CN107206045A

CN107206045A - Identification for the immunogenicity mhc class ii peptide based on immune treatment

Info

Publication number: CN107206045A
Application number: CN201680002764.1A
Authority: CN
Inventors: 布莱恩·J·赫尔尼奇; 加里·K·科斯基
Original assignee: Individual
Current assignee: Individual
Priority date: 2015-07-17
Filing date: 2016-04-07
Publication date: 2017-09-26
Also published as: EP3325005A4; EP3324990A4; CA2992925A1; EP3324990A1; EP3325005A1; CA2992937A1; WO2017014816A1; WO2017014810A1; JP2021043204A; JP2018521953A; JP2018527286A

Abstract

The invention provides with stimulating immune system and can be used for composition, method and the vaccine for the treatment of the malignant tumour related to the overexpression of the albumen of HER 3.Such composition includes the epitope of the albumen of HER 3.

Description

Identification for the immunogenicity mhc class ii peptide based on immune treatment

The cross reference of related application

The application is the patent application serial numbers PCT/US16/21042 submitted on March 4th, 2016 part continuation application, PCT/US16/21042 is the PCT/US15/41034 submitted on July 17th, 2015 part continuation application, PCT/US15/ again 41034 require that following priority and rights and interests again：The U.S.Provisional Serial 62/076,789 that on November 7th, 2014 submits And the U.S.Provisional Serial 62/025,681 that on July 17th, 2014 submits, by the content of each of the above with its full text It is incorporated herein reference.

The application includes the sequence table submitted with ASCII fromat electronics, and is incorporated herein by reference in their entirety.It is described ASCII is replicated, and is created on June 14th, 2006, is named as 319_003_PCT_CIP2_SL, txt, size is 2,666 words Section.

Background technology

In 25-30% breast cancer, and growth factor receptor gene HER2 (human epidermal growth factor receptor-2, also referred to as Neu/erbB2 amplification and overexpression) is related to the high risk of enhanced tumor infiltrating and recurrence and death (Slamon, D. etc., 1987, Science 235:177；Yarden,Y.,2001,Oncology 1:1).The oncogene is encoded The transmembranous receptor tyrosine kinases (RTK) of 185 kilodaltons (kDa).It is used as human epidermal growth factor acceptor (EGFR) family One of four members, HER2 is distinguished with following several ways with other in itself.First, HER2 is orphan receptor.No Identify high-affinity part.Second, to other EGFR family members (HER1/EGFR, HER3 and HER4), HER2 is to be used for shape Into the preferred gametophyte of heterodimer, heterodimer shows high ligand affinity and excellent signaling activity.3rd, total length HER2 carries out proteolysis cutting, discharges soluble, extracellular domain (ECD).The ECD come off is in vitro and in vivo Through show can as total length HER2 activation mechanisms substitute because it leaves the film grappling fragment with kinase activity. Regardless of its expression, several cancer types that the central roles of HER2 in EGFR families signal transduction are participated in it Related, such as breast cancer, oophoroma, colon cancer and stomach cancer (Slamon, D. etc., 1989, Science 244 occur for tumour:707； Hynes, N. etc., 1994, Biochem.Biophys.Acta.1198:165).HER2 can also make tumour cell to some chemistry Therapeutic agent resistant (Pegram, M. etc., 1997, Oncogene 15:537).In view of its important work in tumour generation With HER2 is the important target for the treatment of of cancer.

Mankind's EGF receptor (HER) family of receptor tyrosine kinase adjusts a variety of bioprocess, including cell is bred, moved Move, attack and survive.The family is by four member compositions：EGFR (HER1), HER2 (neu or ErbB2), HER3 (ErbB3) and HER4(ErbB4).So far, it has been reported that 11 kinds of parts, including EGF (EGF), Heparin-binding EGF samples Growth factor (HB-EGF), transforming growth factor α (TGF-α), amphiregulin (AR), epiregulin, β cytokines and tune egg In vain.These parts are directly in conjunction with its homoreceptor, and this causes the formation of acceptor homodimer or heterodimer, and it triggers multiple letters The activation of number pathway.It is overexpressed by activated mutant, acceptor or aberrant ligand discharges, the imbalance of HER- family members causes The development of various human tumors.HER3 is overexpressed in breast cancer, oophoroma and lung cancer, and this hereditary feature with it is bad pre- It is related afterwards.Once being activated by heregulin, HER3 and HER2, EGFR dimerization are to form effective oncogenic receptor heterodimer. In this complex, HER3 preferentially raises PI3 kinases and docks site to its cytoplasm, so as to adjust cell propagation and survive.Arrive So far, it is assumed that HER3 is kinases-inactivation (kinase-inactive), because having in its kinase domain substantially Abnormal sequence signature, and it is assumed that HER3 needs kinases-complete member with HER families to carry out heterodimerization to open Dynamic signal event.It is consistent with this, need HER3 to drive breast tumor cell to breed according to display HER2.However, nearest grinds Study carefully result to show, HER3 can phosphorylation Pyk2, it causes the activation of MAPK paths in human glioma cell.In addition, The specific monoclonal antibodies of HER3 can suppress propagation and the migration of cancerous cell line.It is interesting that display cancer cell leads to recently Cross up-regulation HER3 signal transductions and escape HER families inhibitor for treating, and HER3 suppression is eliminated in breast cancer cell The TAM drug resistance of HER2 drivings.In addition, according to display, to Gefitinib (Iressa) (a kind of EGFR micromolecular inhibitors) The resistance for the treatment of is relevant with HER3 signal activations.

HER3 is the receptor protein played an important role in regulation normal cell growth.HER3 lacks inherent kinase activity, And the presence dependent on HER2 carries out cross-cell membrane transduction signal.Initially during transcription, HER3 Pre-mRNA contains outside 28 Aobvious son and 27 intrones.The complete good HER3mRNA of montage is made up of 28 extrons, and introne has been in the HER3mRNA Come out by montage.

In the past ten years, targeted therapy has become the foundation stone for the treatment of of cancer.Because these receptor tyrosine kinases It can be lacked of proper care in many cancers, so, EGF receptor family member-i.e. EGFR (or HER1) and ErbB2 (or HER2/neu) are Develop into the target had a great attraction.Because being risen in resistance of the mediation to the treatment of HER2 and PI3K paths-guiding main Effect, another member ErbB3 or HER3 of EGF receptor family oncogenic function is carefully investigated recently,.In many not With tumor type, including identified in breast cancer, stomach cancer, colon cancer, carcinoma of urinary bladder and melanoma HER3 activated mutant and/ Or be overexpressed, these indicate a poor overall prognosis in these tumours.

Although being in progress in the field, still do not know using based on cell or based on protein using HER-3 as target To vaccination whether effective immune response can be produced in the mankind.Therefore, this area needs have other be immunized Treatment method is used to treat or prevent the breast cancer and other malignant tumours related to the overexpression of HER-3 albumen.This is specific real The mode of applying meets this demand.

Brief description of the drawings

When read in conjunction with the accompanying drawings, it is better understood with preferred embodiment following detailed description of in the present invention.In order to say Presently preferred embodiment is shown in the bright purpose of the present invention, accompanying drawing.It will be appreciated, however, that the invention is not restricted to accompanying drawing Shown in embodiment accurate arrangement and means.

Fig. 1 shows the immunogenic peptide (SEQ from HER-3 in many patients with activation cd4 t cell ability ID NO：1-3, in the order of presentation).

Fig. 2 shows the HER3 overall situation screenings of the group with 10 fragments of peptides.Fig. 2 also show the HER3 screenings with single peptide (SEQ ID NO：4-7, in the order of presentation).

Fig. 3 shows the HER3 overall situation screenings of the group with 10 fragments of peptides.Fig. 3 also show the HER3 screenings with single peptide (SEQ ID NO：4-7, in the order of presentation).

Fig. 4 shows the HER3 overall situation screenings of the group with 10 fragments of peptides.Fig. 4 also show the HER3 screenings with single peptide (SEQ ID NO：1-3, in the order of presentation).

Fig. 5 shows the IFN-γ yield from different HER3 peptides.

Fig. 6 shows the IFN-γ yield from different HER3 peptides.

Fig. 7 shows the IFN-γ yield from " reverse " screening, since the peptide previously identified, to peptide and HER3 cells Extracellular portion is sensitized.

Fig. 8 shows the IFN-γ yield from " reverse " screening, since the peptide previously identified, to peptide and HER3 cells Extracellular portion is sensitized.

Fig. 9 shows the IFN-γ yield for coming from " reverse " screening in patients, the extracellular knots of the previous unused HER of the patient Structure domain is sensitized, since the peptide previously identified, and peptide and HER3 extracellular domains are sensitized.

Figure 10 shows the IFN-γ yield for coming from " reverse " screening in patients, and previously unused HER is extracellular by the patient Domain is sensitized, since full peptide library, and peptide and HER3 extracellular domains are sensitized.

Figure 11 shows the IFN-γ yield for coming from " reverse " screening in patients, and previously unused HER is extracellular by the patient Domain is sensitized, since full peptide library, and peptide and HER3 extracellular domains are sensitized.

Figure 12 shows the production of the IFN-γ from " reverse " screening of the previously patient of unused HER extracellular domains sensitization Amount, since peptide, is sensitized to peptide and HER3 extracellular domains.

Figure 13 shows the continuous peptide screening in donor #UPCC 15107-24.

Figure 14 shows the continuous peptide screening in donor #UPCC 15107-38.

Figure 15 shows " reverse " sensitization in donor #UPCC 15107-38 and UPCC 15107-24.

Figure 16 shows immunogenicity HER3 epitopes-pulse in donor #UPCC 15107-30 and UPCC 15107-32 DC1 sensitization CD4+Th1 and overcome anti-HER3 immunological tolerances (two patients have the known HER3 peptides to identification and/or Natural HER3ECD anti-HER3 is non-reacted).

Figure 17 shows the immunogenicity CD4+HER3 epitopes for showing MHC II class dystopys.

Figure 18, which shows in the chamber to be placed on the HER-3 CD4+ cells of activation, to be closed on beside HER-3 expression cells, HER-3 CD4+ cells cause apoptosis or the death of HER-3 expression cell breast cancer cells.

Figure 19 shows the HER3 CD4+ peptides mixed using HER3 ECD as tumor antigen identification immunogenicity II classes Method, to produce anti-HER3 Th1 cellular immunities.

Figure 20 shows the checking of the immunogenicity of the CD4+HER3ECD epitopes by " reverse " sensitization identification.Using institute The single peptide shown carries out HER3 ECD screenings.

Figure 21 shows the checking of the immunogenicity of the CD4+HER3 ECD epitopes by " reverse " sensitization identification in addition As a result.

Figure 22 shows the photo of the immunohistochemistry scoring of HER dyeing.

Figure 23 A and 23B are showing the Barrett oesophaguses in low dysplasia (LGD) or high grade dysplasia (HGD) Middle HER families are overexpressed the block diagram (Figure 23 A) of ratio, and different to the height of Barrett lesions (HGD related with cancer) correlation HER families are overexpressed the block diagram (Figure 23 B) of ratio in type hyperplasia or high grade dysplasia without related infiltrating cancer.

Figure 24 A-24C are shown from HDs (healthy donors) to ER IBC/ER^posIBC (estrogen receptor positive wellabilities Breast cancer (IBC)) and TNBCC (three feminine gender IBC) anti-HER3 CD4 Th1 cell responses decline.Accompanying drawing shows systematicness The block diagram (left figure) of the IFN-γ ELISpot analyses of CD4+Th1 cell responses.Patient's group of research is：Healthy donors (HD)； Benign breast biopsy (BD)；The HER2 positives (" HER2^pos") DCIS (DCIS)；HER2 infiltrative breast carcinomas/HER2 is positive Infiltrative breast carcinoma (HER2 IBC/HER2^posIBC)；ERs infiltrative breast carcinoma/estrogen receptor positive wellability Breast cancer (ER IBC/ER^posIBC)；With three cloudy infiltrative breast carcinomas (TN IBC).Table corresponding with the right side of each block diagram It is that individual compares, examines what is obtained by " student " t between within the same time two groups.To all groups of carry out One-way ANOVAs. Figure 24 A show the anti-HER3 CD4 T cells of accumulation by the cell measurement of IFN-γ spot number/million determined by ELISpot Response, its drops to BDs to DCIS to HER2 from HDs significantly^posIBC to ER^posIBC, finally dropping to TN IBC (is respectively 90th, 80,66,79,48,40, p=0.01).Figure 24 B, which are shown, responds storehouse, or the HER3 peptides with positive CD4 Th1 responses Number, drops to BDs to DCIS to HER2 from HDs significantly^posIBC to ER^posIBC, finally to TN IBC (be respectively 1.0, 0.6th, 0.8,0.8,0.5,0.3, p=0.003).Figure 24 C show responsiveness, the percentage of the subject responded with least one kind of peptide Drop to BDs to DCIS to HER2 from HDs than significant^posIBC to ERpos IBC, (be respectively to TN IBC finally 76.7%th, 63.6%, 53.8%, 66.7%, 45.0%, 33.3%, p=0.02).

Figure 25 A and 25B show that the forfeiture of cd4 t cell response is HER3 specific, because the patient in test organizes it Between in terms of lockjaw or AntiCD3 McAb/CD28 stimulations without difference.Accompanying drawing shows the IFN- of systemic CD4+Th1 cell responses The block diagram (left figure) of γ ELISpot analyses.Patient's group of research is：Healthy donors；Benign breast biopsy；HER2 is positive in situ Duct carcinoma；The positive infiltrative breast carcinomas of HER2 infiltrative breast carcinomas/HER2；ERs infiltrative breast carcinoma/estrogen by Body positive infiltrative breast carcinoma；With three cloudy infiltrative breast carcinomas.Table corresponding with the right side of each block diagram is that individual compares, and is led to Cross within the same time " student " t between two groups and examine what is obtained.To all groups of carry out one-way analysis of variances.Figure 25 A are shown In healthy donors, benign breast biopsy, the positive DCISs of HER2, the positive wellability breasts of HER2 infiltrative breast carcinomas/HER2 Between the cloudy infiltrative breast carcinoma of gland cancer, ERs infiltrative breast carcinoma/estrogen receptor positive infiltrative breast carcinoma and three, Do not have statistically in the lockjaw reaction by IFN-γ spot number/200,000 cell measurement determined by ELISpot Significant sex differernce (being respectively 37,30,19,34,24,29, p=0.01).Figure 25 B are shown in healthy donors, benign breast work Inspection, the positive DCISs of HER2, the positive infiltrative breast carcinomas of HER2 infiltrative breast carcinomas/HER2, ERs wellability Between breast cancer/estrogen receptor positive infiltrative breast carcinoma and three cloudy infiltrative breast carcinomas, by ELspot determine by There is no significant property statistically in the AntiCD3 McAb of IFN-γ spot number/200,000 cell measurement/polyclonal stimulations of anti-CD28 Difference (being respectively 688,549,804,699,629,675, p=0.68).

Figure 26 A-26C show that the reaction of anti-HER3 cd4 t cells is related to the recurrence and reaction of new adjuvant chemotherapy, but with pouring Fawn on transfer unrelated.Figure 26 A are four block diagrams, by initially performing the operation under lymph node status (lymph node positive (" LN+ " or " LNpos ") with Lymph Node-negative (" LN- " or " LNneg ")) compared the immune response of IBC patient, it is shown that cumulative acknowledgements do not have Significant sex differernce (above) (being respectively 40,56, p=0.12) statistically, response storehouse (the second figure) (it is respectively 0.4,0.6, P=0.08), responsiveness (the 3rd figure) (being respectively 35.7%, 54.8%, p=0.19) or lockjaw react (figure below) (respectively 22nd, 29, p=0.35).Figure 26 B are four block diagrams, its recurrence by our diagnosis at least 1 year and non-recurrence (no disease) Patient compares the immune response of IBC patient, it is shown that with significant relatively low cumulative acknowledgements (above), (a point ratio is 17,66, p= 0.04), (a point ratio is 0.0,0.6, p for response storehouse (the second figure)<0.05), (a point ratio is 0%, 55.6%, p to responsiveness (the 3rd figure) =0.01), the lockjaw between recurrent and non-recurrent IBC patient reacts no difference (figure below), and (a point ratio is 27,35, p =0.65).Figure 26 C are four block diagrams, its by new adjuvant chemotherapy (pathology complete incidence graph (" pCR ") to residual disease ("< PCR ")) immune response of the response ratio compared with IBC patient.In the patient of new adjuvant chemotherapy is received, have with those<PCR trouble Person compares, and the patient with pCR shows significant higher cumulative acknowledgements (above) (being respectively 144,32, p=0.004), rings Ying Ku (the second figure) (be respectively 0.8,0.4, p=0.05), in pCR and<Responsiveness (the 3rd figure) between pCR patient's immune response (being respectively 80.0%, 27.3%, p=0.10) or lockjaw reaction (figure below) (being respectively 17,59, p=0.15) are without difference.

Figure 27 A-27D are shown in the significant rise of post menopausal HD/BD moderate resistance HER3 cd4 t cells responses, but not with the age, Race or pregnant history and it is different.Figure 27 A are four block diagrams, its by the age (<50 years old and>50 years old) compare the immune of HD patient Response.Cumulative acknowledgements (above) (being respectively 77,103, p=0.25), response storehouse (the second figure) (is respectively 0.8,1.1, p= 0.38), responsiveness (the 3rd figure) (be respectively 72.0,75.0%, p=1.0) or lockjaw reaction (figure below) (be respectively 39,30, P=0.40) without significant difference statistically.Figure 27 B are four block diagrams, pass through ethnic (Caucasian, African U.S. Compatriots and other people) compared the immune response of HD patient.Cumulative acknowledgements (above) (being respectively 87,83,95, p=0.96), response Storehouse (the second figure) (be respectively 0.9,0.7,1.4, p=0.31), responsiveness (be respectively 69.0%, 71.4%, 100%, p= Or lockjaw reaction (figure below) (be respectively 33,51,26, p=0.30) is without significant difference statistically 0.35).Figure 27 C It is four block diagrams, the immune response of HD patient is compared by pregnant history/parity (0 gestation, 1 or more gestation).It is tired Product response (above) (being respectively 82,91, p=0.71), response storehouse (the second figure) (being respectively 1.0,0.9, p=0.62) or response Rate (the 3rd figure) (being respectively 76.5%, 70.8%, p=0.74) does not have significant sex differernce statistically in pregnant history.Sense Interest, compared with least one gestation, the lockjaw reaction (figure below) of nulliparity women is significant higher (respectively 47th, 27, p=0.04).Figure 27 D are four block diagrams, are compared the immune of HD patient by menopausal state (premenopausal with post menopausal) Response.Compared with premenopausal HD/BD, post menopausal HD/BD shows that significant higher cumulative acknowledgements (above) (is respectively every million Individual cell 136,70, p=0.005) and response storehouse (the second figure) (being respectively 1.4 peptides, 0.8 peptide, p=0.03).Post menopausal and Premenopausal HD/sBDs responsiveness (the 3rd figure) (being respectively 90.9%, 66.7%, p=0.23) or lockjaw reaction (figure below) (being respectively 38,28, p=0.37) is without difference.

Figure 28 is ELISpot linear determination figures.By operator, ELISpot determination methods be defined as it is linear and accurate, should Operator by being serially diluted for known anti-HER3 cd4 t cells response by carrying out all surveys of this research in culture medium It is fixed.Cumulative acknowledgements follow linear regression curves, and the curve is from (the every million cells difference of dilution factor 1.0,0.1,0.01,0.001 For 230,35,12,5 points, p<0.0001, r²=0.88).

Detailed description of the invention

Present embodiments provide for the isolated peptides of HER family proteins and other receptor tyrosine kinases.In a reality Apply in scheme that there is provided one or more isolated peptides in HER1, HER3 and c-MET albumen.In one embodiment, peptide Represent HER1 epitope.In one embodiment, peptide represents HER3 epitope.In one embodiment, peptide represents c-MET Epitope.

