and near solitary nodule are good under near infrared light Fluorescence (Figure 22 C and D) in the pulmonary parenchyma region of health.The histopathology of Lung neoplasm is disclosed, and metastatic OSA has by thick fiber Very big bleeding and necrotic zone (Figure 22 E) that capsule is surrounded.IHC shows CD3⁺T cell is gathered around fibrous capsule, tubercle itself Interior T cell is seldom (Figure 22 G and H).Other regions of near-infrared fluorescent identification show the focal region that T cell infiltrates (Figure 22 F, 22I and 22J).T is observed in the big vimentin positive cell peripheral of the exception with significant mitotic figure Cell (Figure 22 K and 22L).These find hint, and single metastatic sarcoma cell can be effective by the tumor specific T cells of intrapulmonary Ground targeting, and ADXS31-164 is provided prevents the mechanism of metastatic lung disease.Dog can well from surgery recovery, and Apneumia metastatic disease is kept 5 months, subsequently in hypodermis (Gegenbaur's cell tumour, II levels；With chondroblast tumour, III Level), there is widely invasion HER2/ in mediastinum (Gegenbaur's cell tumour, II levels) and barrier film (Gegenbaur's cell tumour, III level) Neu+ metastatic diseases.As a result show, although induction of HER2/neu specific T-cells responses, not identifying de- tumour pair Effect, therefore elimination HER2/neu positive metastatic cells are responsible in the induction of HER2/neu specific T-cells, and prevent for a long time Palindromia.This can be supported by following aspect：The time of HER2/Neu- specific T-cells amplification (occurs in 5 dogs and examines Have no progeny about 8 months, at that time many dogs will appear from metastatic disease), and vaccine inoculation and MET cut off latter dog The histopathology of focal t cell response finds in pulmonary parenchyma.

It is that Figure 22 and Figure 23 are provided as a result, it was confirmed that the administration of ADXS31-164 has delayed and/or has prevented metastatic disease, And extend the overall survival phase with the spontaneity osteosarcomatous dogs of HER2+.As can be seen that receiving vaccine from two figures The time-to-live of dog significantly extends, and others are then not up to the median overall survival of those dogs for receiving vaccine.

Although our research confirms that the method prevents the validity of metastatic disease, treating for caring in nursing 5 dogs in, ADXS31-164 vaccine inoculations can not induce the regression of pre-existing severe lung metastatic disease.In a dog In, this fibrous capsule or those cells for seeming to be penetrated around metastasis (metastases) with T cell can not be micro- in the tumour set up Survive in environment related (Figure 22 C).However, in identical dog, large-scale, active division is observed in overall normal pulmonary parenchyma Be intended for around the mesenchymal cell of metastatic OSA cells T cell infiltration focal region, it is surprising that After MET excision, there is no further Lung metastases disease in the dog.These data imply that ADXS31-164 passes through jointly The ability of its powerful innate immune response of induction and adaptive immune response prevents Lung metastases disease, the congenital immunity Response can be sensitized the Apoptosis that metastatic OSA cells occur FAS/FASL mediations, and the adaptive immune response is micro- turn of elimination The form of the HER2/neu specific T-cells of shifting property lung disease.

Conclusion：

When the application is submitted to, there is no Lung metastases disease in 12/18 dog, it was confirmed that in the background of minimal residual disease During administration, ADXS31-164 prevents the metastatic disease with the spontaneity osteosarcomatous experimenters of HER2+.With history HER2/ Neu+ control groups are compared, and vaccine inoculation dog shows that the overall survival phase with conspicuousness statistically increases.HER2/Neu+ The median overall survival of control dog (n=11) is 316 days (p=0.032), wherein the intermediate value of not up to ADSX31-164 treatment dogs is deposited Current.In addition, as a result showing, ADXS31-164 destroys the outer peripheral tolerance (figure in the highly conserved IC1 domains to HER2/neu 26).The amplitude (Figure 18) for applying leucocyte increase in 24 hours in ADXS31-164 is related to survival period, and hint final result part takes Certainly the immune system in dog makes the ability of response to vaccine.Importantly, this research display will be up to 3 × 10⁹Individual CFU's It is safe that ADXS31-164 is applied to the dog with spontaneity OSA, and only causes instantaneous, low-level secondary work when applying With.Additionally, preventing the microcosmic metastatic disease that Lung metastases disease may be partly cell-mediated with CD3+T in lung from eliminating phase Close.Children OSA and other human cancers for expression HER2/Neu is significant for this research.

