CN107533065A - CD4 is monitored in cancer and immune restoration+The method of 1 type t helper cell response - Google Patents

Abstract

The method of diagnosis or treatment with cancer or the mammalian subject for having generation risk of cancer, including：CD4 is produced from antigen presenting cell or its precursor⁺Th1 responses, and the CD4 for causing interferon gamma (" IFN γ ") secretion is produced from the blood sample of the subject⁺T cells；The described anticancer CD4 with detection⁺Th1 responses are to determine whether the response lowers.Recover HER2 specific Cs D4 in HER2 breast cancer patients with positive in need⁺The method of Th1 immune responses, including：BMDC (" DC ") vaccine of the bacterium is administered, it is included with II class MHC binding peptides (" dendritic cell vaccine ") the pulse autologous BMDC in HER2 sources to improve the CD4+Th1 responses of anti-HER 2 of the patient；With the Th1 responses of anti-HER 2 of the patient before and after dendritic cell vaccine is inoculated with are determined to determine the incrementss in the response.

Description

CD4 is monitored in cancer and immune restoration+The method of 1 type t helper cell response

The application is the part continuation application for the Serial No. 14/985,303 submitted on December 30th, 2015, and it is successively The part continuation application for the Serial No. 14/658,095 submitted on March 13rd, 2015, its claim priority successively and by Beneficial to the U.S. Provisional Application of Serial No. 61/953,726 submitted on March 14th, 2014, the full content each so applied It is expressly incorporated herein by reference.The application is also claimed priority and benefited from and carried according to Patent Cooperation Treaty on March 13rd, 2015 The international numbering of friendship is PCT/US 15/20613 international application, and it claims priority and benefited from March 14 in 2014 successively The U.S. Provisional Patent Application for the Serial No. 61/953,726 that day submits, the full content each so applied is by reference to simultaneously Enter herein.

Thank you

The application is developed under being supported in the part that the fund number that NIH authorizes is R01CA096997. Government has certain right for the application.

Technical field

The present embodiment is directed to the gradual loss of immune response in cancer, especially anti-HER2/neu CD4⁺1 type T auxiliary is thin Loss of born of the same parents' (" Th1 ") response in the HER2- breast cancer driven and its recovery, and the diagnostic monitoring method based on it, treatment Method and kit for.

Background technology

The main reason for breast cancer (" BC ") is cancer related mortality rate in world wide.See, Jemal, A. etc., global cancer Disease counts.CA：Cancer clinical doctor magazine 61：69-90(2011).By gene expression mark development, be presently recognized that to Few four kinds of significant tumor of breast phenotypes：Tube chamber A types and tube chamber Type B, basal cell template and human epidermal growth factor receptor-2 Type/neu (" HER2^pos”).See, Perou, CM., etc. Nature406：77-52(2000).HER2 is overexpressed, including about 20- Driving molecule (Meric, F., etc. J.Am Coll.Surg.194 in the kinds of tumors type of 25% breast cancer：488-501 (2002) it is), related with aggressive clinical disease course, the overall poor prognosis of chemotherapy resistance and breast cancer.See, Henson, E.S., Clin.Can.Res.12：845-53 (2006) (" Henson, etc. ") and Wang, G.S., Mol.Med.Rep.6 (4)： 779-82 (2012) (Wang, G.S., etc.).At breast cancer initial stage, HER2 be overexpressed with the invasion of enhancing (Roses, R.E., Deng cancer control measure Sheng Wubiaojis ＆Prev.18 (5)：1386-9 (2009)), tumor cell migration (Wolf-Yadlin, A., etc., molecular system biology 2：54 (2006)), and expression (Wen, the X.F., etc. oncogene of angiogenic factors 25：6986-96 (2006)) it is related, show that HER2 is promoting tumour to occur to play key effect in environment.Although HER2- targets To treatment (i.e.,/ trastuzumab), with chemotherapy therapeutic alliance, significantly improve HER2^posPatient with breast cancer's deposits Motility rate (Piccart-Gebhart., M.J., etc. N.Eng.J.Med.353 (16)：1659-72(2005))(“Piccart- Gebhart, etc. "), the patient of significant proportion the treatment is generated resistance (Pohlmann, P.R., etc., Clin.Can.Res.15：7479-91 (2009) (" Pohlman, is waited ")).Therefore need to differentiate that Endodontic failure high-risk patient is sub- The strategy of group, and improve the new method of HER2- targeted therapy response rates.

There is increasing evidence that improve prognosis in the cellullar immunologic response of strength and breast cancer in tumor microenvironment It is relevant, especially in HER2^posIn hypotype, Alexe., G. are seen, etc. Can.Res.67：10669-76(2007).Therefore, exist Progress is achieved in the decoding of the individual immunity medium of these antitumor actions.Although cytotoxin CD8⁺T lymphocytes (" CTL ") is considered as main effects thing (Mahmoud, the S.M., etc. J.Clin.Oncol.29 of antineoplastic immune in the past： 1949-55 (2011)), promote CTL responses to generate minimum clinic with polypeptide vaccine in the breast cancer of HER2- drivings Influence (Amin., A., etc. cancer immunity immunotherapies .57 (12)：1817-25 (2008)), it may be possible to because CTL functions exist There is no suitable CD4⁺It is non-optimized with the help of T- lymphocytes, according to Bos, R., the report waited, cancer research .70： 8368-77(2010).Except the generation to CTLs and continue it is most important in addition to, CD4⁺T auxiliary (" Th ") cells pass through other Mechanism adjusts antitumor action, including directly cytotoxic destruction tumor promotion, regulates and controls antitumor cell factor response, and increase Strong long time-histories immunological memory (Cintolo, J.A., etc. Future Oncol.8：1273-99(2012)).By promoting immune ball The type conversion of albumen, Th cells also contribute to antitumor humoral immunity and effect B cell response.See, Parker, D.C., Deng Ann.Rev. immunologys 11：331-60 (1993) (" Parker, is waited ").Really, CD4 is produced in tumor microenvironment⁺1 type T Interferon (" IFN ")-γ of auxiliary cell (" Th1 ") infiltration is related to the improvement prognosis of breast cancer.See, Gu-Trantien, C, etc. J.Clin.Inv.123：2873-92(2013).

However, the anti-HER2 CD4 of whole body of the tumor of breast generation in HER2- drivings⁺The effect of Th1 responses is still not clear Really.Still there is an urgent need to the strategy of predicted treatment failure high-risk patient subgroup, and the HER2- targets using trastuzumab are improved To treatment and the method for chemotherapeutic response rate.Therefore, one or more embodiments of the application are intended to tackle and mentioned herein One or more problems.