In some embodiments, the epitope and other receptor tyrosine kinases of the corresponding HER families of protein are to exempt from Epidemic focus.Present embodiment is additionally provided with the composition of the peptide comprising one or more present embodiments.In an implementation There is provided chimeric peptide in scheme, wherein the chimeric peptide includes the peptide of one or more present embodiments.

Include the composition comprising multivalence peptide in one embodiment.Multivalence peptide includes two or more present invention's Peptide.

It is additionally provided with the method for the method for stimulating immune response and the cancer in treatment subject.Additionally provide for controlling Treat the vaccine with prevention disease purposes.As described herein, the peptide of present embodiment can be individually or included in chimeric peptide Trigger immune response.In one embodiment, immune response is humoral response.In another embodiment, immune response It is cell-mediated response.According to some embodiments, peptide of the invention provides protective effect.

In another embodiment, HER3 expression may be used as the mark of tumour progression in gastroesophageal junction precancerous lesion Will thing.

In another embodiment, by determining anti-HER3 CD4 including the use of HER3 MHC II para-immunity originality peptides Th1 forfeiture.

Definition

Unless otherwise defined, all technologies and scientific terminology that otherwise the present invention is used have leads with technology belonging to the present invention The implication identical implication that the those skilled in the art in domain are generally understood.Although with similar or equivalent any side of the present invention Method and material can be used for the experiment or test of the present invention, and method and material that will be preferred be described.

Generally, in terminology used in the present invention and cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization Experimental procedure be known in the art and those conventional.

Standard technique is used for nucleic acid and peptide.Technology and step are generally according to the conventional method and the literature in this area (such as Sambrook and Russell, 2012, Molecular that the various general bibliography provided in the whole text are carried out Cloning,A Laboratory Approach,Cold Spring Harbor Press,Cold Spring Harbor,NY, With Ausubel etc., 2012, Current Protocols in Molecular Biology, John Wiley＆Sons, NY).

The experimental method used in terminology used in the present invention and analytical chemistry described below and organic synthesis is ability Known and those conventional in domain.Standard technique or its change for chemical synthesis and chemical analysis.

As used in the present invention, the implication of each term is associated with its implication in this section below.

The article " one kind " that the present invention is used refers to one or more than one (i.e. at least one) of article grammar object.Example As " a kind of element " refers to an element or more than one element.

When be related to measurable value such as quantity, the duration when, " about " used in the present invention refer to include definite value ± 20%, or ± 10%, or ± 5%, or ± 1%, or ± 0.1% change, because these changes are adapted for carrying out disclosed side Method.

When term "abnormal" is used in organism, tissue, the context of cell or its component, refer to those organisms, Tissue, cell or its component are different from least one observable or detectable feature (such as age, treatment, the time) Organism, tissue, cell or its component of the respective feature of those displays normal (desired).For a kind of cell or tissue class It is that normal or expected feature is probably abnormal for different cell or tissue types for type.

" auxiliary treatment " for breast cancer as used in the present invention refers to that is given after primary treatment (performing the operation) appoints What is treated, to increase the chance of long-term surviving." new auxiliary or new-complementary therapy " is the treatment given before primary treatment.

Term used herein " antigen " or " ag " are defined as triggering the molecule of immune response.This immune response may Be related to antibody generation, or specificity immuning activity cell activation, or both all include.It will be understood by those skilled in the art that appointing What macromolecular, actually including all protein or peptide, can be used as antigen.In addition, antigen can derived from restructuring or Genomic DNA.It will be understood by those skilled in the art that comprising coding trigger immune response protein partial nucleotide sequence or The term " antigen " of any DNA encoding of nucleotide sequence as used in the present invention.In addition, it will be understood by those skilled in the art that anti- Original need not be only by the full length nucleotide sequential coding of gene.It is more than it is readily apparent that the present invention includes but is not limited to use The partial nucleotide sequence of one gene, and these nucleotide sequences carry out different combination with trigger it is required it is immune should Answer.Moreover, it will be understood by those skilled in the art that antigen need not be encoded by " gene ".It is readily apparent that antigen can be synthesized It is producing or can derive biological sample.Such biological sample can include but is not limited to tissue sample, tumor sample, thin Born of the same parents or biofluid.

" antigen presenting cell " (APC) is can to activate the cell of T cell, and it includes but is not limited to monocyte/macrophage Cell, B cell and BMDC (DC).

" the APC " of antigen load or " APC " of antigen pulse is included exposed to antigen and by Antigen-activated APC.Example Such as, APC can the loaded Ag (such as during the culture with the presence of antigen) in vitro.APC can also by exposed to antigen and It is supported in vivo." APC " of antigen load is generally prepared one of in two ways：(1) the small fragments of peptides for being referred to as Antigenic Peptide is straight Connect the outside that " pulse " arrives APCs；Or (2) APC and complete protein or protein particulate are incubated, then complete protein or egg White matter particle is absorbed by APC.These protein are digested small fragments of peptides by APC, and are finally transported and present On APC surfaces.Further, it is also possible to produce the APC of Antigen by the way that the polynucleotides of coding for antigens are imported in cell.

" anti-HER3 responses ", " anti-HER3 CD4 Th1 responses ", " anti-HER3 CD4 t cell responses " etc. refer to specificity For the immune response of HER3 albumen.

Terminology used in the present invention " GVT " refers to a kind of biological effect, and it can subtracting by gross tumor volume Less, tumor cell number purpose reduce, various related to cancer disorder of reduction, the increase of life expectancy or improvement of transfer number Physiological signs are confirmed." GVT " can also be by the prevention first of the peptide, polynucleotides, cell and antibody of the present invention Tumorigenic ability is confirmed.

Terminology used in the present invention " autoimmune disease " is defined as lacking of proper care as caused by autoimmune response.Itself exempts from Epidemic disease disease is inappropriate and overreaction the result to autoantigen.The example of autoimmune disease includes but is not limited to Ai Disen diseases, alopecia areata, ankylosing spondylitis, oneself immunity hepatitis, LADA parotitis, Crohn's disease, glycosuria Sick (I types), malnutrition bullous epidermis, epididymitis, glomerulonephritis, Graves disease syndrome, Ji Lan-Ba Lei Syndrome, Hashimoto's disease, hemolytic anemia, systemic loupus erythematosus, multiple sclerosis, myasthenia gravis, homeliness type day blister Sore, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, chorionitis, Sjogren syndrome, spondyloarthropathy, thyroiditis, Vasculitis, leucoderma, myxedema, pernicious anaemia, intestines colitis etc. of bursting.

Mean any material from same individual such as terminology used in the present invention " autologous ", it then will be by again It is introduced into individual.

Terminology used in the present invention " B cell " is defined as being derived from the cell of marrow and/or spleen.B cell can develop into production The thick liquid cell of raw antibody.

Terminology used in the present invention " cancer " is defined as the hyper-proliferative of cell, and its unique shape-forfeiture is normally controlled Uncontrolled growth is made-caused, lacks differentiation, local organization intrusion and/or transfer.Example include but is not limited to breast cancer, Prostate cancer, oophoroma, cervical carcinoma, cutaneum carcinoma, cancer of pancreas, colorectal cancer, kidney, liver cancer, the cancer of the brain, lymthoma, leukaemia, Lung cancer, germinoma and similar conditions.

" CD4+Th1 cells ", " Th1 cells ", " cell of CD4+T auxiliary types 1 ", " CD4+T cells " etc. are defined as expression A kind of hypotype of the t helper cell of surface protein CD4 and the high-caliber cell factor IFN-γ of generation.Referring also to " T auxiliary is thin Born of the same parents ".

" cumulative acknowledgements " refer to the combination immune response of patient's group, are expressed as all MHC II classes organized from given patient Summation (every 10 analyzed from IFN-γ ELISpot of the reflecting point of binding peptide⁶The point of individual cell forms cell " SFC ").

" DC vaccine inoculations ", " DC is immunized ", " DC1 is immunized " etc. refer to manage immune system using autologous dendritic cell Recognize specific molecular and the strategy of specific response is produced for them.

Term " BMDC " or " DC " are a kind of antigen presenting cells, and it is present in internal, external, in vitro or in place In main or subject, or it may originate from candidate stem cell or monocyte.BMDC and its precursor can be from a variety of lymphs Separated in organ such as spleen, lymph node and marrow and peripheral blood.BMDC has the feature of thin slice (Lamellipodium) Form, the thin slice extends away from BMDC body in a plurality of directions.Generally, the high-caliber MHC of 1 expressed by dendritic cells and Costimulatory molecules (such as B7-1 and B7-2).BMDC can inducing T cell in vitro antigentic specificity differentiation, and can be with Initiate primary T cell response in vitro and in vivo.In the environment of production of vaccine, " DC " of activation is to be exposed to Toll-like The DC of receptor stimulating agent such as lipopolysaccharides " LPS ".The DC of activation can with or can not be loaded with antigen.

" disease " is a kind of health status of animal, and wherein animal can not maintain homeostasis, and if disease does not have Improve, then the health of animal may proceed to deteriorate.

" imbalance " in animal is a kind of health status, and wherein animal is able to maintain that homeostasis, but the health of animal It is good when situation is not as lacking of proper care.Untreated, imbalance not necessarily causes the further reduction of animal health status.

If the disease of patient experience or the reduction of the seriousness or frequency of at least one S or S of imbalance, disease Or imbalance is " mitigation ".

" effective dose " or " therapeutically effective amount " is used interchangeably in the present invention, and is referred to as described in the present invention effectively Realize the amount of compound, preparation, material or the composition of particular biological result.Such result can include but is not limited to logical Cross the suppression for the virus infection that any suitable method in this area is determined.

As used in the present invention, term " endogenous " refers to from or resulted from appointing in organism, cell, tissue or system What material.

As used in the present invention, term " external source " refers to what is introduced or produce from organism, cell, tissue or its exterior Any material.

" HER receptor " is the receptor protein tyrosine kinase for belonging to HER receptor family, including EGFR (ErbB1, HER1), HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4) acceptor.HER receptor is generally comprised：Ectodomain, it can be tied Close HER ligand and/or molecule dimerized with another HER receptor；Lipophilic transmembrane domain；Conservative intracellular tyrosine swashs Enzyme domains；With the carboxyl terminal signal transduction domain with the several tyrosine residues that can be phosphorylated.HER receptor can be with It is " native sequences " HER receptor or its " amino acid sequence variation ".Preferably, HER receptor is native sequence human's HER receptor.

" HER approach " refers to the signaling transduction network mediated by HER receptor family.

" HER activation " refers to activation or the phosphorylation of any one or more of HER receptor.Generally, HER activation causes signal (such as the tyrosine residue in its intracelluiar kinase domain phosphorylation HER receptor or substrate polypeptide by HER receptor is led for transduction Cause).HER activation can be combined to mediate by HER ligand with the HER dimers comprising target HER receptor.HER ligand with The combination of HER dimers can activate the kinase domain of one or more HER receptors in dimer, so as to cause one or more The phosphorylation of the phosphorylation of tyrosine residue in HER receptor and/or the tyrosine residue in other substrate polypeptide, for example Akt or MAPK intracellular kinases.

" HER2 " is the member of human epidermal growth factor acceptor (" EGFR ") family.People mammary gland of the HER2 in about 20-25% It is overexpressed, and is expressed in many other cancers in cancer.

“HER2^pos" be a kind of breast cancer and many other types of cancer classification or molecular isoform.HER2 positive mesh Preceding intensity 2+ or 3+ by FISH (FISH) gene magnifications determined and pathological staining is defined.

“HER2^neg" be defined by the shortage of the FISH gene magnifications determined, and can in most cases wrap Include 0 to 2+ pathological staining scope.

" HER3 " and " ErbB3 " refers to disclosed receptor polypeptides, for example, U.S. Patent number 5,183,884 and 5,480, 968 and Kraus et al. PNAS (USA) 86:9193-9197(1989).

" HER3 ectodomains " or " HER3ECD " refer to the HER3 domains positioned at outside, and it is anchored to cell Film or in the circulating cycle, including its fragment.In one embodiment, HER3 extracellular domain can include four structures Domain：Domain I, domain II, Domain III and domain IV.In one embodiment, HER3ECD comprising amino acid/11- 636 (numbering includes signal peptide).In one embodiment, comprising amino acid 328-532, (numbering includes HER3 Domain IIIs Signal peptide).

" HER3 immunogenic peptides ", " HER3 binding peptides ", " HER3 epitopes " as used in the present invention etc. refer to be derived from or The MHC II class peptides of sequence and its equivalent based on HER3 albumen, particularly HER3ECD.HER3 peptides can activate many patients CD4 T cells.The peptide can be used for dendritic cells pulsed and instruct T cell to recognize HER3.HER3 is in triple negative breast cancer Middle expression, and can be in ER^posThe resistance for anti-estrogen is assigned in breast cancer.HER3 is also expressed in other cancers, Including melanoma, lung cancer, colon cancer, prostate cancer and metastatic brain tumor.According to preferred embodiment, four are identified as follows Plant HER3 immunogenic peptides (epitope) or binding peptide：

P12 (peptide 56-70):CEVVMGNLEIVLTGH(SEQ ID NO:4)；

P81 (peptide 401-415):SWPPHMHNFSVFSNL(SEQ ID NO:5)；

P84 (peptide 416-430):TTIGGRSLYNRGFSL(SEQ ID NO:6)；With

P91 (peptide 451-465):AGRIYISANRQLCYH(SEQ ID NO:7).

" homologous " used in the present invention refers between two polymer molecules, such as between two nucleic acid molecules, such as two Between individual DNA molecular or two RNA molecules, or the subunit sequence similarity between two peptide molecules.When both two molecules In subunit position when being occupied by identical monomelic subunit, if for example, each DNA position is by gland in two DNA moleculars Purine is occupied, then they are complete in the position or 100% is homologous.Percent homology between two sequences be matching or The direct function of homologous position number, if for example, the half of position is (for example, on polymer length in two compound sequences Ten subunits in five positions) be homologous, then two sequences are 50% identical, if 90% position, such as 10 In 9 matchings or homologous, then two sequences there is 90% homology.For example, DNA sequence dna 5'ATTGCC3' and 5' TATGGC3' has 50% homology.

In addition, when the present invention using term " homology " or " homogeneity " to refer to nucleic acid and protein when, should be construed as For the homology or homogeneity applied to nucleic acid and amino acid sequence level.

Term " excess proliferative disease " is defined as the disease as caused by cell hyperproliferation.Exemplary excess proliferative Disease includes but is not limited to cancer or autoimmune disease.Other excess proliferative diseases may include such as vascular occlusion, again Narrow, atherosclerosis or inflammatory bowel disease.

As used in the present invention " teaching material " include can be used for pass on the compositions and methods of the invention serviceability publication, Record, chart or any other expression medium.The teaching material of kit of the present invention can be as adhere to containing the present invention nucleic acid, In the container of peptide and/or composition, or transport together with the container containing nucleic acid, peptide and/or composition.Or, teaching material can Separately to be shipped with container, it is therefore an objective to which recipient's collaboration uses teaching material and compound.

" immune response " as used in the present invention refers to the activation of the host immune system introduced in response to antigen, such as lactation The activation of the immune system of animal.Immune response can be the form of cell or humoral response, or both have concurrently.

" separation " refers to from native state change or remove.For example, the nucleic acid or peptide that are naturally occurring in living animal It is not " separation ", but the identical nucleic acid or peptide that are partially or wholly separated with the coexisting materials of its native state are " to separate ".The nucleic acid or protein of separation can exist in the form of substantially purifying, or may reside in non-natural environment, example Such as host cell.

Term used herein " regulation " refers to and does not receive to treat or be not used the response water in the subject of compound It is flat to compare, and/or with other side is identical but subject of untreated in response level compared with, should in mediation subject Answer horizontal detectable increase or decrease.The term includes disturbing and/or influence natural signals or response, so that tested Beneficial therapeutic response is mediated in person, the preferably mankind.

Defined " measurement " of CD4+Th1 responses by analyzing the anti-HER3 CD4+Th1 immune responses of each subject group Or " measurement of immune response "：(a) total anti-HER3 responses (be expressed as pair >=subject of a kind of immunogenic peptide response hundred Divide ratio)；(b) storehouse (average (n) for being expressed as the immunogenic peptide of each tested group of identification) is responded；Cumulative acknowledgements (table (c) The reflecting point from each tested group of 4 MHC II class HER3 immunogenic peptides is shown as (to analyze from IFN-γ ELISpot Every 10⁶The point of individual cell forms cell " SFC ") summation).

" peptide ", " protein " or " polypeptide " as used in the present invention can refer to the catenation sequence of amino acid, and can be Natural, synthesis or natural and synthesis modification or combination.

As used in the present invention, " colony " includes referring to the separation including homogeneity, substantially homogeneity or foreign cell culture Culture.Generally, " colony " can also be considered as " separation " cell culture.

Receptor tyrosine kinase (" RTK ") is the high-affinity cell of many peptide growth factors, cell factor and hormone Surface receptor.RTK people's EGF receptor (" HER ") family adjusts a variety of bioprocess, including cell is bred, migrates, attacks and deposited It is living.Family is made up of four member HER1 (ErbB1), HER2 (neu or ErbB2), HER3 (ErbB3) and HER4 (ErbB4).

As used in the present invention, " recombinant cell " is the host cell for including recombination of polynucleotide.

" responsiveness " or " anti-HER3 response rates " is used interchangeably in the present invention, it is intended that 4 kinds of HER3 immunogenicities of response The percentage of at least one kind of subject in peptide.

" response storehouse " is defined as the average (" n ") of the HER3 immunogenic peptides of each subject group identification.

" sample " or " biological sample " as used in the present invention refers to the biomaterial from subject, includes but is not limited to Organ, tissue, allochthon, blood, blood plasma, saliva, urine and other body fluid.Sample can be from subject obtain it is any come The material in source.

As used in the present invention " signal 1 " typically refers to be delivered to the first biochemical signals of T cell from the DC of activation.Signal 1 by the antigen offer in DC surface expressions, and is sensed by φt cell receptor by T cell.

As used in the present invention " signal 2 " typically refer to by DC provide to T cell secondary signal.Signal 2 is by activating " costimulation " molecule on DC is provided, typically CD80 and/or CD86 (although known have other costimulatory moleculeses), and is passed through Surface receptor CD28 is sensed by T cell.

As used in the present invention " soluble protein that signal 3 " typically refers to be produced by the DC that activates (is usually cell The factor) produce signal.These signals are sensed by the acceptor on T lymphocytes.3rd signal designation T cell should be obtained Which phenotype or functional character is obtained best to handle current threat.

Term used herein " specific binding " refers to molecule, and such as antibody recognizes and combines another molecule or spy Levy, but substantially nonrecognition or other molecules or feature in combination sample.

Term " subject ", " patient ", " individual " etc. are used interchangeably in the present invention, and are referred to and be applicable to the present invention Any animal of methods described or its either external or in situ cell.In certain non-limiting embodiments, patient, Subject or individual are people.

Terminology used in the present invention " targeted therapy " refers to the treatment of cancer using medicine or other materials, and it, which is disturbed, participates in The certain target molecules of growth of cancer cells, but generally normal cell is not almost damaged to realize antitumous effect.Compared to it Under, traditional cytotoxic chemotherapy agents act on all cells actively divided.It is special in breast cancer treatment monoclonal antibody Be not Herceptin/Using HER2/neu acceptors as targeting.

Terminology used in the present invention " T cell " is defined as participating in the thymus-derived thin of the immune response of various kinds of cell mediation Born of the same parents.

The term " T auxiliary " that is used on cell of the present invention refer to that those skilled in the art can recognize that including different cell classes The subgroup of the lymphocyte (white blood corpuscle or white blood cell) of type.Especially, effect is included according to t helper cell disclosed by the invention Th cells (such as Th1, Th2 and Th17).These Th cells secretion can stimulate other leucocytes or with other leucocyte phase interactions Cell factor, protein or peptide.