Additionally, we are shown here, the administration of the ADXS31-164 of 3.3 × 10^9 CFU is up in dog middle dosage is Safety, short-term or Long term cardiac toxicity are not result in although induction of HER2/neu specific immunities.Including cardiac toxic It is related to the administration of a large amount of HER2/neu specific T-cells in target, de- tumour side effect, or when trastuzumab is same with anthracycline When occur when using.We reduce any potential risk of cardiac toxic using the standard chemotherapy regimen without Doxorubicin.

Our research confirms that ADXS31-164 can prevent the Lung metastases disease of the dog with OSA.These results confirm The security of the dog with OSA and beyond example time-to-live, and prevent with HER2/ to study ADXS31-164 The ability of the metastatic disease of the patient of neu expression tumour (including children's osteosarcoma and breast cancer) has paved road.

Although some features of the present invention have been illustrated and described herein, now one of ordinary skill in the art will think To many modifications, displacement, change and equivalents.It will thus be appreciated that appended claims are intended in the present invention True spirit in all such modifications and variations.

Claims

1. a kind of method that HER2/neu for treating experimenter expresses tumour growth or cancer, methods described includes applying comprising weight The step of group is attenuated listerial composition, nucleic acid of the recombinant attenuated Listeria comprising encoding recombinant polypeptide, wherein The recombinant polypeptide includes the HER2/neu chimeric antigens for being fused to other polypeptide, wherein the nucleic acid molecules include coding institute The first ORFs of recombinant polypeptide is stated, wherein the nucleic acid molecules also the second ORFs comprising encoding metabolic enzyme, And the endogenous gene being wherein mutated in the chromosome of the complementary recombinant listeria bacterium bacterial strain of the metabolic enzyme.

2. method according to claim 1, wherein the composition includes about 3.3 × 10⁹Individual listerial Listeria Dosage.

3. method according to claim 1, wherein the experimenter is people or canid experimenter.

4. method according to claim 3, wherein the people experimenter is children and adolescents or adult.

5. method according to claim 1, wherein the fused polypeptide is applied into the experimenter prevents the tumour Interior escape mutant.

6. method according to claim 1, wherein the HER2/neu chimeric antigens are comprising at least 5,9,13,14 or 17 The chimeric HER2/neu of the people of people's MHC I class epi-positions of individual plotting.

7. method according to claim 1, wherein the chimeric HER2/neu is chimeric canid HER2/neu.

8. method according to claim 1, wherein the nucleic acid molecules are integrated into the Listeria genome.

9. method according to claim 1, wherein plasmid of the nucleic acid molecules in the recombinant listeria bacterium vaccine strain In, and wherein described plasmid is stably maintained at the recombinant listeria bacterium vaccine strain in the case where there is no antibiotic selection In.

10. method according to claim 1, wherein the recombinant listeria bacterium includes the mutation in actA virulent genes.

11. methods according to claim 1, wherein the other polypeptide is selected from：A) non-haemolysis LLO albumen or N- ends Fragment, b) PEST sequences or c) ActA fragments.

12. methods according to claim 1, wherein the metabolic enzyme of second ORFs coding is alanine Racemase or D- aminotransferases.

13. methods according to claim 1, also comprising independent adjuvant.

14. methods according to claim 12, wherein the adjuvant includes granulocyte/macrophage colony stimulatory factor (GM-CSF) albumen, the coding nucleic acid molecule of GM-CSF albumen, saponarin QS21, monophosphoryl lipid A or without methylated Containing CpG ODN.