The content of the invention

In a broad aspect, the invention provides diagnosis or treatment with cancer or to have the lactation that risk of cancer occurs The method of animal subjects, including：From antigen presenting cell (" APCs ") or its precursor and from the subject's CD4 in blood sample⁺The anticancer CD4 of T- Hemapoiesis circulation⁺Th1 responses, it causes interferon gamma (" IFN-γ ") point Secrete；The described anti-cancer CD4 with detection⁺Th1 responses are to determine whether the response lowers.

In a further aspect, the generation step further comprises：Separated from the blood sample unexpanded outer All blood monocytes (" PBMCs ")；With composition pulse PBMCs and APC- precursors monocyte therein, the composition bag Include based on the immunogenicity II class MHC binding peptides for making the haunted cancer types of subject, so as to activate the CD4 in PBMC⁺ Th1 cells are with secretion of gamma-IFN；The detecting step includes the secreted IFN-γ of detection.

At alternative aspect, the generation step further comprises：The CD4 that will be purified in subject's sample⁺T Cell and from subject's sample, APC prematurities with composition pulse or ripe BMDC (" DCs ") it is common Culture, the composition are included based on the immunogenicity II class MHC binding peptides for making the haunted cancer types of subject, so as to Activate CD4⁺T cell is with secretion of gamma-IFN；The detecting step includes the secreted IFN-γ of detection.

On the other hand, the cancer is selected from by breast cancer, the cancer of the brain, carcinoma of urinary bladder, the cancer of the esophagus, lung cancer, cancer of pancreas, liver Cancer, prostate cancer, oophoroma, colorectal cancer and stomach cancer or its be combined formed group.

On the other hand, the cancer is expression HER2.

Another further aspect, the cancer are HER2- positive breast cancers, and the subject is female human, and described immune Originality II class MHC binding peptides are based on HER2 molecules.

In a preferred embodiment, described composition further comprises HER2 II class MHC hla binding peptides, and it includes：

Peptide 42-56 (SEQ ID NO：1)；Peptide 98-114 (SEQ ID NO：2)；Peptide 328-345 (SEQ ID NO：3)；Peptide 776-790(SEQ ID NO：4)；Peptide 927-941 (SEQ ID NO：5)；With peptide 1166-1180 (SEQ ID NO：6).

In a preferred embodiment, the IFN-γ is determined using IFN-γ enzyme-linked immunospot assay (" ELISPOT ") Produce.

On the other hand, HER2- specific Cs D4 is recovered in the HER2- breast cancer patients with positive for have demand⁺Th1 is immunized The method of response, including：To the dendritic cell vaccine (DC vaccine) of patient therapeuticallv's effective dose, the dendron Shape cell vaccine includes the autologous dendritic cell (" BMDC with immunogenicity HER2 II class MHC binding peptide pulses Vaccine inoculation ", DC vaccination) to improve the CD4 of anti-HER 2 of the patient⁺Th1 responses；With according to above-mentioned side The generation in face and detecting step determine the anti-HER2 CD4 before and after patient vaccination's dendritic cell vaccine⁺Th1 Response is to determine the incrementss in the response, wherein the restoration methods further comprise：By one or more attached Time interval is added to implement generation and the detecting step of above-mentioned aspect to determine the anti-HER 2 of patient after inoculation dendron shape vaccine CD4⁺Th1 responses recover state, are recovered with monitoring the response.

On the other hand, the method for screening breast cancer or other cancers individual, including：According to the generation and inspection of above-mentioned aspect Survey step method come detect individual anti-HER2 CD4⁺Th1 responses, to determine compared with healthy individuals, whether the response Lower.

On the other hand, screening has the individual method that breast cancer or other risk of cancer occurs, including：According to above-mentioned side The generation in face and the method for detecting step detect the individual anti-HER2 CD4⁺Th1 responses, with determination and healthy individuals Compare, whether the response lowers.

On the other hand, predict whether HER- breast cancer patients with positive is non-to the standard of such as chemotherapy and trastuzumab The good method of immunization therapy response, including：The patient is detected according to the method for the generation of above-mentioned aspect and detecting step Anti- HER2 CD4⁺Th1 responses.

On the other hand, in positive-aggressive breast cancer of HER2- treated with chemotherapy and trastuzumab (“HER2^pos- IBC ") method that new mammary gland event is predicted in patient, including：According to the generation of above-mentioned aspect and detecting step Method determine the anti-HER2 CD4 of the patient⁺Th1 responses, to determine whether the response lowers.

On the other hand, predict HER2- breast cancer patients with positive through lower rectal cancer trastuzumab and chemotherapy (" T/ C ") after HER2- positive breast cancers pathology response method, including：According to the generation of above-mentioned aspect and detecting step Method determines anti-HER2-CD4 in the patient after T/C treatments⁺The degree of Th1 responsiveness, with determine the response whether be with The complete response of lower rectal cancer pathology correlation (infiltrative breast carcinoma of pathology noresidue after surgery) it is notable elevated anti- HER2 CD4⁺Th1 responses, or the relatively low response that the complete response of right and wrong pathology is related, and further, in the trouble In the case of the non-pathology complete response of person, the anti-HER2 CD4 of the patient⁺Th1 responses are able to by DC vaccine inoculations Recover.

In another broad aspect, the side of diagnosis or treatment with cancer or the mammalian subject for having generation risk of cancer Method, including：Blood is obtained from the subject；In conjunction with inhibition, blood testing is carried out to determine anticancer CD4⁺Th1 should The suppression answered；From selected from by the targeting cancer therapies such as DC vaccines, trastuzumab, chemotherapy, the common cancers such as operation and radiation Select to apply the cancer drug of effective dose to the subject in the formed group for the treatment of.

On the other hand, HER2 is monitored after the targeting additional chemotherapy of breast cancer treatment is completed^posThe side of-IBC patients Method, to assess the risk for recurring the cancer, including：After the treatment is determined according to the generation of above-mentioned aspect and detecting step The anti-HER2 CD4 of patient⁺The degree of Th1 response rates is to determine the anti-HER2 CD4 of the response⁺Whether the notable downward of response Recurrence or higher anti-HER2 CD4 with the cancer⁺Th1 responses are relevant with non-recurrence.Enter on the one hand, the targeting is controlled Treat to include and the patient is administered in the medicine trastuzumab.

In order to more fully understand exemplary embodiment, and other and its further feature and advantage, reference is described below, With reference to accompanying drawing, the scope required by the present embodiment is pointed out by appended claims.

Brief description of the drawings

When reading in conjunction with the accompanying drawings, the specific descriptions of following preferred embodiment may be better understood.In order to say Bright embodiment, is shown in the drawings presently preferred embodiment.It should be appreciated, however, that preferred embodiment be not to be limited by it is attached The precise alignment and means of the embodiment shown in figure.