The term " t helper cell " that is used in the present invention on cell, " helper cell ", " Th cells " etc. represent ability The subgroup for the lymphocyte (white blood corpuscle or white blood cell) including different cell types that field technique personnel can recognize that.Especially, T Auxiliary cell is effector T cell, and its major function is activation and the work(for promoting other B and T lymphocytes and/or macrophage Energy.Helper cell is divided into two kinds of Main Subtypes of referred to as " Th1 " or " 1 type " and " Th2 " or " 2 type " phenotype.These Th cells Secrete cytokines, protein or peptide, the cell factor, protein or peptide can stimulate other leucocytes or with other leucocyte phases Interaction.

" Th1 cells ", " CD4+Th1 cells ", " CD4+T aids in 1 type cell ", " CD4+T cells " as used in the present invention Etc. the mature T cells for referring to express surface glycoprotein CD4.When the surface of antigen presenting cell (" APC ") such as BMDC When the MHC II quasi-molecules of upper expression are to CD4+T auxiliary cells presentation peptide antigen, CD4+T auxiliary cells are activated.When passing through When MHC- antigenic compounds activate CD4+T auxiliary cells, cell factor such as the interferon-γ (" IFN- of its secreting high levels γ”).Such cell for be present in host cell it is some cause disease microorganism be considered as it is highly effective, And it is to pass to the antitumor reaction that some pathogenic microorganisms lived in host cell and cancer are resisted in human cancer Important.

" Th17 T cells " as used in the present invention refers to produce high-caliber cell factor IL-17 and IL-22 and recognized To be highly effective T cell for the pathogenic microorganisms survived on mucomembranous surface,.

" therapeutically effective amount " is the amount of the compounds of this invention, and it improves the symptom of disease when being applied to patient.Composition " is controlled The amount of the compounds of this invention for the treatment of effective dose " is according to compound, morbid state and its seriousness, the age of patient to be treated etc. Change.Therapeutically effective amount can be considered the knowledge and present disclosure of himself come routinely by those of ordinary skill in the art It is determined that.

Term " treatment " refers to treatment or prevention measure of the present invention." treatment " method is to needing this treatment Subject applies the composition of the present invention, such as subject with disease or imbalance, or may finally obtain such disease Or the subject of imbalance, in order to prevent, cure, postpone, reduce seriousness or improve the one or more of imbalance or recurrence imbalance Symptom, or survival in order to extend subject make it exceed the expected time-to-live in the case of not this treatment.

" three is negative " and " TN " breast cancer refer to any to ERs (" ER "), PgR (" PR ") and HER2 survey Try as negative breast cancer cell.

Terminology used in the present invention " vaccine " is defined as a kind of material for being administered to and triggering immune response after animal, wherein dynamic The preferred mammal of thing, the more preferably mankind.After subject is introduced, vaccine can trigger immune response, including but not limited to resist Body, the generation of cell factor and/or other cell responses.

" variant " refers to that, by the insertion of amino acid, missing or conservative replacement, amino acid sequence changes but retained extremely The peptide or polypeptide of a kind of few bioactivity.Variant can also refer to the egg with the reference basic identical amino acid sequence of protein White matter, has the amino acid sequence for retaining at least one bioactivity with reference to protein.The conservative replacement of amino acid, i.e., with having The different aminoacids of similar quality (such as hydrophily, the degree of charging zone and distribution) are replaced amino acid and recognized in the art To be usually directed to small change.These small changes can identify partially by the hydrophilic index of amino acid is considered, (Kyte etc., J.Mol.Biol.157 as understood in the art:105-132(1982)).The hydrophilic index of amino acid is based on it The consideration of hydrophobicity and electric charge.Known in the art, the amino acid of similar hydropathic index can be substituted and still retaining protein Function.In one aspect, it is substituted with hydrophilic index for ± 2 amino acid.The hydrophily of amino acid can be used for disclosing By the substitution for causing protein to retain biological function.Consider the hydrophily of the amino acid in peptide, it allows to calculate the peptide most Big local average hydrophilicity, United States Patent (USP) US4,554,101 reported useful measure with antigenicity and immunogenicity well Correlation, entire contents are incorporated herein by reference.The substitution of amino acid with similar hydrophilicity score can cause peptide to retain life Thing activity, such as immunogenicity, as known in the art.It can be replaced with amino acid of the hydrophilicity value each other in ± 2.Amino The hydrophobicity index and hydrophilicity value of acid are influenceed by the specific side chain of the amino acid.It is consistent with the observation result, with biology The 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor that function is consistent is understood to the side of the relative similarities depending on amino acid, particularly those amino acid Chain, as shown in by hydrophobicity, hydrophily, electric charge, size and other performances.

Scope：Through the disclosure, various aspects of the invention can be presented with range format.It should be appreciated that scope form Description just for the sake of convenient and succinct, and be not necessarily to be construed as the dumb limitation to the scope of the invention.Therefore, scope Description should be considered as having specifically disclosed all possible subrange and should in the range of single number.For example, right The description of such as 1 to 6 scope should be considered as having specifically disclosed subrange, such as from 1 to 3, from 1 to 4, from 1 to 5, From 2 to 4, from 2 to 6, from 3 to 6 etc., and the single number in the range of this, such as 1,2,2.7,3,4,5,5.3 and 6, and no matter How is the width for the scope being described, and this explanation is all suitable for.

Detailed description of the invention

Present embodiments provide for the immune combination of the peptide comprising HER family proteins and other receptor tyrosine kinases Thing.In one embodiment, the invention provides the peptide of the separation in one or more HER-1, HER-3 and c-MET albumen. In one embodiment, peptide of the invention can be used for triggering immune response.The composition of peptide comprising the present invention can be used as just The prophylactic treatment agent of beginning protection, and can be used as treating the therapeutic agent of ongoing situation.

Present invention also offers the method for treating or preventing cancer.Such method is included to subject in need Using the present invention peptide or peptide combination the step of.Cause the induction of antineoplastic immune using this peptide.Therefore, the present invention is provided The method of inducing antitumor immunity in subject, methods described include to subject apply the present invention peptide or peptide combination with And pharmaceutical composition and by its derivative cell composition the step of.

Present invention resides in the method for inducing T cell response in mammal.It is thin that this method includes administration specificity induction T The antigen presenting cell (APC) of born of the same parents' propagation.In one embodiment, method includes applying peptide pulse using the present invention Dendritic cell vaccine so that specificity induction be directed to corresponding to the peptide antigen T cell propagation.

In one embodiment, it can be used for culture amplification T cell with the APC of the peptide pulse of the present invention.Expanded using APC T cell, once sufficient amount of T cells with antigenic specificity is obtained just to give the T cells with antigenic specificity so obtained and feed Newborn animal, so that the inducing antigen-specific T cell response in mammal.

The present invention includes the DC of activation preparation.In one embodiment, the purity of DC preparations is more than 90%.Another In individual embodiment, DC preparations are fully active.For example, activating DC with DC activation schemes, including make DC and TLR activator (examples Such as LPS) contact into line activating.In another embodiment, to actuate different from other enhancings the 3rd signals for the treatment of by calcium thin The combination of the DC activation schemes (such as activator) of intracellular cytokine activates DC.

The present invention includes activating ripe, antigen load the DC that scheme is activated by any DC.The DC of the present invention is produced The cell factor and chemotactic factor (CF) of required level.In one embodiment, the invention provides pulse and the side of active cell Method, thus cell activated state is kept after freezen protective.The DC preparations of the present invention are had an advantage that, come from single leucocyte Exclusion (from patient collect) cell effectively freezen protective be initial vaccine plus multiple " reinforcement " dosage (such as 10 or It is more) form, not any special cell treatment facility or further requirement quality control test in the case of, its It can be thawed as needed in teletherapy position.

The invention further relates to these activation DC freezen protective, with remain thaw after they present antigen in and In various cell factors and the aborning effect and the mode of function of chemotactic factor (CF) so that freezen protective and then thaw swash DC living has Clinical efficacy as fresh harvest and the DC of activation.

As expected in the present invention, it is used to produce the invention provides one kind and freezen protective is stronger to T cell in generation The method that the aspect of signal has the DC of excellent function, and therefore produce the more effectively vaccine based on DC.By effectively freezing Such cell is preserved, can store and the sample that thaws is used to use later, so as to reduce the repeated isolation during production of vaccine The need for elutriation process.DC can be freezed, it is an advantage that then it thaws in the later stage, as it means that single-wheel vaccine Production is segmented into fraction, and freezing is left, and then gives patient one at a time in a few weeks longer, several months, the course for the treatment of of several years, To give " reinforcement " vaccine inoculation of booster immunization.

The present embodiment also includes expressing using HER3 before the cancer as gastroesophageal junction (also referred to as Barrett oesophaguses) The purposes of the mark of tumour progression in lesion.The mark has prognosis and therapeutical uses in wellability stomach oesophagus cancer.

Composition

The invention provides the isolated peptides of HER family proteins and other receptor tyrosine kinases.In an embodiment party In case, the invention provides one or more isolated peptides in HER-1, HER-3 and c-MET albumen.In an embodiment In, peptide of the invention represents the epitope of corresponding HER or c-MET albumen.In some embodiments, corresponding HER or c-MET The epitope of albumen is immunogenicity.

The invention provides the composition of the peptide comprising one or more present invention.Present invention also offers comprising a kind of or The composition of a variety of chimeric peptides.In one embodiment, chimeric peptide includes one or many of corresponding HER or c-MET albumen Individual epitope.

In addition, the invention provides the composition with one or more multivalence peptides.These multivalence peptides include two or many Individual epitope of the invention.

The present invention including the use of the present invention composition subject moderate stimulation immune response method and treating cancer Method, additionally provides the vaccine for treating and preventing purposes.The epitope of the present invention can be wrapped individually or as described herein It is contained in chimeric peptide and causes immune response.In one embodiment, immune response is humoral response.In another embodiment In, immune response is cell-mediated response.According to some embodiments, epitope of the invention or peptide provide protective effect.

In one embodiment, HER-3 epitopes of the invention or other peptides include：

P11-13 (peptide 51-75):KLYERCEVVMGNLEIVLTGHNADLSFLQW(SEQ ID NO:1)；

P81-83 (peptide 401-425):SWPPHMHNFSVFSNLTTIGGRSLYN(SEQ ID NO:2)；

P84-86 (peptide 416-440):TTIGGRSLYNRGFSLLIMKNLNVTS(SEQ ID NO:3)；

P12 (peptide 56-70):CEVVMGNLEIVLTGH(SEQ ID NO:4)；

P81 (peptide 401-415):SWPPHMHNFSVFSNL(SEQ ID NO:5)；

P84 (peptide 416-430):TTIGGRSLYNRGFSL(SEQ ID NO:6)；

P91 (peptide 451-465):AGRIYISANRQLCYH(SEQ ID NO:7).

The HER-3 peptides or any peptide of the present invention can be cyclisation or linear.When cyclisation, epitope can be closed with any Suitable mode is cyclized.For example, disulfide bond can be formed between in the cysteine (Cys) of selection, to provide desired confirmation. It is believed that the formation of cyclisation epitope, which can be provided, improves the conformation of humoral response, so as to improve protecting effect.

By SEQ ID NO：The HER-3 epitopes of 4 definition represent the position 56-70 of HER-3 albumen.By SEQ ID NO：5 determine The HER-3 epitopes of justice represent the position 401-415 of HER-3 albumen.By SEQ ID NO：The HER-3 epitopes of 6 definition represent HER-3 The position 416-430 of albumen.By SEQ ID NO：The HER-3 epitopes of 7 definition represent the position 451-465 of HER-3 albumen.

As described herein, HER-3 epitopes of the invention also include peptide, and it is the function by the SEQ ID NOs peptides defined Equivalent.This functional equivalent has the sequence changed, wherein one or more of corresponding HER-3 epitope sequences amino Acid is substituted, or wherein one or more amino acid lack or be added to corresponding reference sequences from corresponding reference sequences.Example Such as, 1 to 3 amino acid can be added to amino terminal, carboxyl terminal or both.In some instances, HER-3 epitopes are glycosyls Change.

In other examples, HER-3 epitopes can be reverse-reversible type isomers of HER-3 epitopes.Inversely-reversion is repaiied Decorations include the reverse of all amido links in peptide backbone.This reverse can be by the direction of reversed sequence and by using D- amino Acid rather than l-amino acid overturn the chirality of each amino acid residue to realize.This reverse-reversible type isomeric forms can be with Retain flatness and the conformation limitation of at least some peptide bonds.

Nonconserved amino acid substitution and/or conservative replacement can be carried out.When substituted amino acid and the phase in reference sequences When answering amino acid and having similar structure or chemical property, substitution is conservative 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.For example, conserved amino acid Substitution is included with another one aliphatic of substitution or hydrophobic amino acid, such as alanine, valine, leucine and different bright ammonia Acid；With the amino acid of another one hydroxyl of substitution, such as serine and threonine；It is residual with another one acidity of substitution Base, such as glutamic acid or aspartic acid；With another one amide containing residue of substitution, such as asparagine and glutamine；With Another one aromatic moieties of substitution, such as phenylalanine and tyrosine；With another one alkaline residue of substitution, for example, rely ammonia Acid, arginine and histidine；Replace a p1 amino acid, such as alanine, serine, threonine, first sulphur ammonia with another Acid and glycine.

In some instances, lack and add the amino terminal of one of the sequence of peptide positioned at the present invention, carboxyl terminal or Both.For example, HER-3 epitopes equivalent has at least 70% homogeneity with corresponding HER-3 epitope sequences, at least 80% is same Property, at least 85% homogeneity, at least 90% homogeneity, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homogeneity.10 amino acid of each reference sequences are at least 90% identical sequence has no more than 1 change, i.e., any missing, addition or the combination of substitution.By using this area The amino acid sequence of variant and reference sequences are compared to determine homogeneity percentage by the program known or developed.

Functional equivalent is longer than corresponding HER-3 epitope sequences, and functional equivalent can have and wild type HER-3 eggs HER-3 epitope sequences and the sequence at least 90% identical sequence positioned at HER-3 epitope sequences flanks in white.

The functional equivalent of HER-3 epitopes can be by modifying the sequence of epitope, and then determining gained polypeptide stimulates immune The generation of the ability of response, such as antibody is identified.Such antibody can be found in a variety of body fluid include serum and ascites. In brief, humoral sample is separated from warm-blooded animal (such as people), is necessary to determine whether to exist for it to HER-3 polypeptides Antibody.It is being enough to allow to be formed under the condition and time of immune complex between peptide and protein specific antibody, will Body fluid and HER-3 polypeptide cultures, then preferably use elisa technique and are measured.

According to other embodiments of the present invention there is provided chimeric peptide and include the compositions of one or more chimeric peptides. According to various embodiments, HER-3 epitopes are connected comprising HER-3 epitopes, another epitope and with another epitope by chimeric peptide Joint.In one embodiment, other epitopes can include but is not limited to another HER-3 epitope, HER-1 epitopes, HER-2 Epitope and c-Met epitopes.It is also understood that any suitable joint can be used.For example, according to used epitope, HER-3 Epitope may be coupled to the amino or carboxyl terminal of another epitope.The position of other epitopes and selection are depending on HER-3 epitopes Architectural feature, either α spirals or β-bend or chain.

In one embodiment, joint can be that length is about 2 to about 15 amino acid, about 2 to about 10 amino acid Or the peptide of about 2 to about 6 amino acid.Chimeric peptide can be linear or cyclisation.In addition, HER-3 epitopes, other epitopes and/ Or joint can be reverse-inverted versions.Therefore, HER-3 epitopes can be reverse-inverted versions.Or, HER-3 epitopes and Other epitopes can be reverse-inverted versions.In another example, HER-3 epitopes, other epitopes and joint can be inverse To-inverted versions.

In another embodiment, peptide of the invention can in the form of in the mixture rather than chimeric peptide shape Formula.Under any circumstance, the composition of the invention comprising peptide can be that (such as dendron shape is thin for pulse antigen presenting cell Born of the same parents) to produce the useful reagent of cell vaccine.In another embodiment, the composition of the invention comprising peptide can be used The useful immunogene produced in induction of antibodies.The composition of the present invention can be additionally used in immunized subject and delay or pre- preventing tumor Development.The composition of the present invention can be used in vaccine providing protecting effect.

According to the other embodiment of the present invention, there is provided mixed comprising two or more peptides of the invention or chimeric peptide The composition of compound.In some instances, the HER-3 epitopes of each in two or more chimeric peptides are different. In other examples, one of HER-3 epitopes are selected from SEQ ID NOs：1-7.

The peptide of the present invention, including chimeric peptide, can use well known technology to prepare.It is, for example, possible to use recombinant DNA technology Or chemical synthesis, synthetically prepared peptide.The present invention peptide can using it is separately synthesized or as by two or more peptides constitute it is longer Peptide systhesis.The peptide of the present invention is preferably separation, i.e., substantially free of other naturally occurring host cell proteins and its Fragment.

The peptide and chimeric peptide of the present invention can use commercially available peptide synthesizer to synthesize.For example, Kaumaya et al. is in " De Novo”Engineering of Peptide Immunogenic and Antigenic Determinants as Potential Vaccines, in Peptides, Design, Synthesis and Biological Activity (1994), the 133-164 pages " described in chemical method can use, its by quote be incorporated into herein.For example, HER-3 Epitope can be synthesized to form chimeric peptide with another epitope synteny.Peptide symthesis can use Fmoc/t-But chemistry progress.Peptide It can be cyclized in any suitable manner with chimeric peptide.It is, for example, possible to use differentially protected cysteine residues, iodine oxidation, Add water and the removal to promote Acm groups and obtain disulfide bond with disulfide bond and/or the chloro- sulfoxide method of silicyl is formed.

Peptide and chimeric peptide can also use cell free translation system and derived from the DNA construct for encoding epitope or peptide RNA molecule is produced.Or, epitope or chimeric peptide are by using expression vector transfection host cell, and then inducing polypeptide is thin in host Express to prepare in born of the same parents, this expression vector includes the DNA sequence dna for encoding corresponding epitope or chimeric peptide.Produce, pass through for restructuring It is thin that the recombinant precursor of one or more sequences comprising coding epitope, chimeric peptide or its variant is introduced host by conventional method Born of the same parents, the transfection of the conventional method such as calcium phosphate transfection, DEAE- glucans mediation, microinjection, cation lipid-mediation Transfection, electroporation, transduction, scraping, trajectory introduce or infect.

The peptide of the present invention can contain modification, such as glycosylation, oxide side chain or phosphorylation；As long as the modification is not destroyed The bioactivity of peptide.Other modifications include mixing D- amino acid or other can be used for such as serum half-life of increase peptide Amino acid simulant.

The peptide of the present invention can be prepared as combination, and it includes two or more peptides of the invention, as disease Vaccine, for example, cancer.Peptide in the mixture or can use standard technique to be conjugated each other.For example, peptide can be with single more Peptide sequence is expressed.Peptide in combination can be with identical or different.

The present invention should also be construed to include peptide (or encoding its DNA) of the invention " mutant ", " derivative " and " variant ", wherein mutant, derivative and variant are (or when referring to its nucleosides of coding in one or more more amino acid During acid sequence, in one or more base-pairs change) in change peptide so that the peptide (or DNA) of gained with it is as described herein Sequence is different, but with there is identical biological property with peptide disclosed herein.

The present invention also provides the polynucleotides of at least one peptide of coding, and the peptide, which is selected from, has SEQ ID NOs：Appoint in 1-7 The peptide of one or more sequences.Nucleotide sequence includes being transcribed into RNA DNA sequence dna and translates into the RNA sequence of peptide.According to other Embodiment, polynucleotides of the invention are inferred from the amino acid sequence of the peptide of the present invention.As known in the art, due to Redundant codon, the polynucleotides of several replacements are possible, while retaining the bioactivity of translated polypeptide.

In addition, the present invention includes the nucleic acid of separation, this nucleic acid coding has basic homology with peptide disclosed by the invention Peptide.Preferably, the nucleotide sequence for encoding the nucleic acid of the separation of the peptide of the present invention is " substantially homologous ", i.e., about 60% is same Source, more preferably from about 70% is homologous, and even more preferably about 80% is homologous, and more preferably from about 90% is homologous, and even more preferably about 95% is same Source, the nucleotide sequence of the nucleic acid of the separation of the even more preferably about 99% same peptide for coming from the coding present invention.