15. methods according to claim 1, wherein the tumour is HER2/neu positive tumors, and wherein described cancer Disease is HER2/neu expression cancers.

16. methods according to claim 1, wherein the cancer is osteosarcoma, oophoroma, cancer of the stomach, central nervous system (CNS) cancer or Ewing's sarcoma (ES).

17. methods according to claim 16, wherein the osteosarcoma cancer is canid osteosarcoma.

18. methods according to claim 16, wherein the osteosarcoma is children's osteosarcoma.

A kind of 19. methods of the enhancing immune response for expressing HER2/neu tumour growth or cancer for causing experimenter, it is described Method includes the step of applying the composition comprising recombinant attenuated Listeria bacterial strain, the recombinant attenuated Listeria bacterial strain bag Nucleic acid containing encoding recombinant polypeptide, wherein the fused polypeptide includes the HER2/neu chimeric antigens for being fused to other polypeptide, First ORFs of the wherein described nucleic acid molecules comprising the coding recombinant polypeptide, wherein the nucleic acid molecules are also comprising volume In second ORFs of code metabolic enzyme, and the chromosome of the complementary recombinant listeria bacterium bacterial strain of wherein described metabolic enzyme The endogenous gene of mutation.

20. methods according to claim 1, wherein the composition includes about 3.3 × 10⁹Individual listerial Liszt Microbial inoculum amount.

21. methods according to claim 19, wherein the experimenter is people or canid experimenter.

22. methods according to claim 21, wherein the people experimenter is children and adolescents or adult.

23. methods according to claim 19, wherein the fused polypeptide is applied to described with HER2/neu expression The experimenter of tumour prevents the escape mutant of the intra-tumor.

24. methods according to claim 19, wherein the HER2/neu chimeric antigens be comprising at least 5,9,13,14 or The chimeric HER2/neu of the people of people's MHC I class epi-positions of 17 plottings.

25. methods according to claim 19, wherein the chimeric HER2/neu is chimeric canid HER2/neu.

26. methods according to claim 19, wherein the nucleic acid molecules are integrated into the Listeria genome.

27. methods according to claim 19, wherein matter of the nucleic acid molecules in the recombinant listeria bacterium vaccine strain In grain.

28. methods according to claim 19, wherein the plasmid is stably tieed up in the case where there is no antibiotic selection Hold in the recombinant listeria bacterium vaccine strain.

29. methods according to claim 19, wherein the recombinant listeria bacterium includes the mutation in actA virulent genes.

30. methods according to claim 19, wherein the other polypeptide is selected from：A) non-haemolysis LLO albumen or N- are last End fragment, b) PEST sequences or c) ActA fragments.

31. methods according to claim 19, wherein the metabolic enzyme of second ORFs coding is the third ammonia Sour racemase or D- aminotransferases.

32. methods according to claim 19, also comprising independent adjuvant.

33. methods according to claim 32, wherein the adjuvant includes granulocyte/macrophage colony stimulatory factor (GM-CSF) albumen, the coding nucleic acid molecule of GM-CSF albumen, saponarin QS21, monophosphoryl lipid A or without methylated Containing CpG ODN.

34. methods according to claim 19, wherein the tumour is HER2/neu positive tumors, and wherein described cancer Disease is HER2/neu expression cancers.

35. methods according to claim 19, wherein the cancer is osteosarcoma, oophoroma, cancer of the stomach, central nervous system (CNS) cancer or Ewing's sarcoma (ES).

36. methods according to claim 35, wherein the osteosarcoma cancer is canid osteosarcoma.

37. methods according to claim 19, wherein the osteosarcoma is children's osteosarcoma.

38. methods according to claim 19, wherein the immunity for expressing the HER2/neu tumour or cancer should Answer the immune response including the secondary Dominant Epitopes to the HER2/neu albumen.