Fig. 1 is the hierarchical structure chart for representing to include patient/donor group in reference example research described here.Frame Lattice are labeled A-H.The time point of sash G and sash H therapeutic scheme and blood drawing is represented with red callout box.Especially, exist The HER2 of T/C treatments^posIn-IBC sashes (G), patient receives any one in lower rectal cancer T/C, then by performing the operation simultaneously Using adjuvant trastuzumab as terminating；Patient is from operation-preferential method and using adjuvant T/C as terminating.Complete adjuvant Trastuzumab ＜ is drawn blood for 6 months or >=6 months.

Fig. 2 shows BMDC (" DC ") vaccination strategies.By leukopheresis and elution, by patient's Monocyte is separated from other leucocytes first.By these monocytes in serum free medium (" SFM ") with grain Granulocytemacrophage colony stimulating factor (" GM-CSF ") and interleukin (" IL ") -4 are cultivated to be changed into immature tree Prominent shape cell (" IDCs " or " iDCs ").Then these cells and 6 kinds of HER2II class MHC binding peptides are entered into horizontal pulse, and added Add interferon (" IFN ")-γ and lipopolysaccharides (" LPS ") to complete ripe and activation, so as to real before patient is injected back into Now whole DC activation are DC 1s.See, Fracol, M., etc. Ann.Surg.Oncol.20 (10)：3233(2013).As for HLA- A2^posPatient, the cell and I class MHC binding peptides for having half enter horizontal pulse, in addition a semicell and different MHC1 class binding peptides Enter horizontal pulse.

Fig. 3 A and 3B are the charts for showing ELISPOT test bay precision.Research for Fig. 3 A comes from, in ELISPOT There is the sample of 5 donors (being represented with different symbols) of known different anti-HER2 reactivities in 3 days in experiment Carry out three parallel repetitions.For with HER2 ectodomains (" ECD ") peptide mixer (peptide 42-56 (SEQ ID NO：1)), peptide 98-114(SEQ ID NO：And peptide 328-345 (SEQ ID NO 2)：3) donor of stimulated in vitro, for the anti-HER2 of its accumulation Th1 responses (" average SFC (spot formation cell)/2x10⁵Cell ") drafting average coefficient of variation (" average CV "), error bars table Show the standard deviation (" SD ") between each repetition.For each donor as the measure that made a variation between experiment (with different symbol tables Show), Fig. 3 B show Th1 responses (" the average SFC/2x10 for its accumulation⁵Cell ") draw in independent each three groups of examinations in day Standard deviation (" SD ") between testing.SD linear regression, show to return with parallel dotted lines 95% are put caused by connecting line expression Believe section.

Fig. 4 shows the chart of explanation ELISPOT linear relationships.HER2- reaction donors from two high responses (supply Body #1, (triangle) and donor #2 (circle)) triplicate PMBC (" PBMCs ") be diluted to step by step come From in the PBMCs of known heterologous non-HER2 respondents (it is identical PBMC donors for all experiments), HER2 is used ECD peptide mixers (peptide 42-56 (SEQ ID NO：1), peptide 98-114 (SEQ ID NO：And peptide 328-345 (SEQ ID NO 2)： 3)) it is stimulated in vitro.It is that each dilution point subtracts and do not have stimulated background in ELISPOT experiments.

Fig. 5 A-5D show anti-HER2 CD4⁺Th1 responses and IgG1/IgG4 reactivities are in HER2^posTumor of breast is sent out Raw gradually loss.Fig. 5 A show systemic CD4⁺T cell and anti-HER2 CD4⁺The IFN-γ ELISPOT analyses of Th1 responses Column diagram (left side)；Correspondingly, the post-hoc scheffep- values between patient's group compare on the side of column diagram (right side) Show.Patient's group of research is：HD (healthy donors)；BD (benign breast biopsy)；HER2^negDCIS；HER2^neg IBC (non-suspicious HER2^neg(HER2 0 and 1+) infiltrative breast carcinoma)；HER2^pos DCIS(HER2^posDCIS)；With HER2^posIBC (stage I/II HER2^posInfiltrative breast carcinoma).The column diagram at top shows overall anti-HER2 response rate (%100) (patient is more than 1% to the response rate for reacting peptide) (also referred to as " anti-HER2 response rates ")；Middle cylindricality diagram Gone out reaction peptide average (n) (average number for the reaction peptide (n) that the patient in group reacts as integrally) (also by Referred to as " response group storehouse ")；The column diagram of bottom shows averagely total SFC/10⁶Cell is (from each tested group of all 6 kinds of II The summation (every 10 from IFN-γ ELISPOT analyses of class MHC combination reactive polypeptide spots⁶The spot formation cell of cell " SFC ")) (also referred to as " cumulative acknowledgements ") (all variance analysis p ＜ 0.001).When passing through anti-HER2 response rates, response group storehouse When being assessed with cumulative acknowledgements, in HER2^posTumor of breast shows CD4 in occurring⁺Gradual loss (that is, the HD/ of Th1 responses BD→HER2^pos-DCIS→HER2^pos-IBC).In HER2^neg- DCIS and HER2^neg- IBC (IHC 0/1+) and HD/BD are tested There is no the difference for finding Th1 responses in person.Fig. 5 B pass through ELISPOT (cumulative acknowledgements (average total SFC/2x10⁵Cell)) show With shown in Fig. 5 A in identical patient group respectively and the HER2 of T/C treatments^pos(" T/C " means song to-IBC patients group Appropriate monoclonal antibody and chemotherapy) in IFN-γ secretion,.As a result every 2x10 in box palpus figure is passed through⁵The median of cell ± Interquartile range (" IQR ") IFN-γ SFC is represented.Fig. 5 C pass through donor age (age ＜ 50 compares age >=50) (upper left Side), menopausal state (premenopausal to compare post menopausal) (upper right side), ethnic group (white people compare other ethnic groups) (lower left side) and bosom The column diagram for showing HD/BDs moderate resistance HER2 Th1 cumulative acknowledgements differences of pregnant (0 compared to >=1 time pregnancy) (lower right side) layering. In each Th1 modules, as a result passing ratio or average value (± SEM) represent.Fig. 5 D are shown for restructuring HER2 The ELISA results of the seroreaction activity of ECD peptides.ELISA is determined with the OD value under the conditions of 1: 100 serum-dilution (" OD ") shows and (scatter diagram of packet, shows mean OD value with horizontal line).In HD (circle/left side), HER2^pos- DCIS is (square Shape/centre) and HER2^posAnti- HER2 IgG1 antibody levels (top side) and anti-are determined in-IBC (triangle/the right) patient HER2 IgG4 antibody levels (bottom side) (pass through unpaired t-test or the variance point detected using post-hoc scheffe Analysis, * * * p ＜ 0.001, as criterion).Compared with healthy donors (HDs), anti-HER2 IgG1 and IgG4 antibody level exists Show significantly to raise in HER2pos-DCIS patient, decay in HER2pos-IBC patient.