It should be clearly understood that the scope of the present invention includes homologue, analog, variant, derivative and salt, including it is shorter With longer peptide and polynucleotides, and the peptide and polynucleotides analog replaced with one or more amino acid or nucleic acid, And amino acid known in the art or nucleic acid derivative, non-natural amino or nucleic acid and synthesizing amino or nucleic acid, provide these Modification must keep the biological activity of initial molecule.Specifically, any work of active peptide is disclosed according to the principle of the present invention Property fragment and extension, conjugate and mixture.

The present invention should be interpreted that including any and all core that separates homologous with nucleic acid of the present invention and referenced Acid, as long as these homologous dnas have the bioactivity of peptide disclosed by the invention.

It will be understood by those skilled in the art that peptide and its any modified forms of the nucleic acid of the present invention including the coding present invention RNA or DNA sequence dna, include DNA or RNA chemical modification, and it causes nucleotides sequence to be listed in it without cell or when itself and cell It is more stable when related.The chemical modification of nucleotides can be used for strengthening the efficiency that is absorbed by cell of nucleotide sequence or its thin The efficiency expressed in born of the same parents.The present invention covers any and all modification combination of nucleotide sequence.

Further, any amount of program is used for recombinant DNA method well known in the art and produces the present invention's Mutant, derivative or the variant form of protein, such as in Sambrook and Russell (ibid), and Ausubel et al. description Method.By changing the DNA sequence dna of coded polypeptide, the method that amino acid change is introduced in peptide or polypeptide is known in this field , and be also described in detail in these places, elsewhere with paper.

Encoding the nucleic acid of the peptide of the present invention can mix in suitable carrier, for example, retroviral vector.These carriers It is well known in the art.In cell needed for nucleic acid or carrier containing them can be effectively transferred to, the cell is preferred from Patient.Advantageously, the invention provides a kind of ready-made composition, it allows the cell of quick modification patient itself (or another The cell of kind of mammal) modified cells of property are killed with excellent cancer cell rapidly and easyly to produce.

Carrier

In other related fields, the present invention has including coding one or more is selected from SEQ ID NOs：The group of 1-7 compositions In sequence peptide seperated nuclear acid.

In one embodiment, the present invention includes the nucleotide sequence of the peptide of the one or more present invention of coding, and it can be grasped It is connected to the nucleic acid for including promoter/regulatory sequence with making so that nucleic acid is preferably able to the table for the protein for instructing nucleic acid to encode Reach.Therefore, the present invention includes being used for the expression vector that exogenous DNA is introduced into cell and expressed with exogenous DNA in cell And method, such as Sambrook et al. (2012, 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) it is described.By desired polynucleotides mix carrier in And the selection of carrier is it is known in the art that as described in Sambrook et al. (ibid) and Ausubel et al. (ibid).

Polynucleotides can be cloned into polytype carrier.However, the present invention should not be construed as limited to it is any Specific support.On the contrary, the present invention should be construed to include being readily available in this area and/or known a large amount of carriers.For example, this The polynucleotides of invention can be cloned into carrier, and carrier includes but is not limited to plasmid, phasmid, phage-derived thing, animal Virus and clay.Carrier of special interest includes expression vector, replicating vector, probe generation vectors and sequencing vector.

In a particular embodiment, expression vector is selected from viral vector, bacteria carrier and mammalian cell carrier.In the presence of Many includes the expression vector system of at least partially or fully above-mentioned composition.What it is based on protokaryon and/or eucaryote carrier is System can be used for the present invention to produce polynucleotides or its homeopeptide.Many such systems are commercially broadly available.

Further, expression vector can be provided to cell in the form of viral vector.Viral vector technology is that this area is public Know, and such as Sambrook et al. (2012) and in Ausubel et al. (1997) and other virology and molecular biology Described in handbook.Can be used as carrier virus include but is not limited to retrovirus, adenovirus, adeno-associated virus, herpesviral and Slow virus.Generally, suitable carrier contains at least one organism functional replication orgin, promoter sequence, conveniently Restriction endonuclease site and one or more selected markers (referring to WO01/96584, WO01/29058 and the U.S. are special Profit number is 6,326,193).

In order to which at least one module expressed in the required nucleotide sequence of the present invention, each promoter is used to position RNA The initiation site of synthesis.Its most well known example is TATA boxes, but in some promoters of TATA boxes are lacked, such as lactation The promoter of animal terminal deoxynucleotidyl transferase gene and the promoter of SV40 genes, covering initiation site discrete in itself Element helps to fix original position.

Other promoter element, i.e. enhancer, adjust the frequency of transcription initiation.Generally, these promoter elements are located at Initiation site upstream 30-110bp region, although having shown that many promoters also include the function in initiation site downstream recently Element.Interval between promoter element is typically flexible so that can be kept when element is inverted relative to each other or is moved Promoter function.In thymidine kinase promoter, the interval between promoter element can increase before activity is begun to decline To 50bp.According to promoter, it can be seen that Individual elements can synergistically or independently work with activated transcription.

Promoter can be to the natural related promoter of gene or polynucleotide sequence, such as by separating positioned at code area The promoter that the 5' non-coding sequences of section and/or extron upstream can be obtained.This promoter is properly termed as " endogenous ".Class As, enhancer can be and the natural related enhancer of the polynucleotide sequence positioned at the sequence downstream or upstream.Or, lead to Cross coded polynucleotide fragment being placed under restructuring or the control of allogeneic promoter and obtain some advantages, the promoter refers to Promoter generally not related to polynucleotide sequence in its natural surroundings.Restructuring or heterologous enhancer also refer in its natural surroundings In the usual enhancer that is not combined with polynucleotide sequence.Such promoter or enhancer can include the promoter of other genes Or enhancer, and from the promoter or enhancer of any other protokaryon, virus or eukaryotic separation, and non-" naturally deposit " promoter or enhancer, the i.e. different elements containing different transcriptional regulatory districts, and/or change the mutation of expression.Except closing Outside into the nucleotide sequence for producing promoter and enhancer, can use recombinant clone and/or nucleic acid amplification technologies (including PCRTM), and with reference to composition disclosed by the invention (United States Patent (USP) 4,683,202, United States Patent (USP) 5,928,906) sequence is produced. Further, it is also possible to which control sequence can also be used by predicting, this control sequence carves guide sequence in non-core organelle such as mitochondria, leaf Transcribe and/or express in green body etc..

Naturally, it is important that cell type, organelle and organism are effectively instructed using promoter and/or enhancer In DNA section expression.The technical staff of biology field typically knows how to use promoter, enhancer and cell Type combination is used for protein expression, for example, see Sambrook etc. (2012).Used promoter can be composing type, group Knit specificity, induction type and/or under proper condition be used for instruct introduce DNA section high level expression be it is useful, for example Be conducive to large-scale production of recombinant proteins and/or peptide.Promoter can be heterologous or endogenous.

The promoter sequence of example is early stage cytomegalovirus (CMV) startup immediately in the EXPERIMENTAL EXAMPLE that the present invention is provided Subsequence.The promoter sequence is strong constitutive promoter sequence, and it can drive any multinuclear being operably connected with it The high level expression of nucleotide sequence.It is also possible, however, to use other constitutive promoter sequences, including but not limited to simian virus 40 (SV40) early promoters, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) LTR (LTR) promoter, Moloney viral promotors, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous meat It is tumor virus promoter, and people's gene promoter, such as, but not limited to actin promoter, Myosin promoter, blood red Protein promoter and muscle creatine promoter.In addition, the present invention, which should not necessarily be limited by, uses constitutive promoter.Inducible promoter It is considered as the part of the present invention.Polynucleotide sequence table can be started by being provided in the present invention using inducible promoter The molecular switch reached, when expecting this expression, it is operably connected, or the closing expression when that need not express.Induction The example of type promoter includes but is not limited to metallothionein promoter, Glucocorticoid promoter, progesterone promoter and Fourth Ring Plain promoter.In addition, the present invention includes the use of tissue-specific promoter, the promoter is only active in required tissue.

For the expression of the nucleotide sequence of the peptide of assessing the coding present invention, expression vector in cell to be imported can be with Containing selected marker or reporter gene or both, in order to from the cell mass for attempting to transfect or infect by viral vector Expression cell is identified and selected in body.In other embodiments, selectable marker can be carried on single DNA fragmentation simultaneously For cotransfection program.Both selected marker and reporter gene can be enabled in host with the appropriate side joint of regulatory sequence Expressed in cell.Useful selectable marker is known in the art, and including such as antibiotics resistance gene, such as neo Deng.

Reporter gene is used to identify the cell of potential transfection and the function for evaluating regulatory sequence.Coding is easy to what is determined The reporter of protein is well known in the art.Generally, reporter be not present in recipient organism or tissue in or by The gene of recipient organism or tissue expression, and encoding proteins matter, the property that this protein expression is easily detected by some Matter (such as enzymatic activity) is showed.The expression of reporter is that the right times after DNA to be introduced to recipient cell are determined.

Suitable reporter can include the plain enzyme of coding fluorescence, beta galactosidase, chloramphenicol acetyltransferase, secretion Alkaline phosphatase or green fluorescence protein gene gene (see, for example, Ui-Tei et al., 2000FEBS Lett.479：79- 82).Suitable expression system is known, and well known technology can be used to prepare or commercially-available.Inside missing is built The partial digested generation that body can use the internal restriction sites of uniqueness or pass through nonuniqueness restriction site.Then can be by Construct is transfected into the cell for showing high-caliber siRNA polynucleotides and/or expression of polypeptides.Generally, with minimum 5' sides The construct in pterion is accredited as promoter, and this minimum 5' flanking region shows the highest level expression of reporter.It is such to open Sub-area may be coupled to reporter gene and for the reagent for the transcriptional capability for assessing regulation promoter driving.

Vaccine

In one embodiment, the present invention relates to the vaccine of the peptide comprising the present invention.The vaccine of the present invention can be provided Any combinations of particular peptide, the particular peptide is to need the cancer in the subject for the treatment of for specific prevention or treatment.

The vaccine of the present invention can be with inducing antigen-specific T cell and/or high titre antibody response, so as to induce or trigger Cancer or tumour or to expression antigen cancer or the immune response of tumor response for expression antigen.In some embodiment party In case, induction or the immune response triggered can be cell, body fluid or cell and humoral immune response.In some embodiments In, induction or caused cellullar immunologic response can include interferon-γ (IFN-γ) and/or tumor necrosis factor α (TNF-α) Induction or secretion.

In one embodiment, the present invention relates to anti-cancer vaccine.Vaccine can include one or more cancer antigens.The epidemic disease Seedling can prevent tumour growth.The vaccine can reduce tumour growth.The vaccine can prevent the transfer of tumour cell.Depend on Cancer antigen, vaccine can be with targeted therapy breast cancer, liver cancer, prostate cancer, melanoma, leukemia, head and neck cancer, spongioblast Knurl, recurrent respiratory papilloma, cancer of anus, cervix cancer, cancer of the brain etc..

In a specific embodiment, vaccine can be by inducing come the removing of mediate tumor cell or preventing to give birth to Long, induction mode includes：(1) antibody needed for humoral immunity is produced by B cell response；(2) increase cytotoxic T is thin Born of the same parents such as CD8+ (CTL) is to attack and kill tumour cell；(3) t helper cell response is increased；(4) and IFN-γ and TFN- α are passed through Or preferably all above-mentioned substance increase inflammatory reactions.Vaccine can make no tumor survival increase by 30%, 31%, 32%, 33%, 34%th, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%44% and 45%.Vaccine can be after immune By tumor quality reduce 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%th, 43%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%th, 59%.

Compared with the cellullar immunologic response in the subject for not applying vaccine, vaccine can be in the subject for applying vaccine Increase about 50 times to about 6000 times of cellullar immunologic response, about 50 times to about 5500 times, about 50 times to about 5000 times, about 50 times about 100 times to about 6000 times, about 150 times to about 6000 times, about 200 times to about 6000 times, about 250 times to about 6000 times or about 300 Again to about 6000 times.In some embodiments, compared with the cellullar immunologic response in the subject for not applying vaccine, vaccine can With apply vaccine subject in increase about 50 times of cellullar immunologic response, 100 times, 150 times, 200 times, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 Times, 1000 times, 1100 times, 1200 times, 1300 times, 1400 times, 1500 times, 1600 times, 1700 times, 1800 times, 1900 times, 2000 Times, 2100 times, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 Times, 3200 times, 3300 times, 3400 times, 3500 times, 3600 times, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 Times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 Again, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times.

Compared with the IFN-γ level in the subject for not applying vaccine, vaccine can increase in the subject for applying vaccine Plus about 50 times to about 6000 times of interferon gamma (IFN-γ) level, about 50 times to about 5500 times, about 50 times to about 5000 times, about 50 Times to about 4500 times, about 100 times to about 6000 times, about 150 times to about 6000 times, about 200 times to about 6000 times, about 250 times extremely About 6000 times or about 300 times to about 6000 times.In some embodiments, the IFN-γ water in the subject with not applying vaccine It is flat to compare, vaccine can make the subject using vaccine about 50 times of IFN-γ level increase, 100 times, 150 times, 200 times, 250 Times, 300 times, 350 times, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times, 1000 times, 1100 times, 1200 times, 1300 times, 1400 times, 1500 times, 1600 times, 1700 times, 1800 times, 1900 times, 2000 times, 2100 times, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, 3500 times, 3600 times, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times.

The vaccine of the present invention can have the feature needed for effective vaccine, such as safe so that vaccine does not cause disease in itself Disease is dead；Prevent disease；Induce neutralizing antibody；Inducing protective T cell response；And it is easy to administration, few side effects, biology surely Qualitative high and per dosage cost is low.Vaccine can be by realizing some in these features containing cancer antigen as discussed below Or all.

Load the generation of (pulse) immunocyte

The present invention includes being exposed to the peptide of antigen or the present invention or thin with antigen or peptide " pulse " of the invention Born of the same parents.For example, APC (such as DC) can load Ag in vitro, such as by the cultured in vitro in the presence of antigen, or by exposed to Antigen In vivo culture.

Those skilled in the art also will readily appreciate that APC can by APC exposed to antigen for a period of time by way of quilt " chopping ", the time is enough to promote the antigen to present on APC surfaces.For example, APC, which can be exposed to, is referred to as Antigenic Peptide The antigen of small peptide pieces, its direct " pulse " to APC outside (Mehta-Damani et al., 1994)；Or APC can be with Complete protein or protein particulate culture, are then absorbed by APC.These whole albumen are digested to small fragments of peptides by APC, And finally carry and present on APC surfaces (Cohen et al., 1994).The antigen of peptide form can pass through mark of the present invention Accurate " pulse " technology is exposed to cell.

It is not intended to any particular theory, the antigen of external source or autoantigen form is processed by the APC of the present invention To retain the immunogenic forms of antigen.The immunogenic forms of antigen mean can quilt to produce by fragmentation processing antigen Immunocyte (such as T cell) recognizes and stimulates the antigen forms of immunocyte.Preferably, this external source or autoantigen are logical Cross the protein that APC is processed into peptide.The related peptide produced by APC can be extracted and purify for use as immunogenic composition. It can also be used for inducing the protein tolerance to being handled by APC by the APC peptides handled.

The APC of Antigen, or be referred to as the present invention " pulse APC " is by being in vitro or in vivo exposed to APC What antigen was produced.In the case of APC in vitro pulse, APC can be layered on culture dish, and with enough amounts and enough Time is exposed to antigen, to allow antigen binding APC.Realizing that antigen is combined required amount and time with APC can be by using It is known in the art or disclosed method is determined in addition herein.Other methods well known by persons skilled in the art, for example, be immunized Determine or combination mensuration, available for the presence that antigen on APC is detected after antigen.

In another embodiment of the present invention, APC can be transfected with carrier, and this carrier allows APC expression specificities Protein.Then it can will be processed and be presented on cell surface by the APC protein expressed.Then can be by the APC of transfection As immunogenic composition, to produce the immune response to the protein by vector encoded.

As discussed elsewhere herein, carrier can be prepared with including specific polynucleotides, this specific polynucleotides Coding and expression need the protein of immunogenic response.Preferably, using retroviral vector infection cell.More preferably Ground, is used for infection cell using adenovirus vector.

In another embodiment, carrier can be encoded by modification virus carrier by the Receptor recognition on APC Protein or part thereof targets APC, thus the endocytosis that occupying start carrier of the carrier to APC acceptors, it is allowed to process The antigen encoded with the nucleic acid presented by viral vector.Virus can be originated in by the nucleic acid of Viral delivery, when the expression on APC When, it encodes virus protein, is then processed and presented on APC MHC acceptors.

As expected in the present invention, a variety of methods can be used for transfecting polynucleotides into host cell.Method include but Be not limited to calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, dispersion system of colloid (i.e. macromolecular be combined Thing, Nano capsule, microballoon, pearl and the system based on lipid, including oil in water emulsion, micella, mixed micelle and liposome).This A little methods are well known in the art, and described in disclosed document, to cause those skilled in the art to be able to carry out These methods.

In another embodiment, the polynucleotides of coding for antigens can be cloned into expression vector, and can be by Carrier is introduced into APC, otherwise produces the APC of load.The method for introducing cell by various types of carriers and by nucleic acid can obtained It is discussed in the open source literature obtained.For example, expression vector can be transferred to host thin by physics, chemistry or biological means In born of the same parents.See, e.g., (2012, the Molecular Cloning such as Sambrook：A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and (1997, the Current Protocols in such as Ausubel Molecular Biology, John Wiley＆Sons, New York).Introduce the expression of the polynucleotides comprising coding for antigens Carrier produces what pulsed cell will be appreciated that.

The present invention includes the various methods for pulse APC, including but not limited to protein, cDNA or mRNA forms Intact antigen loads APC.However, the present invention should not be construed as limited to the particular form of the antigen for pulse APC.Phase Instead, the present invention includes the other methods known in the art for being used to produce the APC of Antigen.Preferably, limited with coding The mRNA transfections APC of antigen.Suitable primer and the reverse transcriptase-polymerase chain reaction being coupled with responsive transcription can be used (RT-PCR) it is quick in vitro to produce the mRNA for corresponding to gene outcome known to its sequence.Provided with mRNA transfections APC excellent In the advantage of other antigen load technologies for producing pulse APC.For example, from microcomponent (i.e. tumor tissues) cloning RNA Ability APC use is expanded in the vaccine inoculation of a large amount of patients.

Antigen composition for can be used as vaccine, antigen composition must be in cell, tissue or mammal (for example People) the middle immune response induced for antigen.As used in the present invention, " immune composition " can include antigen (such as peptide or many Peptide), the nucleic acid of coding for antigens (such as Antigen Expression Vectors) or expression or the cell for presenting antigen or cellular component.Specific real Apply in scheme, all or part or its immunologic function that antigen composition included or encoded any antigen of the present invention are of equal value Thing.In other embodiments, antigen composition is in the nucleic acid comprising other immunostimulant or the such reagent of coding In mixture.Immunostimulant includes but is not limited to extra antigen, immunomodulator, antigen presenting cell or adjuvant.At it In its embodiment, one or more other reagents are with any combinations and antigen or immunostimulant covalent bonding.Some In embodiment, antigen composition is conjugated to or comprising HLA Anchor motifs amino acid.

As expected in the present invention, vaccine can be different in terms of the composition of its nucleic acid and/or cellular component.Unrestricted In property example, the nucleic acid of coding for antigens can also be prepared together with adjuvant.Of course it is to be understood that various groups of the present invention Compound can further include other component.For example, one or more vaccine components may be embodied in lipid or liposome. In another non-limiting examples, vaccine can include one or more adjuvants.It is of the invention according to present disclosure Vaccine and its various components can be prepared by any method disclosed herein or known to persons of ordinary skill in the art and/or Using.

It should be appreciated that the antigen composition of the present invention can be prepared by methods well known in the art, include but is not limited to The chemical synthesis purified in the other products chemically reacted by synthesis in solid state and by HPLC, or be by translating in vitro Expression encodes the nucleotide sequence (such as DNA sequence dna) of the peptide comprising antigen of the present invention or polypeptide and produced in system or living cells.Separately Outside, antigen composition can include the cellular component separated from biological sample.By antigen composition separate and dialyse extensively with Remove one or more undesirable small molecular weight molecules and/or freeze more easily to be configured to required carrier.It should also manage Solve, other amino acid, mutation, chemical modification for being prepared in vaccine component etc. (if any) do not disturb anti-preferably substantially Original identification epitope sequences.

Antigen presenting cell is treated

The present invention includes producing APC colonies (such as BMDC for presenting peptide of the present invention in its surface；DC side) Method, it can be used subsequently to treatment.This method can in vitro be carried out from the cell sample that patient obtains.Therefore, with The APC that this mode is produced can prepare the medicament for treating or preventing cancer.Cell should be received by the immune system of individual, Because they come from the individual.The cell produced by this way is delivered to them initially from the individual that it is obtained, so that shape Into the treatment embodiment of the present invention.

DCs is derived from the versatility monocyte as antigen presenting cell (APC).DCs is generally to deposit in peripheral tissues , wherein they are produced to capture antigen.After antigen capture, antigen is processed into small peptide and shifts to Secondary Lymphoid by DCs Organ.In lymphoid organ, DCs presents Antigenic Peptide to nave T cell, so as to trigger the signal cascade of polarization T cell differentiation.Cruelly After dew, there is the antigen molecule combined with MHC I classes or II class binding peptides in DCs, and activate CD8⁺Or CD4⁺T cell (Steinman, 1991, Annu.Rev.Immunol.9：271-296；Banchereau etc., 1998, Nature392,245- 252；Steinman, etc. 2007, Nature 449：419-426；Ginhoux etc., 2007, J.Exp.Med.204：3133- 3146；Banerjee etc., 2006, Blood 108：2655-2661；Sallusto etc., 1999, J.Exp.Med.189：611- 614；Reid etc., 2000, Curr.Opin.Immunol.12：114-121；Bykovskaia etc., 1999, J.Leukoc.Biol.66：659-666；Clark etc., 2000, Microbes Infect.2：257-272).

DCs is responsible for the induction of adaptive immune response, coordinates and adjust, and is additionally operable to coordinate the effector of congenital mode Communication between the accommodation mode of immune system.These features cause DCs to turn into the strong candidate of immunization therapy. DCs have by macrophage increase the unique ability being sampled with receptor-mediated encytosis to environment (Gerner etc., 2008,J.Immunol.181:155–164；Stoitzner etc., 2008, Cancer Immunol.Immunother 57: 1665-1673；Lanzevecchia A.,1996,Curr.Opin.Immunol.8:348-354；Delamarre etc., 2005, Science,307(5715):1630-1634)。

DCs also needs to ripe signal to strengthen its antigen presentation capability.DCs is by providing extra ripe signal for example TNF-α, CD40L or Ca2+ oscillations transduction agent raise surface molecular (such as CD80 and CD86 (also referred to as secondary signal molecule)) Express (Czerniecki etc., 1997 .J.Immunol.159:3823-3837；Bedrosian etc., 2000, J.Immunother.23:311-320；Mailliard etc., 2004, Cancer Res.64,5934-5937；Brossart etc., 1998,Blood 92:4238-4247；Jin etc., 2004, Hum.Immunol.65:93-103).Have determined including TNF-α, The mixture of the cell factor of IL-1 β, IL-6 and prostaglandin E2 (PGE2) have make DCs ripe ability (Jonuleit etc., 2000,Arch.Derm.Res.292:325-332).DC can also be ripe with Calcium ionophore before with antigen pulse.

Except pathogen identification receptor, such as PKR and MDA-5 (Kalali, 2008, J.Immunol.181:2694- 2704；Nallagatla etc., 2008, RNA Biol.5 (3):140-144), DCs also contains a series of receptors, referred to as Toll-like Acceptor (TLR), it can also sense the danger from pathogen.When these TLR are triggered, induce a series of sharp in DCs Change living, this causes the maturation and signal transduction ((Boullart etc., 2008, Cancer of T cell Immunol.Immunother.57(11):1589-1597；Kaisho etc., 2003, Curr.Mol.Med.3 (4):373-385； Pulendran etc., 2001, Science293 (5528):253-256；Napolitani etc., 2005, Nat.Immunol.6 (8): 769-776).DC can activate and extend the various arms of cell-mediated response, and such as NKT γ-delta T cells and alpha-beta T are thin Born of the same parents, and once activate, DCs keeps its immunocompetence (Steinman, 1991, Annu.Rev.Immunol.9:271-296； Banchereau etc., 1998, Nature 392:245-252；Reid etc., 2000, Curr.Opin.Immunol.12:114- 121；Bykovskaia etc., 1999, J.Leukoc.Biol.66:659-666；Clark etc., 2000, Microbes Infect.2:257-272)。

Present invention also offers the method for one or more inducing peptide antigen presenting cells (APC) using the present invention.APC Can be by inducing the BMDC from PMBC, then in vitro/in vitro or one with the present invention in vivo Plant or a variety of peptides contact (stimulation) to induce.When the peptide of the present invention is applied into mammal in need, in mammal Immune inducing in vivo has the APC of the peptide of the present invention, and this peptide is fixed on APC.Or, can after the peptide of the present invention is fixed into APC Using by cell as vaccine administration to subject.For example, in vitro apply may comprise steps of：APC is collected from mammal, And contact APC and the peptide of the present invention.

The present invention also provides APC, and it presents the compound formed between one or more peptides of HLA antigens and the present invention. Contact the APC obtained by the nucleotides of the peptide with the present invention or the such peptide of coding and be preferably derived from and be used as treatment and/or prevention Target subject, and can as vaccine individually or with other drugs be administered in combination, other drugs include the present invention Peptide, allochthon or T cell.

The invention provides stimulate APC (preferably DCs) under immunization therapy within a context to stimulate in mammal The composition and method of immune response.DCs can by using the present invention peptide or peptide combination of stimulation they be manipulated, and make DCs is ripe, so that they are in mammal moderate stimulation antineoplastic immune in need.

In one embodiment, present invention resides in the method for inducing T cell response in mammal.Methods described bag Include and apply APC, for example DC, wherein the APC is by making APC combine contact with the peptide or peptide of the present invention and be activated, so as to produce Load the APC of peptide.

In one embodiment, the present invention relates to produced new APC and its for especially expand required T cell, Activation T cell, specific amplification T cell and amplification and the thorn for being related to and using the peptide load APC of the present invention and the T cell of peptide Swash related many therapeutical uses.In some cases, the DCs that OCT4 is stimulated can be used for extending peptide-specific T-cell.

Found the present invention relates to one kind, it is that the DC for combining contact with the peptide or peptide of the present invention can be used for induction peptide specific The amplification of T cell.It would be recognized by those skilled in the art that being considered as initiation or load peptide with the DCs that the peptide of the present invention is contacted 's.The DCs of present invention load peptide can be used for triggering the immune response for required antigen (such as HER-3).Therefore, the present invention is negative The DCs for carrying peptide can be used for the treatment disease related to HER-3 imbalance expression.

The method for treating disease

Present invention additionally comprises treatment and/or prevention by pathogenic microorganism, Autoimmune Disorders and/or excess proliferative disease The method of caused disease.

It can be included by using the disease that treat or prevent of the present invention by virus, bacterium, yeast, parasite, primary moved Disease caused by thing, cancer cell etc..The present invention pharmaceutical composition may be used as broad sense immunopotentiator (DC activate composition or System), therefore available for treatment disease.The medicine composite for curing of the present invention and/or the Exemplary diseases of prevention can be used The including but not limited to infection of viral aetiology, such as HIV, influenza, bleb, virus hepatitis, Epstein-Barr virus, polio, Viral encephalitis, measles, varicella, papillomavirus etc.；Or bacterial etiology infection, such as pneumonia, tuberculosis, syphilis；Or post The infection of infested aetology, such as malaria, trypanosomiasis, leishmaniasis, trichomoniasis, amcbiasis.

The pharmaceutical composition (DC, expression vector, expression construct of transduction etc.) of the present invention can be used to treat or prevent Cancer before or proliferative state, include but is not limited to the cancer such as polyp of colon, Crohn disease, ulcerative colitis, breast lesion Preceding or proliferative state.

The cancer of the invention of the composition treatment of the present invention can be used, including but not limited to primary or metastatic is black Melanoma, gland cancer, squamous cell carcinoma, adenosquamous carcinoma, thymoma, lymthoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymph Knurl, Hodgkin lymphoma, leukaemia, uterine cancer, breast cancer, prostate cancer, oophoroma, cancer of pancreas, colon cancer, multiple marrow Knurl, neuroblastoma, human primary gastrointestinal cancers, the cancer of the brain, carcinoma of urinary bladder, cervix cancer etc..

Other excess proliferative diseases including but not limited to rheumatoid of the DC activation systems treatment of the present invention can be used It is property arthritis, inflammatory bowel disease, osteoarthritis, liomyoma, adenoma, lipoma, hemangioma, fibroma, vascular occlusion, narrow again Narrow, atherosclerosis, precancerous lesion (such as adenomatous hyperplasia and prostatic intraepithelial neoplasia), carcinoma in situ, oral hairy are white Spot or psoriasis.

The autoimmunity sexual maladjustment of the composition treatment of the present invention can be used to include but is not limited to AIDS, A Disenshi Disease, adult respiratory distress syndrome (ARDS), allergy, anaemia, asthma, atherosclerosis, bronchitis, cholecystitis, Crohn disease, burst Ulcer colitis, atopic dermatitis, dermatomyositis, diabetes, pulmonary emphysema, erythema nodosum, atrophic gastritis, glomerular kidney Inflammation, gout, Graves disease, IHES, IBS, lupus erythematosus, multiple sclerosis, severe flesh Powerless, myocardium or pericarditis, osteoarthritis, osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis, chorionitis, Sjogren syndrome and autoimmune thyroiditis；Complication, haemodialysis and the extracorporal circulatory system of cancer；It is virus, bacterium, true Bacterium, parasite, protozoan and invermination；And wound.

In treatment method, the administration of the present composition can be " preventative " or " therapeutic " purpose.When preventative When ground is provided, composition of the invention is provided before any symptom, but in a particular embodiment, in one or more diseases There is provided vaccine to prevent further symptom development or prevent existing symptom from becoming worse after shape breaking-out.The preventive administration of composition For preventing or improving any subsequent infection or disease.When with therapeutic offer, in the paresthesia epilepsy of infection or disease Or pharmaceutical composition is provided afterwards.Therefore, the present invention can be expected before pathogenic agent or morbid state or in infection Or disease starts to provide afterwards.

The effective dose of composition by be the selected result for realizing enhancing immune response dosage, and such dosage It can be determined by those skilled in the art as general matter.For example, for siberian crabapple of the medical needle to cancer or pathogen The effective dose of system defect can cause the dosage needed for immune system activation, cause occur antigen-specific when exposed to antigen Property immune response.The term is also synonymous with " sufficient amount ".

Effective dose for any concrete application can be according to the disease or imbalance such as treated, the specific group applied Compound, the size of subject and/or the factor such as disease or the seriousness of imbalance and change.Those of ordinary skill in the art can be with The effective dose of empirically determined particular composition of the invention, without excessive experiment.

Bacterin preparation

Present invention additionally comprises the bacterin preparation suitable for immunization therapy.In certain embodiments, bacterin preparation is used for pre- Anti- and/or treatment disease, such as cancer and infectious diseases.In one embodiment, patient is applied according to the present invention Vaccine for prevention and/or treating cancer can be carried out before or after the surgical operation of cancer is removed, for treating Carry out, and before or after the radiotherapy for treating cancer and its appoint before or after the chemical therapeutic method of cancer What combination is carried out.In other embodiments, bacterin preparation can combine or combine with another composition or drug products and apply For patient.It should be appreciated that the present invention can be also used for the cancer in prevention individual, this individual no cancer but trouble is likely to be at Risk of cancer.

The administration of cancer vaccine prepared in accordance with the present invention is widely used in prevention or treating cancer, and the cancer is to a certain degree The selection of the upper antigen formation part depended on to cancer vaccine.The cancer that can be suitably treated according to the practice of the present invention includes But it is not limited to lung cancer, breast cancer, oophoroma, cervix cancer, colon cancer, head and neck cancer, cancer of pancreas, prostate cancer, stomach cancer, bladder Cancer, kidney, osteocarcinoma, liver cancer, esophagus knurl, brain tumor, testicular tumor, hysteroma and various leukaemia and lymthoma.

In one embodiment, tumour or cancer cell to be treated can be derived from according to the vaccine of the present invention.For example, In the treatment of lung cancer, lung carcinoma cell will be treated to produce lung cancer vaccine as described above.Similarly, mammary gland Theratope, knot Intestinal cancer vaccine, pancreatic tumor vaccine, stomach Theratope, bladder Theratope, kidney vaccine etc., according to practice, will be used as immunotherapeutic agent Produced and applied, to prevent and/or treat the tumour or cancer cell that produce vaccine.

In another embodiment, as mentioned, can also be by collecting by pathogen stream according to the vaccine of the present invention Enter the related antigen in culture medium to be produced to treat the various infectious diseases of influence mammal.Due to identical by causing In the immunogenicity of different types of organism expressing and the type of protective antigens of disease exist heterogeneity, can by from The organism storehouse of the different important antigen of expression prepares vaccine to prepare polyvaccine.

In another embodiment of the present invention, vaccine can be administered to inguinal lymph nodes by being injected in tubercle In.Or, according to vaccine targets, vaccine can be through intracutaneous or subcutaneous administration in the four limbs, arm and leg of patient under consideration.Although This method is gratifying generally for melanoma and other cancers (including prevention or treatment infectious diseases), but It can use other methods of administration, for example, intramuscular or enter blood flow.

In addition, vaccine can be together with adjuvant and/or immunomodulator to the anti-of the activity and patient for being strengthened vaccine Should.Such adjuvant and/or immunomodulator are it will be appreciated by those skilled in the art that and holding in obtainable open source literature Easily description.

As expected in the present invention, and depending on produced vaccine classes, if it is desired, can be by biological anti- Answer and cultivate cell in device or fermentation tank or other containers or device suitable for raised growth cell to expand the yield of vaccine.At this In the device of sample, periodically, culture medium is frequently or continuously collected, with before these materials or antigen degradation, from culture medium Reclaim any material or antigen.

If desired, the device or composition comprising vaccine or antigen for being produced and being reclaimed according to the present invention, this device or Composition is suitable to continue or interval discharges, in fact, implanting or being locally applied in vivo, so that this material is relatively slow Or time controlled released is in vivo.

Other steps in prepared by vaccine can meet the requirement of specific vaccine with individuation.Such additional step ability Field technique personnel should be appreciated that.For example, the antigen-like material of some collections can be concentrated and use detergent in some cases Handle and ultracentrifugation is to remove transplanting alloantigen.

HER3 expresses the biomarker of diagnosis and the treatment as disease

In another embodiment, HER3 expression may be used as with Barrett oesophaguses and Height Anomalies structure (HGD) biomarker of the invisible invasive disease in patient.The present invention consider in addition be using HER3 or CMET as The therapeutic agent of target, it can provide the secondary prevention of stomach oesophagus cancer in some patients.

These methods of the present invention be definitely not all include, and be adapt to application other methods for Those of ordinary skill is obvious.In addition, the effective dose of composition can be by similar to effect needed for known performance Compound is further estimated.

Embodiment

The present invention is described in further detail by reference to following examples.There is provided what these embodiments were merely to illustrate that Purpose, and be not intended to it is restricted, unless otherwise prescribed.Therefore, the present invention is certainly not understood as limited to following implementation Example, but should be understood to cover becomes obvious any and all changes shape due to the teachings that the present invention is provided Formula.

It is not described any further, it is believed that those of ordinary skill in the art can use preceding description and following illustrative implementation Example is manufactured and using the present invention, and the method for practice calls protection.Therefore, following examples point out the excellent of the present invention Embodiment is selected, and is not necessarily to be construed as limiting remainder of this disclosure in any way.

Embodiment 1：Create the peptide vaccine of other receptor tyrosine kinases for causing breast cancer and other solid cancers

Contrived experiment is directed to the alternative medicine of those patients for being identified as BRCA carriers of mutation to develop.Namely Say exist for mammary cancer risk in heredity, substitute that is seeking two point mastectomies young patient Unsatisfied demand.

The women for being mutated BRCA1/BRCA2 with mastocarcinoma gene has 70% lifetime risk for developing into breast cancer, And BRCA1 carriers of mutation often develops triple negative breast cancer.Contrived experiment is to develop the vaccine for the group and evaluate it Immune induction experiment in security this be vaccine inoculation be used for initially prevent breast cancer first time attempt.It will also include BRCA2 carriers of mutation prevents estrogen receptor positive breast cancer with the polyvaccine for seeing whether the usable present invention.

Contrived experiment is to study the expression of the receptor tyrosine kinase in breast cancer and DCIS from BRCA carriers of mutation. It was observed that the tumour early stage from BRCA carriers of mutation is usually overexpressed c-MET oncogene and HER-3, and from not mutated Or the tumour expression HER-2 and HER-3 of sporadic patient.This is important, because according to the disclosure of the present invention, now The target of known cancer immunization therapy can be used for exploitation to be used for the vaccine of sporadic and BRCA carriers of mutation.This is using immune Response prevents targetedly first distinguishing characteristics.Therefore, the present invention include for vaccine development composition and method and It is used for the purposes for preventing the substitute as bilateral mastectomy.

Create peptide vaccine

HER families are made up of four kinds of related signaling molecules-HER-1, HER-2, HER-3 and HER-4 of participation kinds cancer. The known overexpression that HER-2 is found in 20% to 30% breast cancer.Provided herein is result prove other HER family members Participate in early stage and infiltrative breast carcinoma and other cancers.For example, HER-1 is expressed in a small number of breast cancer, typically three is negative Breast cancer.C-MET is the growth factor receptors of the recurrence for many cancers for participating in activation HER-3.HER-3 is in colon cancer, prostatitis It is overexpressed in gland cancer, breast cancer and melanoma.HER-3 is damaged and breast carcinoma in substantial amounts of DCIS.Receiving HER-2 In some patients of vaccine, HER-3 can be detected in residual DC IS in operation.As the result of these discoveries, except breast HER-2 in gland cancer, the possibility using these molecules as target is considered as beneficial.

The immunogenic peptide (Fig. 1 and Fig. 2) from HER-3 identified is as follows：

P11-13 (peptide 51-75):KLYERCEVVMGNLEIVLTGHNADLSFLQW(SEQ ID NO:1)；

P81-83 (peptide 401-425):SWPPHMHNFSVFSNLTTIGGRSLYN(SEQ ID NO:2)；

P84-86 (peptide 416-440):TTIGGRSLYNRGFSLLIMKNLNVTS(SEQ ID NO:3)；

P12 (peptide 56-70):CEVVMGNLEIVLTGH(SEQ ID NO:4)；

P81 (peptide 401-415):SWPPHMHNFSVFSNL(SEQ ID NO:5)；

P84 (peptide 416-430):TTIGGRSLYNRGFSL(SEQ ID NO:6)；With

P91 (peptide 451-465):AGRIYISANRQLCYH(SEQ ID NO:7).

Provided herein is result prove that these peptides can activate the CD4 T cells of many patients.The peptide can be used for pulse BMDC simultaneously trains T cell to recognize HER-3.HER-3 is expressed in triple negative breast cancer, and can be in ER positive breasts The resistance for anti-estrogen is assigned in cancer.HER-3 also in other cancers express, the cancer include melanoma, lung cancer, Colon cancer, prostate cancer and metastatic brain tumor.It is not intended to any particular theory, the intracellular portion from molecule Peptide be also likely to be favourable.

The disclosure provided based on the present invention, the method for the immunogenic peptide based on identification HER-3 can screen and reflect Make the immunogenic peptide of HER-1 and c-MET receptor tyrosine kinase molecules.The immunogenic peptide of the present invention, which can be used for preparing, to be used In the multivalence preventative vaccine of breast cancer and other cancers.