Fig. 6 is shown for HD (healthy donors)；BD (benign breast biopsy)；HER2^negDCIS；HER2^neg IBC (non-suspicious HER2^neg(HER2 0 and 1+) infiltrative breast carcinoma)；HER2^pos DCIS(HER2^posDCIS)；With HER2^posIBC (stage I/II HER2^posInfiltrative breast carcinoma) the single HER2 peptides of patient to HER2^posDuring tumor of breast occurs The CD4 of accumulation⁺Contribution immune Th1 does not reflect immune sculpture (immune sculpting).Use HER2 ectodomains (" ECD ")-restricted peptides and intracellular domain (" ICD ")-restricted peptides.Show ECD peptides 42-56 (" ECD p42-56 ") (SEQ ID NO：1) (upper left corner)；ECD peptides 98-114 (" ECD p98-114 ") (SEQ ID NO：2) (left portion) and ECD peptides 328-345(“ECD p328-345”)(SEQ ID NO：3) (lower left corner) and ICD peptides 776-790 (" ICD p776-790 ") (SEQ ID NO：4) (upper right corner)；ICD peptides 927-941 (" ICD p927-941 ") (SEQ ID NO：5) (right middle)；And ICD Peptide 1166-1180 (" ICD p1166-1180 ") (SEQ ID NO：6) the Th1 reaction implementations in (lower right corner).Individual is special Property peptide response is by ELISPOT with every 2x10⁵The average IFN-γ SFC descriptions of individual PMBC (PBMCs).Th1 is anti- Implementations are answered to show in HER2^posTumor of breast occur moderate resistance HER2Th1 immunity continuous conspicuousness progressively decline (HD → BD→HER2^neg→DCIS→HER2^neg-IBC→HER2^pos-DCIS→HER2^pos- IBC) (by variance analysis, all p ＜ 0.005).As a result represented with average value ± SEM.

Fig. 7 shows the minimum time rate of change of the anti-HER2 Th1 responses of donor.For paired HD (Green triangle types；N=4) and HER2pos-IBC subject's (blue square of untreated before；N=4) draw from the blood obtained at a distance of at least six moon The anti-HER2 Th1 cumulative acknowledgements (left side) matched caused by sample with donor and response group storehouse (right side).With the time observation HD and The HER2 of untreated before^posThe minimum Th1 response variabilities in vivo that-IBC subjects supply.

Fig. 8 A-8E show HER2^pos- IBC moderate resistance HER2 Th1 loss is not due to lack immunocompetence or immune Suppress the enhancing of phenotype, but with IFN-γ: the functional shift of IL-10- production phenotypes is relevant.Fig. 8 A are by determining accumulation (meaning is average total SFC/10 for Th1 responses⁵Cell) production of IFN-γ is shown to recall in (recall) IFN-γ ELISPOT The stimulant of tetanus toxoid or Candida albicans.As a result every 2x10 in box palpus figure is passed through⁵Median corresponding to cell ± interquartile range (" IQR ") IFN-γ SFC is represented.From HER2^posThe PMBC of-IBC patients, with health Without significant difference, the T/C that still receives that the patient did not received treatment either is controlled the PMBC of donor Treat.Fig. 8 B, top side, using from HD, HER2^pos- IBC (stage I/II) and HER2^pos- IBC s/p T/C be (T/C- treatments Patient) the representational flow cytometry that shows of PMBC of patient dyed to determine their immune table Type.Bottom histograms are shown respectively and illustrated CD4⁺(CD3⁺CD4⁺) (top dyeing) or CD8⁺(CD3⁺CD8⁺) T cell (bottom Dyeing) relative scale, which respectively show patient group CD4⁺(CD3⁺CD4⁺) T cell (left side) and CD8⁺(CD3⁺CD8⁺)T- The relative scale of cell (right side)：HD (dark post), stage I/II HER^pos- IBC (intermediolateral column) and HER2^pos-IBC s/p T/ C (light post).From HER2^posThe PBMC of-IBC patients, from the PBMC of healthy donors without significant different, the patient without By be do not received treat or receive what T/C was treated,.Fig. 8 C, top side, using from HD, HER2^pos- IBC (stage I/ ) and HER2 II^pos- IBC s/p T/C PMBC shows representational flow cytometry dyeing to determine them Immunophenotype.Relatively low column diagram is shown respectively and represented regulatory T cells (" T_reg“；CD4⁺CD25⁺FoxP3⁺) (top contaminates Color) and marrow source property suppression cell (" MDSC "；CD11b⁺CD33⁺HLA-DR^-CD83^-) (bottom dyeing) relative scale, patient's group Regulatory T cells (" T_reg″；CD4⁺CD25⁺FoxP3⁺) (left side) or marrow source property suppression cell (" MDSC "；CD11b⁺CD33⁺ HLA-DR^-CD83^-) (right side) relative scale：HD (dark post), stage I/II HER^pos- IBC (intermediolateral column) and HER2^pos- IBC s/p T/C (light post).From HER2^posThe PBMC of-IBC patients, no matter patient be before untreated or pass through T/C is treated, from healthy donors without significant different.Fig. 8 D show that circulation HER2- specificity IL-10 secretion will not suffer from Change between person's group.By ELISPOT, from HER2^posThe PMBC of-IBC patients, the patient is either not Received the (" HER2 for the treatment of^pos- IBC ") still receive the (HER2 that T/C is treated^pos- IBC s/p T/C), exist with healthy donors Anti- HER2 IL-10 secretions, with overall anti-HER2 response rate (top), are organized storehouse (middle part) and tired without significant difference Product response (bottom) is assessed.As a result passing ratio or average value ± SEM are represented.Fig. 8 E are shown in HER2^posTumor of breast Relative HER2- specificity IFN-γ and IL- secretions in generation.In HD, HER2^pos- IBC (untreated before), and HER2^posTo the accumulation matched with donor across six kinds of HER2 II class peptides in-IBC s/p T/C (T/C treatments) patient IFN-γ secretion and IL-10 secretions (SFC/10⁶Cell) it is compared.The column diagram passes through the SFC tributes across patient's group The percentage (percentage described by column diagram) offered is HER2 antigens-idiosyncrasy (top side) and positive control (CD3 or CD8/ 28) (bottom side)；(IFN-γ secretion (green)；IL-10 secretes (red)) show HER2- specificity IFN-γ and IL-10 Relative scale.From healthy donors to the HER2 for receiving or not receiving T/C- treatments^pos- IBC patients, HER2- specificity IFN-γ with IL-10 relative scale significantly reduces.Absolute IFN-γ: IL-10 secretions are than value changes respectively from 6.6: 1 (HDs) to 0.97: 1 (T/C treatments) and 0.74: 1 (HER2^pos- IBC) (top side).For positive control (the anti-anti- CD3/CD28 of CD3/) (bottom side) IFN-γ: significant relative change is not observed in IL-10 secretions.