The result that the present invention is provided shows the identification that HER-2 sister's albumen is acted in breast cancer.These sister's eggs In vain can be with efficient targeting, it is possible to develop the vaccine of other solid tumors.Peptide available for targeting HER-1 and HER-3 has been developed .In DCIS, especially, specific anti-HER-1, HER-2 in patient before and after vaccine inoculation have been identified With HER-3 responses, it provides support for the exploitation available for prevention early-stage cancer or the polyvaccine for treating the women for having DCIS. The composition of the present invention can be used for treating other cancers, including but not limited to colon cancer, melanoma, brain tumor, lung cancer, ovary Cancer and other tumours.

Melanoma

Melanoma is a kind of aggressiveness cutaneum carcinoma, if be not found early, it may be possible to fatal.Use standard dendron Shape cell vaccine is tested in mouse, and wherein BMDC is engineered causes about 70% melanoma to show Mutain (BRAF).Mouse is protected to be attacked from melanoma cells with the inoculation of these BMDCs, it was demonstrated that can develop Vaccine for melanoma.Be not intended to it is any particular theory, BRAF and HER-3 targeting combination can be used for treat black Plain knurl and other cancers, including but not limited to solid cancer, such as colon cancer, cancer of pancreas and lung cancer and other intestines and stomach swell Knurl.

In addition, it has been shown that Melanoma Tumor escapes immunosurveillance using B cell, can it is taken as that eliminating some B cells To improve treatment.It can assess whether that tumor microenvironment is changed into the response of Th1 types can help prevent escape with contrived experiment.

In some cases, vaccine of the invention can be used for the melanoma that treatment has been spread.In some cases, this hair Bright to provide the treatment for being used for eliminating remaining cell, the remaining cell is generally resistant to drug therapy.

Embodiment 2：Come from the CD4 for MHC II classes for the tumour antigen in vaccine inoculation-mix⁺The identification of peptide is new Strategy

Although cytotoxicity CD8+T lymphocytes (CTL) are considered as the main effects thing of antineoplastic immune in history, But individually strengthen CTL responses with CD8+ vaccines in various tumor types and generate uncertain clinical effectiveness, it may be possible to Because helping lower CTL function suboptimums without enough CD4+T lymphocytes.CD4+T auxiliary 1 type (Th1) cell secretion INF- γ/ TNF-α, induced tumor aging and apoptosis.Therefore, CD4+ epitopes are successfully mixed into cancer vaccine and builds and produce lasting antigen Specific C D4+ immune is still a challenge.Using HER3 extracellular domain (ECD) as candidate, " cancer drives (oncodriver) " tumour antigen, is tested to identify immunogenicity HER3 CD4+ peptides, and it shows that II classes are promiscuity and produce The raw anti-HER3 CD4+ to contain in vaccine constructs are immunized.

The material used in these experiments and method will now be described.

Material and method

Contrived experiment identifies the HER3 CD4+ peptides that immunogenicity II classes mix using HER3 ECD as tumour antigen, To produce anti-HER3 Th1 cellular immunities.

Experimental program is summarized

5 amino acid in the library of the long peptides of 15-mer, peptide are created from HER3 ECD overlapping.These peptide pulses are arrived and confessed On the DC of the monocyte derived of body, and it is ripe for 1 type polarization (DC1；IL-12 secretes) phenotype.Harvest DC1 and with purifying CD4+ T cell is co-cultured, and the CD4+T cells have the subject of known anti-HER-3 Th1 responses in our DCIS vaccine researches. Measure, will identify using the big storehouse with 10 peptides, and by interferon gamma (IFN-γ) secretion to CD4+T cells Process is gradually reduced to single reactive epitope such as.When screening 5-6 subject, identify 4 peptides and seem in most of donors Reaction, this 4 peptides are HER3_56-70(SEQ ID NO：4)、HER3_401-415(SEQ ID NO：5)、HER3_416-430(SEQ ID NO：And HER3 6)_451-465(SEQ ID NO：7).Identify and the CD4+T cell recognitions of HER3 extracellular domains are not reacted The subject of property, and enter horizontal pulse to its DC1 with 4 kinds of HER3 peptides, and the DC1 of pulse is cultivated one week together with cd4 t cell, Then reactions of the DC1 of test pulse to the reactivity of HER2 peptides and to extracellular HER3 albumen.In all cases, at least 1 Individual peptide causes the identification to the peptide in pulse to monocyte and whole HER3 albumen, is indicated above initial sensitization and occurs in vitro. Also show, healthy donors can react to these peptides and there is the triple negative breast cancer that anti-HER3 Th1 responses are lost Reacted in patient.Referring to Gala, K. etc., Clin.Cancer Res 2014；20:1410-1416 and Datta, J. etc., “Progressive Loss of Anti-HER2 CD4+T-helper Type 1Response in Breast Tumorigenesis and the Potential for Immune Restoration " .OncoImmunology (will go out Version).

Experimental program main points are as shown in figure 19：

The library of fragments of peptides comprising 123 overlapping 15 amino acid lengths is from HER3 extracellular domains (ECD) Produce, the fragments of peptides is overlapping by 5 amino acid.

BMDC derived from autologous monocyte from donor (DC) is fast by GM-CSF, IFN-γ and LPS It is rapid-result it is ripe be 1 type polarization (DC1 → IL-12 secretions) phenotype, and with related peptide (such as HER3 ECD or HER3 CD4+ peptides, as schemed institute Show) pulse.By preparing IL-12, DC1 polarization Th1 responses.

The DC1s of the harvest and CD4 of purifying⁺T cell is sensitized together in the co-cultivation of 8-10 days.

The CD4+T cells (it is largely expected to turn into antigentic specificity) of sensitization are stimulated with immature DC (iDC) again, should Immature DC (iDC) is carried out with specific C D4+ peptides (such as HER3 libraries peptide cluster) interested or the control of incoherent II classes peptide Pulse.

Then the supernatant from these cocultures is harvested.Measured by IFN-γ ELISA, if IFN-γ is produced Amount is at least twice of unrelated control, then Th1 responses are considered as antigentic specificity.

The Clinical immunology laboratory of hospital of the University of Pennsylvania carries out HLA-DR, DP, DQ parting to contributor, to comment The MHC II types for estimating CD4+Th1 responses are promiscuity.

The library of the fragments of peptides comprising 123 overlapping 15 amino acid lengths is produced from HER3-ECD.From donor DCs maturations are DC1 derived from autologous monocyte, and use HER3-ECD pulses.The DC1 of harvest and the CD4 T cells of purifying are total to Culture.After 10 days, then stimulate the CD4 T cells confrontation immature DC (iDC) of sensitization, the immature DC (iDC) HER3 texts Storehouse peptide cluster or incoherent CD4 control peptides enter horizontal pulse.Measured by IFN-γ ELISA, if IFN-γ yield is unrelated At least twice of control, then Th1 responses are considered as antigentic specificity.

3 steps of experiment point are carried out：1) for having known anti-HER3 ECD anti-after the DC1 vaccines of inoculation HER2 pulses The patient with breast cancer of answering property, identifies its immunogenicity CD4+ peptides；2) by " reverse " method for sensitizing, confirmed in identical patient The immunogenicity of these peptides；3) there is the patient of known anti-HER3 ECD anergies after vaccine inoculation, its CD4+ is identified Peptide, whether to observe cell to natural HER3 ECD sensitization, so as to overcome/eliminate autoantigen (such as HER3) tolerance.

Result of the test is illustrated

The step sizing of HER3 ECD peptide libraries can be by the immune of the CD4+Th1 cell recognitions of HER3 ECD sensitization to identify Munogenic epitopes

Th1 sensitizations initially are carried out in 5 patient with breast cancers with known anti-HER3 ECD reactivity, it is single to identify Immunogenicity HER3 CD4+ epitopes.To achieve it, the CD4+Th1 of HER3 ECD sensitizations is successively again by 10 peptide cluster (1- 10th, 11-20 etc.) stimulate, then 3- peptides cluster (1-3,6-6,7-10 etc.) is narrowed, and finally obtain single immunogenicity HER3 peptides. Representativeness screening is shown in Fig. 2,13 and 14.Four kinds of immunogenic peptide-HER3 (56-70) (SEQ ID NO：4)、HER3 (401-415)(SEQ ID NO：5)、HER3(416-430)(SEQ ID NO：6) with HER3 (451-465) (SEQ ID NO：7) Repeatably identified and mixed across HLA-DR, DP and DQ hypotype.When using by the four kinds of HER3 identified peptide pulses DC1 makes the Th1 cell sensitization from the reactive donors of 4 non-HER3, and the then iDC of challenge identification HER3 ECD pulses When, all donors not only show successful sensitization to individual immunity originality HER3 peptides, and can recognize natural HER3-ECD.

The result that the present invention is provided, which proves to be identified with the DC1 of peptide library pulse derived from overlapping tumour antigen, to be used for Mix II class peptides in the exploitation of CD4 T cell vaccines.During this investigation it turned out, immunogenicity HER3 CD4 peptides are effectively overcomed pair The immunological tolerance of autologous tumor antigens.In vaccine construction, these HER3 CD4 peptides can be applied to carrying HER3 and cross table In the cancer patient reached.In addition, these results represent a kind of new strategy, it uses DC1-Th1 platforms, for quick and can weigh Identify that the II classes from any tumour antigen mix immunogenicity CD4 epitopes again, for cancer immunotherapy.Table 1 below is shown There is the Preliminary Identification result of immunogenicity CD4+HER3 ECD peptides in the patient of known anti-HER3 reactivity.Table 2 is shown The amino acid sequence for the four kinds of immunogenicity HER3 CD4+ epitopes identified by step sizing.

Tetra- kinds of immunogenic peptide-HER3 of table 1-_56-70(SEQ ID NO：4)、HER3_401-415(SEQ ID NO：5)、 HER3_416-430(SEQ ID NO：6)、HER3_451-465(SEQ ID NO：7)-can be weighed in the donor being previously sensitized to HER3ECD Multiple identification

Donor #	HER3_56-70	HER3_401-415	HER3_416-430	HER3_451-465
					15107-38	√			√
15107-24	√	√	√
					15107-26	√	√	√	√
26113-03	√	√	√	√
					15107-31			√	√

The amino acid sequence of table 2- immunogenicity HER3 CD4+ epitopes

HER3_56-70	CEVVMGNLEIVLTGH(SEQ ID NO:4)
		HER3_401-415	SWPPHMHNFSVFSNL(SEQ ID NO:5)
HER3_416-430	TTIGGRSLYNRGFSL(SEQ ID NO:6)
		HER3_451-465	AGRIYISANRQLCYH(SEQ ID NO:7)

By " reverse " sensitization, confirmation-such as single epitope to the immunogenicity of the CD4+HER3 ECD epitopes of identification is quick The CD4+Th1 of change recognizes natural HER3 ECD ability

Figure 15 is shown in the donor with known HER3 ECD reactivity, and CD4+T cells are by respective donor specific Immunogenicity HER3 epitopes-pulse DC1 sensitizations, and with respective HER3 epitopes and the iDC of natural HER3 ECD pulses again Stimulate.

Figure 20 and 21 shows other results of " reverse " sensitization

Seemed to eliminate the immune self tolerances of anti-HER3 with the DC1 of the immunogenicity HER3 epitopes-pulse CD4+Th1 being sensitized Property

As shown in figure 16, when the DC1 using the HER3 peptide pulses with four kinds of identifications makes to come from the non-reactions of four HER3 ECD Property donor CD4+Th1 cell sensitizations, and then the iDC of challenge identification HER3 ECD pulses when, it was demonstrated that all donors are not Only single HER3 epitopes are shown and are successfully sensitized, and also demonstrate recognizable natural HER3 ECD.

CD4+HER3 epitopes show that MHC II classes are promiscuity

Extracellular domain (ECD) using HER3 is tested as " carcinogenic driving " tumour antigen of candidate, to reflect Determine immunogenicity HER3 CD4+ peptides, the peptide shows the anti-HER3 CD4+ that II classes are promiscuity and generation is available for vaccine constructs It is immune, as shown in figure 17.

Carry out the peptide of self tumor antigen

The result that the invention of this hair is provided is proved：

● it can be identified and be developed for CD4+T cell vaccines with the DC1 of peptide library pulse derived from overlapping tumour antigen Promiscuity MHC II class peptides.

● immunogenicity HER3 CD4+ peptides effectively overcome the immunological tolerance to autologous tumor antigens.

These results represent a kind of new strategy, and it uses DC1-Th1 platforms, come for quick with repeatably identification Mix immunogenicity CD4 epitopes from the II classes of any tumour antigen, for cancer immunotherapy.

Using these HER3 CD4+ peptides, it is necessary to enter to the patient for carrying the cancer that HER3 is overexpressed in vaccine construction Row research.

Embodiment 3：HER3 expression be gastroesophageal junction precancerous lesion in tumour progression mark

RTK including HER family members, which is overexpressed in wellability stomach oesophagus cancer, has prognosis and therapeutic potential.In canceration RTK expression in glandular stomach esophageal injury is not yet widely studied.

Barrett oesophagus, or there is in distal esophagus metaplasia columnar epithelium, it is prone to adenocarcinoma of esophagus (Cameron, A.J. etc., Gastroenterology 109 (5):1541-6(1995)).Although from anomaly sxtructure to wellability The histology transformation of malignant tumour is fully characterized, but the carcinogenesis in metaplasia cell is related to the hereditary change not exclusively understood. Several nearest reports have determined that ErbB-2 (HER2) expression in the Barrett foods with anomaly sxtructure In the hypotype of pipe lesion.(Almhanna, K., etc. Appl.Immunohistochem.Mol.Morphol.July 16 is 2015) (Almhanna waits)；Fassan, M., etc. Histopathology 61 (5):769-76 (2012) (Fassan waits)；With Rossi, E., etc. J.Cell.Mol.Med.13 (9B):3826-33 (2009) (Rossi, etc.)).In addition, the speed of HER2 expression Rate is related to dysplastic degree, imply that tumour occur in relational approach.

Include breast cancer, lung cancer and human primary gastrointestinal cancers (Yokata, J. etc., Lancet 1 in many more conventional malignant tumours: 765-767 (1986)) and oesophagus stomach cancer in have confirmed that the overexpressions of RTK molecules, the RTK molecules include HER families Member (HER1, HER2 and HER3) and cMET, mesenchyma-epithelium transforming factor.The mirror that HER2 is overexpressed in breast cancer subgroup It is to use that fixed, HER2, which is overexpressed with the more relevance and HER2 of the biology of wellability and the efficient targeting of monoclonal antibody, Critical event (Joensuu, H., etc. N.Eng.J.Med.354 (8) in the progress of the targeted therapy for the treatment of solid tumor: 809-20(2006)).This experience provides the foundation for the further effort of other malignant tumours of targeting RTK molecular therapies.

HER2 be overexpressed is confirmed in a small number of stomach cancers, and in metastatic position with Herceptin to it Targetted, its influence to result it is moderate (Bang, Y.J., etc., Lancet 376 (9742) 687-97 (2010) (Bang, Deng)).Compared with more distal end sdenocarcinoma of stomach, HER2 be overexpressed proximal gastric and stomach oesophagus junction it is frequent (Rajagopal, I., Deng J.Clin.Diagn.Res.9 (3):EC06-10 (2015)), and it has been entered with Herceptin in metastatic position Row targeting, its influence to result is moderate, and (Bang is waited and Fichter, C.D., etc. Int.J.Cancer 135 (7):1517- 30 (2014) (Fichter, etc.)).In most of researchs, the expression of HER1 and HER3 in stomach cancer is related to poor prognosis (Kandel, C., etc. J.Clin.Pathol.67 (4):307-12 (2014) and Hayashi, M., etc. Clin.Cancer Res.14(23):7843-9(2008)).CMET overexpression is related to the poor prognosis in adenocarcinoma of esophagus, and cMET is relied on The suppression of the signal transduction of property have adjusted HER1 and HER3 activity (Liu, X., etc. Clin.Cancer Res.17 (22): 7127-38(2011)).These data provide for the RTK expression further made great efforts to characterize in stomach oesophagus stomach cancer and precursor lesion Theoretical foundation.RTK expression is identified in oesophagus stomach cancer and precursor lesion.Research described below is intended to be characterized in stomach oesophagus connection Abnormality in RTK expression, with make great efforts identification be used for treat and primary prevention potential target.

Method

After the institutional review board approval of the University of Pennsylvania, to there is Barrett anomalies of esophagus from 73 The clinography and tissue of patient's (low anomaly sxtructure (LGD), n=32, or Height Anomalies structure (HGD), n=59) of structure Learn sample and carry out retrospective analysis.By endoscopic biopsy and mucosa resection the sample from storage from 2003 to 2012 Formalin fix and the tissue block of FFPE is loaded on slides (Fisher Scientific, Waltham, MA) at 5 μm Section, then dewaxes and rehydrated.All biopsy materials are to HER1 (clone H11；1:50；DAKO), HER2 (HercepTest, DAKO, Carpinteria, CA) and HER3 (clone RTJ.2；1:30；Santa Cruz Biotechnology, Dallas, TX) (Leica Bond-III instruments) carries out immunostaining, and is commented by single virologist under microscope (Leica Bond-III) Valency.Film 3+HER dyeing is considered as positive, as seen by fig. 22 >=10% tumour cell in film 2+HER2 contaminate Color.When there are enough tissues can use, cMET immunohistochemical stainings are carried out in 42；In >=50% tumour cell It is positive that moderate or strong film, which are dyed,.RTK is overexpressed, anomaly sxtructure-gland cancer for assess pairing related to clinical data The relevance of biopsy specimen and infiltrating cancer or the diagnosis to the gland cancer of subsequent biopsy specimen and the relevance of infiltrating cancer.

Statistical analysis

All analyses are tested using double tails.Descriptive statistic is represented as in the frequency and continuous variable of classified variable It is worth (interquartile range (IQR)).Pearson's χ 2 or Fisher are accurately examined and Wilcoxon rank tests are respectively used to point Analysis classification and continuous variable.P value≤0.05 is considered to have statistical significance；All tests are all bilaterals.Use SPSS V22.0 is analyzed (IBM, Armonk, NY).

As a result

By immunohistochemical method identify and analyze altogether 73 have minuent Barrett anomalies of esophagus structures (n= 32) or height Barrett anomalies of esophagus structure (n=59) patient HER1, HER2, HER3 and cMET expression.In group The position age is 65 years old (IQR 60-73 Sui)；81.9% is male, and 87.5% is white people.Drinking rate is 14.3% in group, Active smoking rate is 6.3%, and 55.6% is former smoker, and 26.4% has pernicious family history.In clinic and the population system of measurement There is no significant difference between LGD and HGD groups in meter variable, it is as shown in table 3 below.

Table 3 has the demography and Clinical symptoms of Barrett anomalies of esophagus structures group, and rudimentary and senior Single argument compares

Anomaly sxtructure patient

Compared with low anomaly sxtructure (LGD), Height Anomalies structure (HGD) and HER1 (22.4% couple of 3.1%, p= 0.016), HER2 (5.3% couple of 0.0%, p=0.187) and (45.6% couple 9.4% of HER3<0.001) overexpression is related.

Have 6 Foci wellability adenocarcinoma of esophagus patients related to anomaly sxtructure lesion, all these generation all with HGD (HGD：10.2% contrast LGD：0.0%, p<0.001) it is associated.Separately there are 9 patients in subsequent biopsy specimen (HGD： 17.0% contrast .LGD：0.0%, p=0.017) on be diagnosed as wellability adenocarcinoma of esophagus.With the trouble without wettability carninomatosis stove Person (71.4% couple of 38.6%, p=0.032) compares, HER3, rather than HER1 or HER2 (HER1 (26.7% to 20.5%, p= 0.616) and HER2 (14.3% to 2.3%, p=0.077) increase) overexpression it is associated with HGD lesions, as Figure 23 A with Shown in 23B.

CMET overexpression is observed in 18 in 42 (42.9%) evaluate samples, and compared with LGD samples, In HGD observe more cMET (58.3% couple of 36.7%, p=0.200), and most often with HER3 (62.5% HER3 Positive sample and 38.2% HER3 ' negative ' specimens (p=0.212)) coexpression.In HER1 positive (p=0.729) or HER2 sun Similar trend is not observed in property (p=NA) sample.There is one to make a definite diagnosis infiltrating cancer in 42 (5.6%) patients；CMET exists (p=0.243) is overexpressed in the patient.

Discuss：

The analysis of RTK expression in the anomaly sxtructure lesion connected to stomach oesophagus is confirmed：(1) HER family proteins exist Raised in Barrett anomalies of esophagus structures；(2) frequency that HER families and cMET are overexpressed is proportionate with anomaly sxtructure degree； (3) HER protein upregulations, particularly in anomaly sxtructure lesion, the incidence increase to mediated invasion cancer is related.

Therefore, HER3 may be used as the biological mark of the invisible invasive disease of the patient with Barrett oesophaguses and HGD Will thing.In addition, the secondary prevention of stomach oesophagus cancer can be provided in patient's subgroup by the therapeutic agent of targeting of HER3 or CMET.

Commenting to the HER2 that is overexpressed in a small number of cases previously was had been limited to the assessments expressed of HER in Barrett oesophaguses Estimate, referring to Almhanna et al., Fassan et al. and Rossi et al..HER2 overexpressions are present in 3.3% biopsy in this research In sample, the speed being overexpressed less than HER1 or HER3.This pattern is connected in cancer with HER family proteins in invasive stomach oesophagus Expression it is consistent, wherein HER3 is overexpressed more common (Fichter etc.) than HER2.HGD, HER3 eggs are proceeded to from LGD White overexpression increase, particularly HER3 is frequently over-expressed and is represent novelty, although do not have unexpected discovery. The homologous and Heterodimerization drive signal activation of HER receptor；Multiple members of HER families are observed in other tumor types The overexpression that clusters.In combination, c-MET the positive regulators HER1 and HER3 of activation¹¹Activity.In fact, these acceptors it Between interaction for the multivalence treatment method using a variety of RTK as target provide theoretical foundation (Baselga, J., etc., N.Eng.J.Med.366(2):109-19(2012)；Waddell, T., etc. Lancet Oncol.14 (6):481-489 (2013))。

Notebook data also implies that the chance for targetting secondary prevention stomach oesophagus cancer is not developed also.It is in situ in breast duct in the past Targeting HER2 expression as target and is provided with promising result (referring to U.S.Published in cancer (DCIS) Application US 2015/0323547A1；U.S.Ser.No.14/985,303filed December 30,201；, Datta, J. etc., OncoImmunology 4:8e1022301(2015)DOI:10.1080/2162402X.2015.1022301； Datta, J. etc., Breast Cancer Res.17 (1):71(2015)).This method be still for gastrointestinal cancer away from From remote target.Nevertheless, the treatment method of Barrett oesophaguses at present, including endoscopic excision and ablation mode and Radical surgery has obvious limitation.The alternative strategy for reducing the incidence of disease and reducing the risk of infiltrating cancer is desirable 's.The analysis of this RTK expression in the anomaly sxtructure lesion that stomach oesophagus is connected is confirmed：(1) HER family proteins are with different Raised in the Barrett oesophaguses of normal structure；(2) frequency that HER families and cMET are overexpressed is proportionate with anomaly sxtructure degree； (3) HER protein upregulations, particularly in anomaly sxtructure lesion, the incidence increase to mediated invasion cancer is related.

Therefore, HER3 may be used as the biological marker of the invisible invasive disease with Barrett oesophaguses and HGD patient Thing.In addition, the secondary prevention of stomach oesophagus cancer can be provided in patient's subgroup as the therapeutic agent of target using HER3 or CMET.

In a word, notebook data is indicated in the high-grade anomaly sxtructure lesion of gastroesophageal junction, particularly with concealment In the high-grade anomaly sxtructure lesion of the gastroesophageal junction of property infiltrating cancer between HER3 frequent overexpression and vicious transformation Relation.These discoveries can prove there is more positive management method for expressing HER3 anomaly sxtructure lesion, and be HER3 Following application of the therapeutic agent of targeting in early stage disease settings provides theoretical foundation, this be skilled addressee readily understands that 's.

We illustrated in HER-2 in the past^posNatural anti-HER-2 CD4 Th1 progressive in tumor of breast generating process Loss.The forfeiture of this response with to lower rectal cancer lack pathology complete incidence graph (" pCR ") it is relevant, and with breast cancer relapse Elevated risk is related, and can be recovered with vaccine inoculation.The following examples 4 have studied to be resisted during tumor of breast occurs HER3 Th1 responses whether there is similar loss.

Embodiment 4：Anti- HER3 CD4 Th1 forfeiture is occurred in that in tumor of breast generation and negatively correlated with result

The overall summary of embodiment 4

We illustrated in HER2 in the past^posNatural anti-HER2 CD4 Th1 progressive is damaged in tumor of breast generating process Lose.The forfeiture of this response with to lower rectal cancer lack pathology complete incidence graph (" pCR ") it is relevant, and with the liter of breast cancer relapse High risk is related, and can be recovered with vaccine inoculation.The embodiment have studied whether there is during tumor of breast occurs The loss of similar anti-HER3 Th1 responses.

The peripheral blood from 131 subjects is have collected, including healthy donors (" HD "), benign breast disease (" BD ") are suffered from Person, DCIS patient (" DCIS ") and infiltrative breast carcinoma (" IBC ") patient.Pass through Enzyme-linked Immunosorbent Assay (ELISpot) Determination method test analyzes immune to the immune response for the four kinds of different HER3 immunogenic peptides identified in example 1 above and 2 The all of response measure.

Anti- HER3 responses from HDs to IBC have significant decline.Three feminine gender (" TN ") IBC are in all three immune parameters With minimum response.In three kinds of immune parameters, HDs has than ER^posBoth IBC and TN IBC patients are significantly higher to be immunized Response.It is interesting that HER2^posIBC shows the immune response similar to HDs and BDs.With the patient or right do not recurred then The patient that lower rectal cancer has pCR is compared, and pCR patient is lacked with recurrent breast and to lower rectal cancer with aobvious Write lower anti-HER3 CD4 Th1 responses.

Thus, it is found that the anti-HER3 of CD4 Th1 tumor of breast occur during lose, be most significantly in TN IBC, this The therapeutic choice of group is limited and significantly worse to the prognosis of HER3 overexpressions.These find to have attempt to recover this response to prevent The meaning only recurred.

Background

Almost eighth women will can develop breast cancer in life at it.Wherein, overexpression HER2 cancer with Higher far-end transfer rate is related to overall poor prognosis.Herceptin (a kind of monoclonal antibody for HER2) draws Enter the significant progression free survival phase and Overall survival for extending HER2 positive cancer patients, these indicate HER2 in regulation breast Key effect (Giordano S.H., etc. J.Clin.Oncol. (2014) in gland cancer progress:JCO-2013[Published online before print May 5,2014,doi:10.1200/JCO.2013.54.0948])。

Immune system plays a crucial role in the tumour of regulation HER2 expression.Before it has been shown that from health volunteer to HER2^posDCIS to HER2^posIBC, but do not include HER2^negUnder IBC natural anti-HER2 cd4 t cells response has progressively Drop.In addition, relatively low anti-HER2 immune responses are related to subsequent breast cancer relapse, and higher anti-HER2 immune responses with it is right The pathology complete incidence graph of new adjuvant chemotherapy is related, implies immune system in HER2^posEffect in tumour generation.Referring to Datta, Etc., J., OncoImmunology 4 (10):e1027474.DOI:10.1080/2162402X.2015.1022301(2015) and U.S.Published Application US 2015/0323547A1(collectively hereinafter, " Datta, etc. ").Our groups have developed HER2 dendritic cells pulsed vaccines, and it recovers in DCIS and IBC patients Anti- HER2 CD4 and CD8 t cell responses.Sharma, A., etc. Cancer 118 (17):4354-4362(2012)；Koski, Etc., G.K., J.Immunother.35 (1):54-65(2102)；and U.S.Published Application US 2015/ 0323547 A1。

HER2 is the member of EGFR families, and EGFR families are also to include HER1 and HER3 one group of RTK.Although well-known HER2 itself dimerizations, but effects of the HER3 in signal transduction is not clear, and it may send out with itself and HER2 Raw dimerization.It is escape machine in the patient with breast cancer treated with Herceptin that HER3 and HER2 dimerization, which has been suggested, System.Czopek, J., etc. Contemp.Oncol.17 (5):446-9 (2013) (" Czopek, etc. ") and Bae, S.Y., etc., Breast Cancer Res.Treat.139(3):741-50 (2013) (" Bae, etc. ").Handkerchief trastuzumab is that in the market is nearest A kind of tumour medicine of addition, FDA approvals are obtained as the handkerchief trastuzumab of the tumour medicine of lower rectal cancer as first Suppress HER2/HER3 dimerizations, and when combining with Herceptin for patient with breast cancer, it has therefore proved that have to overall survival Benefit.Jhaveri, K., etc. J.Natl.Compr.Canc.Netw.12 (4):591-8 (2014) and Harbeck, N., etc., Breast Care 8(1):49-55(2013)。

HER3 expression is not clear in breast cancer hypotype, although negative in some ER positives, the HER2 positives and three Its overexpression is observed in (" TN ") hypotype.Moeder, C., etc. Cancer 115 (11):2400-9(2009).It is interested Be, although HER3 be overexpressed may be in HER2^posIt is more conventional in IBC, but its prognostic value is more significant in TN IBC.Although HER3 is in ER^pos/HER2^posExpression in IBC does not influence disease-free survival (" DFS ") or total existence (" OS "), but in TNIBC HER3 expression it is related to worse DFS and 10 year 5 years OS.Bae etc. and Czopek etc..It is worth noting that, being had according to definition The TN IBC patients for having HER3 to be overexpressed select without any classic treatment, with the HER3 TN IBC patients' being overexpressed Considerable part can benefit from recognizable new target drone.Unclear anti-HER3 CD4 Th1 responses whether there is in health In donor and this response whether tumor of breast occur during change.The answer of these problems is sought in this research.

Method

Subject records

Have 131 subjects and meet research standard, and continuously Pennsylvania is registered in the case of informed consent University.This research is by the institutional review board and Abramson Cancer centers of the University of Pennsylvania before subject enters group Approval.In this 131 subjects, wherein the PMBC of 9 patients is not enough to be measured, remaining 122 by Examination person has immune response data for check.In healthy donors (HD, n=30), benign breast disease (BD, n=11), DCIS (n=13), HER2^posIBC (n=21), ER^posInfiltrative breast carcinoma (ERpos IBC, n=20) and three feminine gender IBC (TN IBC, n=27) between the cd4 t cell responses of four kinds of difference HER3 immunogenic peptides is compared.

PMBC is collected

Peripheral blood is collected by venipuncture.By blood with 1：1 ratio dilutes in Hank's buffer solutions or PBS, and By lymphocyte separation medium in conical pipe dilution blood lower leaf.Then by 1200rpm lower densities from The heart 30 minutes, separates blood.Collecting monocytic cell layer, and washed twice in Hank's buffer solutions or PBS.Count cell and with Every milliliter of 10,000,000 cell is resuspended, and is freezed 24-48 hours at -80 DEG C, is then transferred to minus 200 DEG C, wherein cell is kept Storage is until measuring.

Measure anti-HER3 CD4 Th1 responses

Determined by ELISpot, anti-HER3 CD4 Th1 cell responses are measured according to the scheme of manufacturer.In short, with The hole pvdf membrane flat board of 70% Ethanol activation 96, is washed with PBS, is then coated with anti-IFN-γ (anti-IFN-γ) antibody, and 4 DEG C it is incubated overnight.After 24 hours, PBS washing flat boards are used again, are then sealed with the Iscove's culture mediums containing 10% human serum Close 1 hour.By PMBC in 37 DEG C of defrostings, wash, count and thin with 1,000,000 in PBS or Hank's buffer solutions Born of the same parents/milliliter are resuspended, then with every 200,000 cell in hole with four kinds of immunogenicity HER3 peptides：

P12 (peptide 56-70)：CEVVMGNLEIVLTGH(SEQ ID NO:4)；

P81 (peptide 401-415)：SWPPHMHNFSVFSNL(SEQ ID NO:5)；

P84 (peptide 416-430)：TTIGGRSLYNRGFSL(SEQ ID NO:6)；With

P91 (peptide 451-465)：AGRIYISANRQLCYH(SEQ ID NO:7), anti-CD3/CD28 (polyclonal stimulant, Positive control) tetanus toxoid (Santa Cruz Biotechnology, Dallas, TX) or without (non-stimulated control).One Three parts of formula is measured.Flat board is incubated 48 hours at 38 DEG C.After 48 hours, PBS washing flat boards are used, biotin is then added The anti-IFN-γ antibody changed, flat board is incubated 2 hours at 38 DEG C.Plate is washed with PBS again, streptavidin-HRP is added, Plate is cultivated 1 hour at 38 DEG C.Finally, plate is washed with PBS, tmb substrate solution is then added.After 5 minutes, filled with running water Divide washing, stop colour developing.Plate is dried overnight at 4 DEG C.

Immune response is analyzed

By immunodotting software to spot count.Three parameters or measurement are quantified to determine immune response：(1) The response of cumulative acknowledgements or the summation in every million cell to all four HER3 immunogenic peptides in terms of spot, (2) ring There is the peptide number of 20 or more spots in Ying Ku, or each subject, and (3) responsiveness or at least one peptide of response The percentage of subject (it is 20 or more points to define threshold value).Lockjaw response in every 200,000 cell blots and AntiCD3 McAb/CD28 responses in the spot of every 200,000 cells are also by quantification of control.All immune response indexs pass through Graphpad prism software analysis.

As a result

Research object feature

Have 131 subjects and meet research standard, and in University of Pennsylvania's infection from hospital informed consent form.9 The cell that subject is used to analyze is not enough, is left 122 subjects.Wherein, average age was 50 years old, from 25 years old to 83 years old, 72.1% is white people, and 18.0% is non-descendants American, and 9.8% is another race.Subject is divided into five groups：HDs (n=30), BDs (n=11), DCIS (n=13), HER2^posIBC (n=21), ERpos IBC (n=20) 27).In 68 IBC subjects In, 35 (51.5%) are that I-stage, 22 (32.4%) are that II stages, 9 (13.2%) are that III stages, 2 (2.9%) are the IV stages. 52 (76.5%) receive chemotherapy and/or Trastuzumab and/or TAM treatment, and 16 (23.5%) is first treatment.Three Individual DCIS patient and three HER2^posIBC patient receives HER2 dendritic cells pulsed vaccine inoculations.Other characteristic reporters are under In table 4.

The feature of the research object of table 4

The anti-HER3 immune responses of CD4 Th1 cells from healthy contributor to infiltrative breast carcinoma hypotype have reduction

Compare HDs, BDs, DCIS, HER2^pos IBC、ER^posIBC and TN IBC, three kinds of immune parameters all decline, Touched the bottom in TNIBC：Cumulative acknowledgements (90 pairs 80 pairs 66 pairs 79 pairs 48 pairs 40, p=0.01, respectively as shown in fig. 24 a), ring (76.7% pair of Ying Ku (1.0 couples 0.6 couple 0.8 couple 0.8 couple 0.5 couple 0.3, p=0.003, respectively as shown in fig. 24b) and responsiveness 63.6% couple of 53.8% couple of 66.7% couple of 45.0% couple of 33.3%, p=0.02, respectively as shown in Figure 24 C).It is worth noting that, In all three immune parameters of HDs and TN IBC patients：Cumulative acknowledgements (90 pair 40, p=0.002), (1.0 pairs of storehouse of response 0.3, p=0.002) and responsiveness (76.7% couple of 33.3%, p=0.001), these differences not only statistically it is significant more Height, and also above more than one times.Compared with TN IBC patients, there are BDs significant higher cumulative acknowledgements (to be respectively 40 pairs 80, p=0.007), DCIS patient has significant higher response storehouse (being respectively 0.8 pair 0.3, p=0.04), HER2^pos IBC (it is respectively 33.3% couple of 66.7%, p=with significant higher response storehouse (being respectively 0.3 pair 0.8, p=0.01) and responsiveness 0.04)。ER^posIBC patient has next to the lowest anti-HER3 cd4 t cells response, and immune at all three compared with HDs Statistically significantly lower response is shown in parameter：Cumulative acknowledgements (being respectively 48 pair 90, p=0.03), response storehouse (point ratio For 0.5 pair 1.0, p=0.008) and responsiveness (being respectively 45.0% couple of 76.7%, p=0.03).It is worth noting that, HER2^posThe anti-HER3 responses of IBC are not significantly different with HDs, BDs or DCIS subject.

The relatively low anti-HER3 immune responses of CD4 Th1 cells are HER3 specific in infiltrative breast carcinoma patient, and And the extensive defect of immune response can not be attributed to

Lockjaw response and the polyclonal stimulation with AntiCD3 McAb/CD28 responses are analyzed, to compare and control overall immune should The property answered.Determine every 200 by ELISpot, the spot of 000 cell is understood, CD4Th1 cell anti-tetanus responses HDs, BDs、DCIS、HER2^pos IBC、ER^posIt (is respectively 37 pairs 30 pairs 19 pairs 34 pairs 24 not have difference between IBC or TN IBC patients To 29, p=0.65, as shown in fig. 25 a).Importantly, the anti-tetanus response between HDs and TN IBC patients is similar (37 pair 29, p=0.37), but this two groups of patients have most different anti-HER3 CD4 Th1 cell responses.Equally, with anti- There is no most of subjects in difference, each group that there is spot firm too much to develop in CD3 CD28 polyclonal stimulation with As for can not count.In those of countable, HDs, BDs, DCIS, HER2^pos IBC、ER^posIBC or TN IBC patients (point Wei not be 688 pairs 549 pairs 804 pairs 699 pairs 629 pairs 675, p=0.68, see Figure 25 B) between without statistically significantly difference.

Prognosis and feature of the anti-HER3 CD4 Th1 cell responses of infiltrative breast carcinoma patient to tumor invasion are related

In order to determine whether anti-HER3 cd4 t cells response is related to the feature of tumor invasion, by being drenched in initially performing the operation Fawn on state (lymph node positive (" LN^pos"), Lymph Node-negative (" LN^neg")), diagnosis and reaction for new adjuvant chemotherapy are extremely The recurrence of the patient of few 1 year and non-recurrence state (pathologic complete response (" pCR "), residual disease ("<PCR ")) compare The immune response of IBC patient.In all three parameters, although with LN^negPatient (n=31) compares, LN^posIBC patient (n= 28) there is overall relatively low immune response, but without the significant property of statistics, three parameters are：Cumulative acknowledgements (are respectively 40 pairs 56, p=0.12), response storehouse (being respectively 0.4 couple 0.6, p=0.08) and responsiveness (respectively 35.7% couple of 54.8%, p= 0.19), as shown in fig. 26.It is worth noting that, LN^posSubject has the tested of lymphatic metastasis after including new adjuvant chemotherapy Person.LN after new adjuvant chemotherapy^negSubject eliminated from analysis because do not know before treatment they whether nodosity. In the patient of diagnosis at least 1 year, compared with those patients of holding without disease (n=36), those suffer from recurrent breast The patient of (part or DISTANT METASTASES IN, n=7) shows significantly lower anti-HER3 responses in three immune parameters, and three are exempted from Epidemic disease parameter is：Cumulative acknowledgements (being respectively 17 pair 66, respectively p=0.04), response storehouse (are respectively 0.0 pair 0.6, p<0.05) and Responsiveness (being respectively 55.6%, p=0.01 after 0% pair of diagnosis) is as shown in fig. 26b.