Fig. 9 A-9B show anti-HER2 CD4⁺Whole body loss and the transport to HER2 of TH1 subgroups^posThe ratio of breast lesion The cancer week T lymphocytes of example imbalance are unrelated.Fig. 9 A show that two width come from HER2^pos- DCIS lesions (top) and HER2^pos-IBC The photo of the tissue samples (25 μm of post of amplification) of tumour (bottom) representational h and E (" H＆E ") dyeing.Pass through Immunohistochemistry colours, and arrow points to and HER2^poS-DCIS lesions (top) are compared, HER2^pos- IBC tumours (bottom) cancer week Matrix is observed that lymphocytic infiltration is relative and lacked.In appreciable HER2^pos- DCIS (n=14) and HER2^pos- IBC (n=8) In matrix lymphocytic infiltration in the table by photo with low (infringement ＜ 15%), in (15%-24%) and height (＞ 25%) It is quantitative.Fig. 9 B show fourth officer in representational HER2^pos- DCIS (left side) and HER2^posIt is multiple in-IBC (the right) lesion The photo of the result of labelled immune fluorescence.In 5/5 (100%) HER2^posCD4 is observed in-IBC tumours⁺- T cell (green letter Number) substantial lack, wherein main infiltration and matrix lymphatic infiltration be CD8⁺(yellow signal).By contrasting, (4/4 tumour) observes main CD4 in conduit including DCIS⁺- T cell infiltrates.Describe representational HER2^pos- DCIS and IBC lesions；The image of multiple labelling shows above-mentioned corresponding H＆E parts (25 μm of post of amplification).

Figure 10 A-10D show CD4⁺Th1 induces HER2^high, rather than HER2^lowThe mankind and murine mammary carcinoma cells wither Die.Figure 10 A (top side) show the shooting result of the western blot analysis of the caspase-3 mRNA of detection cracking.By SK-BR- 3 cells are with being carried out as follows co-cultivation：Swimming lane 1)-only use complete medium (complete medium)；Swimming lane 2)-only with 10⁶CD4⁺T is thin Born of the same parents (only CD4⁺)；Swimming lane 3 and 4) -10⁶CD4⁺T- cells add 10 respectively⁵HER2II classes peptide (" iDC H ") or incoherent II The immature dendritic cell (" iDCs ") of class BRAF peptides (" iDC B ")-pulse；Swimming lane 5 and 6) -10⁶CD4⁺T- cells are distinguished Plus 10⁵The BMDC 1s of HER2 (" DC1 H ") or BRAF (" DC1 B ")-pulse；Swimming lane 7)-CD4⁺10⁶DC1 H 10⁵+ IFN-γ and TNF-α neutrAb and swimming lane 8)-CD4⁺10⁶DC1 H 10⁵+ IgG Isotype controls Ab.Increased half Guang day The cracking of winter enzyme -3 shows to work as and DC1 H：CD4+T- cells rather than DC1 B, iDC H, or during the co-cultivation of iDC B groups, SK-BR-3 Cell dosage dependence apoptosis.Vinculin, which is used as loading, to be compareed.The Western blotting of displaying is the representative of three experiments.In Between the column diagram (red column) of part show the result represented with average caspase-3 mRNA/vinculin ratio ± SEM, This result shows the protein imprinted corresponding Apoptosis multiple with upper part swimming lane 1-6 (to be determined with Image J softwares Amount).Right figure (black post) (the Western blotting swimming lane 7-8 for corresponding to upper part) in column diagram, these posts represent three The Apoptosis rescue rate of individual experiment/average caspase-3 mRNA/vinculin ratio ± SEM (31.4 ± 5.3%, IFN- γ/TNF-α neutralizes and compares control group).Compared with IgG Isotype controls, CD4⁺：The SK-BR-3 Apoptosis of DC1 H inductions leads to Cross neutralization IFN-γ and TNF-α is significantly rescued.Bottom shows the IFN-γ in respective co-cultivation by ELISA (left side y-axis) (solid post) and TNF-α (right side y-axis) (linear columns) produce accordingly.As a result represented with pg/mL, and it is represented Three experiments.Figure 10 B show " only CD4⁺", " CD4⁺+ DC1 B ", and " CD4⁺+ DC1 H, " the cell photo of groups of cells.It is logical Cross DAPI dyeing and carry out observing apoptosis cell.When with " CD4⁺+ DC1 B " or " only CD4⁺" group compares, in CD4⁺+ DC1 H groups are observed The apoptotic cell (asterisk) increased to number.Column diagram (right figure) shows the percentage of the apoptotic cell of three groups of cells of diagram (multiple), the CD4 related to visual effect⁺The Apoptosis of+DC H groups increases to 25 times.As a result three experiments are represented, and Represented with apoptotic cell percentage ± SEM.Figure 10 C show the shooting result of western blot analysis, wherein HER2^high SK- BR-3, HER2^intermediateMCF-7, and HER2^lowUnanimously maintained in the MDA-MB-231 mankind's BC cells IFN-γ-Ra and The expression of TNF-α-R1 acceptors.Vinculin, which is used as loading, to be compareed.Figure 10 D are shown and undressed control group (no Rx) Or the treatment of the selection single cell factor is compared, in transgenosis muroid HER2^highBreast cancer TUBO (top figure) and MMC15 (HER2^high) in cell (middle), restructuring muroid (" rm ") Th1 cell factor rmIFN- γ and rmTNF- α therapeutic alliance is led Cause dramatically increasing for Apoptosis.In muroid HER2^low/negControlled in cell 4T1 (bottom diagram) with dual rmIFN- γ+rmTNF- α Treat, this effect does not reappear.As a result three experiments are represented, and represent that it is thin by streaming with apoptotic cell percentage ± SEM Born of the same parents' instrument detects PI^pos/ annexin V^posPercentage (* p≤0.05, * * p ＜ 0.01, * * * p ＜ 0.001) obtain.

Figure 11 A-11C are shown by Th1-elaborated cell factors IFN-γ and TNF-α, HER2^highRather than HER2^lowMankind's BC cells are to CD4⁺The Apoptosis of Th1 mediations is sensitive.Figure 11 A (upper diagram) show detection cracking The shooting result of the western blot analysis of caspase-3 mRNA.Using transwell systems, by 50x10³MCF-7 (HER2^intermediate) and 50x10³MDA-MB-231(HER2^low) cell respectively with culture medium (complete medium), only 10⁶CD4⁺ T- cells (only CD4⁺) and 10⁶CD4⁺T- cells+10⁵Each HER2 (DC1 H)-or BRAF control (DC1 B)-pulse DC 1s Co-cultured.Half Guang asparagus fern that is being shown in Western blotting and being represented in following corresponding column diagram (lower) The cracking of enzyme -3 shows working as and DC1 H：CD4⁺When T- cells co-culture, MCF-7 cells (left side) rather than MDA-MB-231 are thin The increase of born of the same parents (right side) apoptosis.Vinculin, which is used as loading, to be compareed.The Western blotting of displaying represents three experiments, and its result is used Ratio ± the SEM of average caspase-3 mRNA/vinculin represents (fold induction for showing Apoptosis).Figure 11 B are shown By 50x10³The protein that SK-BR-3 cells co-culture with the culture supernatant that following operating condition obtains in Figure 10 A prints Mark result photo [only uses complete medium；Only with 10⁶CD4⁺T cell (only CD4⁺)；CD4⁺T- cell+HER2 pulses iDC (“iDC H”)；And CD4⁺T- cell+BRAF- pulses iDC (" iDC B ")；CD4⁺T cell+10⁵HER2- pulse DC1 (" DC1 H”)；And CD4⁺T- cells+10⁵BRAF- pulses DC1 (" DC1 B ")].With DC1 B, iDC H, iDC B, or only CD4⁺'s Group is compared, DC1 H：CD4⁺The caspase-3 mRNA level of the detected cracking of group is of a relatively high.As a result three experiments are represented. Figure 11 C show the caspase-3 mRNA for detection cracking, (left with the TNF-α and IFN-γ culture SK-BR-3 of instruction quantity Side), MCF-7 (centre), and the photo of the Western blotting of MDA-MB-231 cells (right side).Relatively low column diagram corresponds to top The swimming lane of the Western blotting figure of portion's displaying.Compared with untreated control, combined with Th1 cell factors IFN-γ and TNF-α Treatment causes SK-BR-3 (HER2^high；10ng/mLTNF- α+100U/mL IFN-γs) and MCF-7 (HER2^intermediate； 100ng/mL TNF-α+1000U/mL IFN-γs) cell has higher Apoptosis.MDA-MB-231 cells (HER2^low； 200ng/mL TNF-α+2000U/mL IFN-γs) still largely not by dual IFN-γ+TNF-α treatment Influence.As a result three experiments are represented.(* p ＜ 0.05, * * p ＜ 0.001).

Figure 12 A-12E show anti-HER2 CD4⁺Th1 immunity is after HER2- pulses DC1 is immune, rather than in HER2- Discriminatively repaired after targeted therapy,.Figure 12 A be by assessing overall anti-HER2 response rates (top), response group storehouse (in Between), and cumulative acknowledgements (bottom), obtain receiving trastuzumab and chemotherapy (" " t/C- treatments HER2^pos- IBC ") (red) HER2^posThe HER2 of-IBC patients and before untreated^pos- IBC patients (" HER2^pos- IBC no tx ") (black) in CD4⁺Column diagram in Th1 responses.By anti-HER2 response rates (top), organize storehouse (middle part) and cumulative acknowledgements (bottom) illustrate, with The stage I/II HER2 of untreated before^pos- IBC patients (no tx) compare, and anti-HER2 Th1 responses are not in stage I- III HER2^posGlobal amplification is able to after the T/C treatments of-IBC patients (T/C treatments).Compared with no tx (n=5), After HER2- specificity and lockjaw (positive control) stimulate, IFN-γ: the relative scale of IL-10 reacting cells (is described under relatively The percentage of the column diagram of side；IFN-γ：It is solid；IL-10：Diagonal) do not improved in T/C treatments (n=5).Figure 12 B are By assessing overall anti-HER2 response rates (top), response group storehouse (middle part) and cumulative acknowledgements (bottom), obtain in HER2 arteries and veins (" HER2 before and after punching-DC1 is immune^pos- IBC PRE vax ") (black) and (" HER2pos-IBC POST vax ") it is (green Color), HER2^posCD4 each instant in-IBC patients⁺The chart of Th1 responses.(POST vax) is immunized in HER2 pulses-DC1 Afterwards, it can observe that all anti-HER2 Th1 are immunized in 11 stage i HER2pos-IBC (PER vax) patient immediately Measurement significantly improves.However, after lockjaw stimulation, the relative scale of IFN-γ and IL-10 reacting cells (is described under relatively The percentage of the column diagram of side；IFN-γ：It is solid；IL-10：Diagonal) substantially do not change, suffer from PRE vax (n=5) Person compares, and HER2 pulse vaccine inoculations significantly increase IFN-γ and IL：Phase of 10 reacting cells in POST vax (n=5) Comparative example.Figure 12 C show that DC vaccine inoculations and trastuzumab/chemotherapy are made in the anti-HER2Th1 stage match being immunized With.(" No tx ") of untreated before AJCC stages I, (" the T/C treatments ") of T/C treatments, and HER2- pulses DC1 are immunized (“POST-vax”)HER2^posIt is by the anti-HER2 response rates (top) of entirety, response group that matching between-IBC patients, which is compared, What storehouse (centre) and cumulative acknowledgements (bottom) were assessed.After HER2- pulses DC1 is immune, rather than after T/C treatments, Stage I HER2^posThe distinguishing recoveries of the middle Th1 of stage match of-IBC patients are continued.As a result with ratio or average Value ± SEM is represented；(* * p ＜ 0.01, * * * p ＜ 0.001).Figure 12 D and 12E show the CD4 after DC vaccine inoculations⁺Th1 The continuation of immune response.Before DC vaccine inoculations (" PRE VACCINE "), it is inoculated with after DC vaccines immediately (" IMMEDIATEPOSTVACCINE ") and vaccine inoculation >=6 month (" >=6 MO POSTVACCINE "), by stage I/II HER2^posImmune response in-IBC patient is compared.After the period immediately after vaccine inoculation, although at this time point (interrupted arrow) has been initialized or completed in all patient's body whole body chemotherapy, is used for 11 vaccine inoculation >=6 month There are the anti-HER2 CD4 of 9 patients in the patient of assessment⁺The immune lasting amplifications of Th1.Scattered point gives the CD4 in response group storehouse⁺ Th1 reactivities analyze the cumulative acknowledgements (Figure 12 E) of (Figure 12 D) and individual vaccine inoculation subject.