Finally, in the patient for receiving new adjuvant chemotherapy (n=16), pCR groups (n=5) with<PCR groups (n=11) are compared to tool There are significantly higher cumulative acknowledgements (being respectively 144 pair 32, p=0.004) and response storehouse (respectively 0.8 pair 0.4, p=0.05), As shown in Figure 26 C.PCR groups and<Do not united in terms of responsiveness between pCR groups (being respectively 80.0% and 27.3%, p=0.10) Meter learns significant difference.It should be noted that LN^posAnd LN^negPatient's (being respectively 22 couple 29, p=0.35), patients with recurrent and non-patients with recurrent (be respectively 27 couple 35, p=0.65) and pCR and<PCR (being respectively 17,59, p=0.15) does not have difference in lockjaw response. Therefore, it is HER3 specific with CD4 Th1 cell responses relatively low in the more IBC patient of invasive tumor feature.

Healthy donor's feature of anti-HER3 CD4 Th1 cell effects

Anti- HER3 CD4 Th1 responses in HD and BD be according to the age (<50 years (n=25) or >=50 years (n= 16)), race (Caucasian (n=29), non-descendants American (n=12) or other (n=5)), pregnant state (0 (n=17) or 1 It is individual or multiple pregnant (n=24)) and menopausal state (premenopausal (n=30)) be compared.According to age (Figure 27 A), race (Figure 27 B) or previous pregnancy history (Figure 27 C), accumulation reactive polypeptide, response storehouse or responsiveness do not have difference.However, such as Figure 27 D institutes Show, compared with premenopausal women, postclimacteric women have significantly higher cumulative acknowledgements (every million cell 136 pair, 70 points, P=0.005) (above) and response storehouse (being respectively 1.4 pair of 0.8 peptide, p=0.03) (the second figure).Do not united in terms of responsiveness Meter learns significant difference (being respectively 90.9%, 66.7%, p=0.23) (the 3rd figure).Pre-menopausal women and postmenopausal women's is broken Also without the significant difference of statistics (figure below) between cold response, the difference for showing immune response in menopausal state is HER3 special Property.

ELISpot analyses are to be proved to be accurate by linear precision measure

Had verified that before our laboratory, ELISpot determination methods.In order to confirm to carry out all experiments of this research Operator under the experiment accuracy, for being carried out from the serial dilutions of known high anti-HER3 cd4 t cells respondent Linear micrometric measurement.The concentration series of PMBC from 1.0 to 0.1 to 0.01 to 0.001 are diluted in culture medium, And the anti-HER3 immune responses of accumulation are measured with the spot of every million cells.Figure 28 shows, accumulated value respectively from 230,35, 12 to 5 point (p<0.0001, r=0.88) linear decline.

Discuss

The rapid of effect of the immune system knowledge in cancer development, progress and prognosis expands.It has been determined that Immune deficiency state increases the risk of cancer development, is not only so, and to from non-disease to the tumour from viral source The tumour in poison source is also so.Boshoff, C., etc. Nature Rev.Cancer 2:373-82(2002)；Sheil, A.G.,World J.Surg.10:389-96(1986)；Penn,I.,Transplantation 61:274-78(1996)；and Penn,I.,Transplantation 60:1485-91(1995).It it is known that some immunophenotypes are related to breast cancer；Circulation Inflammatory cytokine TNF-α and IL-6 are higher in patient with breast cancer, and low CD4+/CD8+T cell ratio is with more attacking Property breast cancer phenotype it is related, and tumor infiltrating lymphocyte is related to more preferable prognosis in some breast cancer.Alokail, Etc., M.S., Med.Oncol.31 (8):38(2014)doi:10.1007/s12032-014-0038-0；Jai, Y., etc., Med.Oncol.31:981(2014)；And Matsumoto, H., etc. J.Clin.Pathol.doi:10.1136/ jclinpath-2015-202944.However, being lost for the Immune discrimination that specific molecular cancer in other immunocompetences host drives Evidence it is relatively new.Only just show recently, from healthy donor to HER2^posDCIS to HER2^posIBC's is natural anti- HER2 CD4 Th1 cell responses decline, and one of leading research is shown in during tumor of breast occurs for the driving of this species specificity cancer Immune response receive loss.Will be readily appreciated by those of ordinary skill in the art that identification and reason to this certain loss Solution is for specific immunity targeted therapy by with very big potentiality.

This research shows：(1) from HDs to ER^posWith TN IBC, anti-HER3 CD4 Th1 cell effects decline；(2) resist HER3 responses are related to prognosis, and particularly relatively low response is related to recurrence, and higher response and the pCR phases of new adjuvant chemotherapy Close；(3) post menopausal HD has significant higher anti-HER3 immune responses.All these discoveries will have anti-HER3 CD4 Th1 The diagnosis of cell response and clinical application.

Anti- HER3 CD4 Th1 cell responses highest in HD, minimum in TN IBC, wherein TN IBC prognosis is than it The IBC of his type prognosis be overexpressed by HER3 influenceed it is more serious its.Bae etc. and Czopek, J. etc..Although HER3 expression exists It is unknown in the colony of the IBC patient studied at present, but and HER2^posIBC colonies compare, TN IBC and ER^posIBC groups can There can be higher levels of HER3 expression, it shows the response similar to HDs.In fact, our previous studies show to resist HER2 CD4 Th1 cell responses are directly related to HER2 expression；Anti- HER2 CD4 Th1 cells are in HER2^posIn IBC, without It is HER2^negThere is significant decline in IBC.Compared with the breast cancer of expressed receptor, HER3 not only has more to TN IBC prognosis Big influence, and even in TN IBC, compared with HER2 (1+) tumour, it may have bigger to HER2 (0) prognosis Influence.Schmidt, G., etc. Arch.Gynecol.Obstet.290:1221-29(2014).This can be with partial interpretation HER2^posThe similitude of immune response between IBC and HD.If tumour is because HER2 is overexpressed and is bred, to swollen Just not driven for knurl progress makes it escape immunosurveillance.If however, immune system identified HER2 and using HER2 as Targeting, tumour can adapt to HER3 overexpressions, and immune evasion is for the existence of tumour cell just into evolving favorably.

It is interesting that ER^posIBC shows the anti-HER3 CD4 t cell responses similar to TN IBC, and significant is less than HDs or HER2^posIBC anti-HER3 CD4 t cell responses.Although compared with TN IBC, HER3 is expressed in ER^posIn IBC Prognosis conspicuousness is smaller, but evidence shows that HER3 mRNA expression expresses positive correlation with ER.Fujiwara, S., etc. Breast Cancer 21:472-81(2014).This can explain the relatively low immune response observed in the IBC subgroups.

Patient with breast cancer with recurrent disease has relatively low anti-HER3 CD4 compared with keeping the patient without disease Th1 responses, this shows that immunosurveillance is probably the successful important mechanisms of long-term treatment.Patients with recurrent more likely has high HER3 tables The tumour reached, this point be it is possible, the tumour of high HER3 expression in itself with Preventive and the higher wind of worse overall survival Danger is related.Li, Q., etc. Oncology Reports 30:2563-70(2013)；Smirnova, T., etc. Oncogene 31: 706-15(2012)；And Ocana, A., etc. J.N.C.I.105 (4):266-73(2013).In addition, it has been suggested that immune to compile Collect as escape mechanism, tumour cell is optionally eliminated from there through immune system, evolve and exempted from expressing escape until them The molecule cancer driving of epidemic disease identification, such as HER3.Dunn, G.P., etc. Nature Immunology 3 (11):991-8(2008).Cause This, HER3 expression is probably, due to lacking immunosurveillance, to strengthen the risk of recurrence after it.It is interesting that nearest evidence table Bright recurrent tumor may be differed on pathology with primary tumor.Between primary and secondary tumors for progesterone by The inconsistent rate of body is especially high, and the prognosis of any kind of inconsistent all instructions recurrent tumor is poor. Idirisinghe, P.K.A., etc. Am.J.Clin.Pathol.133:416-29(2010)；Broom, R.J., etc., Anticancer Research 29:1557-62(2009)；And Liedtke, C., etc. Annals of Oncology 20: 1953-58(2009).It is believed that comparing the HER3 expression between primary and secondary tumors there is presently no research；HER3 tables Up between primary and recurrent tumor whether it is inconsistent be unknown and HER3 expression whether can represent recurrence Escape mechanism be also unknown.If answer is affirmative, patient of the targeting with low anti-HER3 cd4 t cells response can be with Improve immunosurveillance and help prevent long-term recurrence.

Also imply effect of the immune system in prognosis, have to new adjuvant chemotherapy pCR patient than<PCR patient shows Write higher anti-HER3 cd4 t cells response.Have shown that HER3 signal transductions have mediated the acquired resistance to targeted therapy Property.Sergina, N.V., etc. Nature 445:437-41(2007)；And Frogne, T., etc. Breast Cancer Res.Treat.114:263-75(2009).Imply it is not acquired resistance herein, but initial resistance, anti-HER3 is immunized Greatest benefit is reacted in the potential prognostic markers thing of the patient of lower rectal cancer.Further research should illustrate anti- Whether HER3 immune responses are not only prediction prognosis, but also can intervene to promote the response to treatment.

In our current research, a HD part, particularly postmenopausal women, show higher anti-HER3 reactions.However, Different from the past confrontation HER2 reaction result of study, based on pregnancy history result be do not have it is discrepant.Although biologically, Higher anti-HER2 reactions can be attributed to the exposure that breast is degenerated with subsequent cell protein under immunosurveillance in pregnancy, This change in mammary gland essence is unlikely climacteric.Press, M.F., etc. Oncogene 5:953-62(1990). Have been observed that breast density changes with Hormone change (such as in climacteric) in imaging, this can be simulated in pregnancy Breast is degenerated, and can also simulate the cell protein generally expressed in breast tissue being exposed to immune system.Clendenen, Etc., T.V., Magnetic Resonance Imaging 31:1-9(2013).Another explanation can be by postmenopausal women Higher anti-HER3 immune responses are attributed to risk difference.The expression of various breast cancer cancer drivings is clearly to have age-dependent Property：HER2^posIBC becomes unlikely with the age, and ER^posIBC becomes more likely.Clark, G.M., etc.,J.Clin.Oncol.2:1102-09(1984)；Eppenberger-Castori, S., etc. Int.J.Biochem.and Cell Biol.34:1318-30(2002).In addition, both acceptors are not independent each other, because being with the ER/PR expression at age HER2 dependences, vice versa.Neven, P., etc. Breast Cancer Res.Treat.110:153-59(2008).TN IBC patient, most sensitive group is overexpressed to HER3 with minimum anti-HER3 immune responses and prognosis, the more frequency in pre-menopausal women Occur numerously..and Howlander, the N. such as Bae, etc. J.N.C.I.106 (5):1-8(2014).Therefore, premenopausal HD Asia Group represents the group for the high risk for occurring TN IBC, and post menopausal HD represents the group that alreadys exceed the high risk phase and actual It is upper to represent the relatively low group of the risk with the HER2 and HER3 breast cancer being overexpressed simultaneously.Although it is further noted that TN IBC is more conventional in young woman, but it imply that more preferable prognosis in elderly population, and reason is unknown. Aapro, M. etc., annals of Oncology 23 (6):vi52-55(2012).If higher anti-HER3 immune responses are true It is due in fact the biological mechanism of climacteric group rather than the biological mechanism of risk averse group, it is possible to partial interpretation menopause Afterwards in women TN IBC more preferable prognosis.

Conclusion

This example demonstrates under HDs, BDs and DCIS to ERpos and TN IBC, anti-HER3 CD4 t cell responses Drop.In addition, relatively low anti-HER3 reactions and recurrence and lower rectal cancer<PCR is related, shows this immune response in wellability breast Prognostic can also be played in gland cancer.Most of all, these results reflect the result of previous research, it is shown from HDs To HER2^posDCIS to HER2^posThe decline of IBC natural anti-HER2 immune responses.This similar result is not only hopeful For confirming former discovery, and it is hopeful to play bigger immune system effect in the driving of molecule cancer is found.It is interesting , post menopausal HDs has significantly higher immune response than premenopausal HD, and premenopausal HD groups are generally in the development excessive tables of HER3 High risk is in the breast cancer reached and the mechanism for mediating the risk may be pointed to.Determine that the DC1 vaccines of HER3 pulses connect Kind whether can to HER3 be overexpressed breast cancer development have treatment and/or risk conditioned act on this put it is critically important.Continue Check that immune system drives the effect in Specific cancer to be also no less important in other cancers.

Before conclusion：From healthy donors group to invasive breast cancer group, the anti-HER-3 immune responses loss of CD4 Th1 cells, Most significant is that the prognosis that this group of therapeutic choice be limited and HER-3 is overexpressed is significant worse in TN IBC groups.Anti- HER3 is immunized Response also mitigates the response to treatment and prognosis, and it points to potential immunotherapeutic targets.HER3 is added into DC1 vaccines to be immunized Originality peptide can increase the IBC PATIENT POPULATIONs that can benefit from vaccine inoculation.Most of all, these result verifications were former It was found that, and point to the bigger immune system effect in the driving of molecule cancer is found.

The result of the present embodiment, is that in tumor of breast generation, the anti-HER3 CD4+Th1 of group are notable from HDs group to IBC Loss, it can be interpreted as the cancer that can be used for diagnosis and treatment expression HER3 by those of ordinary skill in the art, especially newborn Gland cancer, particularly three feminine gender IBC.It can be expected that blood testing can be developed to detect the CD4+ of circulation anticancer in subject Th1 responses are so as to utilize these discoveries.Preferably, this blood testing will use HER3 immunogenic peptides, for example, use herein 4 kinds of HER3 immunogenic peptides for enumerating or appointing for immune response, can be induced in patients based on patient's cancer type What its MHC II para-immunity originality peptide., can be with the monitoring of this blood testing with recurrent breast as non-limiting examples Gland cancer and the patient for lacking pCR to lower rectal cancer, to determine its anti-HER3 CD4 Th1 response and correspondingly treat.

The low anti-HER3 responses detected by blood samples of patients test or other method can be resisted by restoration methods, Such as vaccine, and it is preferably based on the BMDC of patient's monocyte derived with HER3 immunogenic peptides pulse/incubation Vaccine, the 4 kinds of HER3 immunogenic peptides wherein used in the HER3 immunogenic peptides such as embodiment of the present invention.This area is common Technical staff will readily appreciate that there is the other manner for recovering patient's immune response.Especially, should for TN IBC patients Group is inherently associated with limited therapeutic choice, the method for measuring it HER3 responses, and if desired, extensive by DC1 vaccines The method of multiple this response may be proved to be very valuable.Anti- HER3 immune responses are also used as needing lower rectal cancer Patient potential prognosis biomarker.The DC1 vaccines of HER3 pulses or other suitable vaccines may be overexpressed to HER3 Breast cancer and other expression HER3 cancer development have treatment and/or risk conditioned effect.The discovery of the present invention can Be understood to can be used for desired by the exploitation present invention for the array blood testing and measure that diagnose and/or treat.

The disclosure of every patent, patent application and publication that the present invention is quoted is integrally incorporated the present invention by quoting In.Although disclosing the present invention by reference to specific embodiment, but it is clear that others skilled in the art can not take off Other embodiments of the invention and change are designed in the case of from true spirit and scope of the present invention.

Claims

1. a kind of isolated peptides, it is selected from the group consisted of：

P11-13 (peptide 51-75)：KLYERCEVVMGNLEIVLTGHNADLSFLQW (SEQ ID NO.1), p81-83 (peptide 401- 425)：SWPPHMHNFSVFSNLTTIGGRSLYN (SEQ ID NO.2), p84-86 (peptide 416-440)： TTIGGRSLYNRGFSLLIMKNLNVTS (SEQ ID NO.3), p12 (peptide 56-70)：CEVVMGNLEIVLTGH(SEQ ID NO.4), p81 (peptide 401-415)：SWPPHMHNFSVFSNL (SEQ ID NO.5), p84 (peptide 416-430)： TTIGGRSLYNRGFSL (SEQ ID NO.6) and p91 (peptide 451-465)：AGRIYISANRQLCYH(SEQ ID NO.7).

2. a kind of immunomodulator, it includes the peptide described in one or more claims 1.

3. a kind of vaccine, it includes the peptide and pharmaceutically acceptable salt described in one or more claims 1.

4. vaccine according to claim 3, it further includes adjuvant.

5. a kind of cell, wherein the cell is contacted with the peptide described in one or more claims 1.

6. cell according to claim 5, wherein the cell is antigen presenting cell.

7. the cell of claim 5, wherein the cell is T cell.

Trigger the method for immune response in subject 8. a kind of, including the group described in claim 1 is applied to the subject Compound.

9. a kind of method for treating cancer in subject, including applied to the subject described in one or more claims 1 Peptide.

10. method according to claim 9, wherein the subject is people and suffers from cancer.

11. method according to claim 10, wherein the cancer is selected from the group consisted of：Breast cancer, oophoroma, Lung cancer, prostate cancer, colon cancer, melanoma, cancer of pancreas, human primary gastrointestinal cancers, the cancer of the brain and its any combinations.

12. a kind of active cell method, including the cell is contacted with the peptide described in one or more claims 1.

13. method according to claim 12, wherein the cell is antigen presenting cell.

14. method according to claim 12, wherein the cell is T cell.

15. it is a kind of for the load peptide of immunization therapy, the production method of the BMDC (DC) of activation, including：

With DC described in the peptide pulse described in one or more claims 1；

The DC is activated with least one TLR activators.

16. method according to claim 15, including make the DC with the intracellular calcium concentration in the DC can be improved Reagent is contacted.

17. method according to claim 15, wherein the reagent includes Calcium ionophore.

18. method according to claim 15, further comprises DC described in freezen protective, wherein when the DC thaws, The DC produces at least one cell factor of effective dose to produce t cell response.

19. the cell produced as the method described in claim 15.

20. a kind of vaccine, it includes the cell produced as the method described in claim 15.

21. vaccine according to claim 20, wherein the vaccine is the form of injectable multiple dose vaccine.

22. a kind of method of the initiation immune response in mammal, including by as the method generation described in claim 20 Cell mass is applied to mammal in need.

23. a kind of method for treating mammalian diseases or imbalance, including by as the thin of the method generation described in claim 20 Born of the same parents group is applied to the mammal of this needs.

24. a kind of tumour for being used to detect in the precancerous lesion at the stomach oesophagus junction surface of the subject with Barrett oesophaguses is entered The biomarker of exhibition, it includes detecting the overexpression of HER3 in the subject.

25. a kind of method for treating the patient for having lost anti-HER3CD4+Th1, including apply at least one agent to the patient The DC1 vaccines of the antigen pulse of monocytic dendritic cell shape cell (DC) precursor from the patient of amount, the list of the patient Monocytic dendritic shape cell precursors HER3MHC II para-immunity originality peptides enter horizontal pulse, and wherein peptide is selected from group consisting of：

P12 (peptide 56-70)：CEVVMGNLEIVLTGH(SEQ ID NO：4)；

P81 (peptide 401-415)：SWPPHMHNFSVFSNL(SEQ ID NO：5)；

P84 (peptide 416-430)：TTIGGRSLYNRGFSL(SEQ ID NO：6)；With

P91 (peptide 451-465)：AGRIYISANRQLCYH(SEQ ID NO：And its any combinations 7).

26. method according to claim 25, wherein the patient has three negative infiltrative breast carcinomas.

27. a kind of method for detecting patient's moderate resistance HER3CD4+Th1 losses, including：With HER3MHC II para-immunity originality peptide arteries and veins The PMBC for coming from the patient is brought, wherein the peptide is selected from group consisting of：

P12 (peptide 56-70)：CEVVMGNLEIVLTGH(SEQ ID NO：4)；

P81 (peptide 401-415)：SWPPHMHNFSVFSNL(SEQ ID NO：5)；

P84 (peptide 416-430)：TTIGGRSLYNRGFSL(SEQ ID NO：6)；With

P91 (peptide 451-465)：AGRIYISANRQLCYH(SEQ ID NO：And its any combinations 7)；It is resulting with detecting Immune response.

28. method according to claim 27, wherein measuring the anti-HER3 CD4 by IFN-γ ELISpot determination methods The detection of Th1 responses.