Figure 13 A-13E show the anti-HER2 Th1 responses lowered after T/C treatments and unfavorable clinic and pathological examination It is related.According to Figure 13 A chemotherapy order (lower rectal cancer compares adjuvant), Figure 13 B- are from trastuzumab treatment is terminated at this The time registered in research (＜ 6 is compared >=6 months)；Figure 13 C- Estrogen Receptor status (ER^posCompared to ER^neg) and Figure 13 D- diseases Stage (I compares III compared to II) of science is classified, and Figure 13 A-13D show the HER2 of T/C treatments^posThe subgroup of-IBC patients Analysis, these patients do not prove out in anti-HER2 response rates (top graph), group storehouse (middle graph) or cumulative acknowledgements (bottom diagram) There is obvious difference.Figure 13 E are shown terminate with T/C after without the HER2 that mammary gland event (" No BE ") occurs^pos- IBC patients Compare, the patient of mammary gland event ("+BE "), which occurs, has the anti-HER2 reactive (the picture left above) significantly lowered and accumulation Th1 anti- Answer (lower-left figure), pathology complete response (pCR) HER2 is reached after lower rectal cancer T/C^posIn-IBC patients, with not having The patient for carrying out pCR compares, and anti-HER2 Th1 response groups storehouse (scheming in right) and cumulative acknowledgements (lower right corner figure) significantly improve.

Figure 14 is 95 HER2 being incorporated into^posThe flow chart of the study population of patient with breast cancer's experiment embodiment 2.All is swollen Knurl is considered as the infiltrative breast carcinoma of HER2 overexpressions in institutional framework (3+ or 2+/FISH- are positive).The time point of blood drawing is used Red callout represents.Trastuzumab and the subsequent median of chemotherapy (T+C)-treatment group are 44 months (IQR 31).

Figure 15-15D show HER2^posThe anti-HER2 CD4 of patient's group of the non-recurrences of-IBC and recurrence⁺The change of Th1 responses It is different.Figure 15 A are shown in HER2^pos- IBC breast cancer donor (untreated [n=22] before, recurrence=[25], and non-recurrence [n=48]), (top side) is layered by HER2 response rates, the IFN- between response group storehouse (middle part), and cumulative acknowledgements (bottom side) γ⁺Anti- HER2 CD4⁺T cell response makes a variation.As a result passing ratio (%) or average value ± SEM are represented.Single factor test method analyzes p Value is aside shown；Post-hoc Bonferroni test * p ＜ 0.05, * * * p ＜ 0.001.Figure 15 B are shown from non-recurrence With the PMBC of recurrence group in immunocompetence without significantly it is different-pass through ELISPOT CD28s anti-to anti-CD3/ The IFN-γ secretion for stimulating (top side) or the tetanus toxoid (lower left side) for recalling stimulation and Candida albicans (lower right side) come Measure.As a result with intermediate value ± interquartile range (" IQR ") IFN-γ SFC/2x10⁵Cell represents.Figure 15 C show Th1 (T- bet⁺IFN-γ⁺) " T-bet " compare Th2 (GATA-3⁺IFN-Y⁺) " GATA-3 " phenotype to it is non-recurrence and recurrence group PBMCs in HER2 peptides-specific IFN-γ⁺The Relative Contribution of cell.In gating CD4⁺After cell (top side), shown in these groups The dyeing of representative；The result of adjacent column diagram is represented at (middle part) with average proportions (the %) ± SEM shown.When passing through anti-HER2 Th1 response rates (left side), after organizing storehouse (centre) and accumulation meter response (the right) assessment, bottom diagram shows that circulation HER2- is special IL-4 secretions do not change between non-recurrence and recurrence group.As a result represented with ratio or average value ± SEM.Figure 15 D pass through stream Formula cell art shows T_reg(CD4⁺CD25⁺FoxP3⁺) relative scale.Show the dyeing (top side) for having representative in group；It is adjacent The result of column diagram is represented at (middle part) with average proportions (%) ± SEM.Bottom side show HER2- specificity IL-10 secretion across It is similar between the non-recurrence of more all three Th1 measurements and recurrence group.As a result represented with ratio or average value ± SEM.

Figure 16 shows treatment HER2 completely^posThe Cox Proportional hazards mould of the disease-free survival (" DFS ") of patient with breast cancer Type, it is layered by anti-HER2 Th1 response rates.Compared to Th1 response patients, non-Th1 responses patient proves obvious even worse risk The DFS of adjustment.

Specific embodiment

It should be appreciated that the present embodiment has been simplified for accompanying drawing, picture and description, it is used to be clearly understood that coherent element with explanation, And for purposes of clarity, eliminate many other elements that may occur in the present embodiment.The ordinary skill people of association area Member should appreciate that these other elements are suitable and/or needs, to implement the present invention.However, because these elements exist Known in the art, and because they do not facilitate a better understanding of the present invention, therefore do not provide to these yuan herein The discussion of element.

In this specification, " one embodiment " or " embodiment " or similar means mentioned describe relevant with embodiment Special characteristic, structure or characteristic include at least one embodiment of the present invention in.Therefore, through the phrase of this specification The appearance in each position such as " in one embodiment " or " embodiment " is not necessarily referring to for identical embodiment.

In addition, in order to promote the understanding to the principle of the invention, referring now to embodiment shown and described herein, and It will be described using specific language.However it will be appreciated that the scope of the present invention will not be thus restricted；Herein with Description or any change of embodiment represented with illustration and further modification and rule of the present invention it is further Using this is conventional for technical staff that the invention relates to the field.The restriction of the scope of the present invention should basis Expression with last right requirement in the patents announced with one or more determines.

Definition

Unless otherwise defined, all technologies used herein and scientific terminology have this area of theme belonging to the present invention general Logical technical staff's is generally understood that identical meaning.Although it can be used for similar or equivalent to any method as described herein and material The present embodiment, preferable method described herein and material.

Generally, the experimental procedure in nomenclature used herein and cell culture, molecular genetics, organic chemistry and nucleic acid Chemistry and hydridization be it is well-known in the art and generally use those.

Use standard law nucleic acid and peptide.Method and steps usually various is joined according to this area conventional method and generally Examine file carry out (for example, Sambrook and Russell, 2102, molecular cloning：Laboratory's handbook, ColdSpringHarborPress, ColdSpringHarbor, NY, and Ausubel etc., 2012, modern molecular biology experiment Technology, John Wiley＆Sons, NY), provided through this document.

Nomenclature used herein and the experimental procedure used in following analytical chemistry and organic synthesis are this areas It is well-known and generally use those.Standard law is changed for chemical synthesis and chemical analysis it.

As it is used herein, the implication of each following term is related to the implication of its part herein.

Article " one kind " used herein refers to herein grammatically one or more than one (namely at least one) Object.For example, " a kind of element " refers to an element or more than one element.

" about " used herein, when refer to such as quantity, duration etc. can measured value when, mean including from standard value ± 20% or ± 10%, or ± 5%, or ± 1%, or ± 0.1% excursion, such change is disclosed for implementing Method is appropriate.

" adjuvant treatment " used herein for breast cancer refers to giving after primary treatment (that is, perform the operation) any For increasing the treatment of long-term surviving possibility." lower rectal cancer " is the treatment given before primary treatment.

Term " antigen " used herein or " ag " are defined as the molecule for causing immune response.The immune response may wrap Including antibody producing or activation specific immune activity cell, or both all includes.It will be understood by those skilled in the art that substantially Any macromolecular including all proteins or peptide, antigen can be used as.Further, antigen can from restructuring or genome DNA is obtained.It will be understood to those of skill in the art that any DNA, including coding trigger the partial nucleotide of immune response protein Sequence or nucleotide sequence, encode the term " antigen " being used herein.Further, it will be appreciated by those skilled in the art that antigen not Need to only rely on full length gene nucleotide sequence and encoded.It is obvious that the present embodiment includes, however it is not limited to, using more than The partial nucleotide sequence of one gene, and these nucleotide sequences are subjected to different combinations to trigger desired be immunized should Answer.Moreover, it will be understood by those skilled in the art that antigen completely without " gene " coding.It is obvious that antigen can produce Either synthesize or obtained from biological specimen.Such biological specimen can include but is not limited to tissue samples, tumor sample, carefully Born of the same parents or biofluid.

" antigen presenting cell " or " APC " is the cell that can activate T cell, includes, but are not limited to monocyte/huge Phagocyte, B cell and BMDC (" DCs ").

" antigen-pulse APC " or " antigen-load APC " include warp-wise antigen exposure and by Antigen-activated APC. For example, during culture existing for antigen, APC can Antigen in vitro.By being exposed to antigen, APC can also be in body Inside it is supported." antigen load APC " is traditionally prepared with one kind in two methods：(1) small fragments of peptides, known as antigen Peptide, by the outside of direct pulse to antigen presenting cell；Or (2) hatch APC and whole protein or protein particulate, then this A little protein or protein particulate are absorbed by APC.These protein small peptide fragment is digested to by APC and finally transport to and submission To APC surfaces.Alternatively, it is also possible to by the way that the polynucleotide of coding for antigens is introduced into cell to produce antigen-load APC.

" anti-HER2 responses " is the immune response that specificity is directed to HER2 albumen.

Term " antitumor action " used herein refer to can by the reduction of gross tumor volume, tumour cell quantity Reduce, the reduction of metastatic tumour quantity, it is contemplated that the increase in life-span or the improvement of a variety of physiological signs related to cancerous condition come The biological effect of display." anticarcinogenic effect " can also be by binding peptide, and polynucleotide, cell and antibody are in the very first time The ability of the generation of pre- preventing tumor is shown.

" Apoptosis " is the process of apoptosis.Caspase-3 mRNA is the death protein being frequently activated Enzyme.

" autologous " used herein is referred to from any thing acquired in the individual identical individual with being introduced into afterwards Matter.

Term " B cell " used herein is defined as the cell obtained from marrow and/or spleen.B cell can develop As the thick liquid cell of production antibody.

" binding peptide ", see " HER2 binding peptides ".

The loss for the characteristics of term " cancer " used herein is defined as uniqueness-normally control, cause not save The growth of system, the cell propagation for lacking differentiation, local organization infiltration, and/or transfer cell propagation.Example includes but is not limited to, Breast cancer, prostate cancer, oophoroma, cervical carcinoma, cutaneum carcinoma, carcinoma of urinary bladder, the cancer of the esophagus, cancer of pancreas, colorectal cancer, stomach cancer, kidney, liver Cancer, the cancer of the brain, lymthoma, leukaemia, lung cancer, germinoma etc..

“CD4⁺Th1 cells ", " Th1 cells ", " CD4⁺T- aids in 1 type cell ", " CD4⁺T cell " etc. is defined as expressing The hypotype of the T- auxiliary cells of surface protein CD4 and the high-caliber cell factor IFN-γ of production.Also see, " t helper cell ".

" cumulative acknowledgements " mean the combined immunization response of patient's group, and it is with all 6 kinds of MHCII from particular patient group The entire quantity of the reaction spot of class binding peptide represents (every 10 from IFN-γ ELISPOT analyses⁶The spot formation of cell is thin Born of the same parents " SFC ").

" DC vaccine inoculations ", " DC is immunized ", " DC1 is immunized " etc. refer to administering siberian crabapple with autologous BMDC Unite so as to identify special molecular and start the strategy of special response to it.

Term " BMDC " or " DC " be present in vivo, in vitro, in vitro either in host or subject or The antigen presenting cell that can be obtained from candidate stem cell or monocyte.BMDC and their precursor can be from a variety of Lymphoid tissue separates, for example, spleen, lymph node, and from marrow and peripheral blood.BMDC has a kind of morphology special Point, its oriented thin slice (lamellipodium) of multiple directions extension away from BMDC body.Typically, 1 expressed by dendritic cells is high Horizontal MHC and costimulatory molecules (for example, B7-1 and B7-2).BMDC can inducing T cell in vitro antigen-specific Property differentiation, and internal and external initial t cell response can be initiated.In the environment of production of vaccine, " DC " of activation is The DC of the Toll-like receptor activator exposure of warp-wise lipopolysaccharides " LPS " etc..The DC of activation may be loaded or may be loaded with Antigen.Also see, " ripe DC ".

" CD-1 polarize BMDC ", " DC 1s " and " 1 type polarization DC " refers to secreting such as IL-12, IL-18 and The Th1 such as IL-23 drive the ripe BMDC of cell factor, and DC 1s are fully able to promote cell-mediated be immunized.At this In preferred embodiment in, with HER2MHC II class binding peptide pulse DC Is.

" ERs (" ER ") is positive " or " ER^pos" cancer be detect estrogen be expressed as the positive cancer.Conversely Ground, for this expression, feminine gender is expressed as " ER is negative " cancer detection.The analysis of ER states can be by known in the art Any method implement.

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

" HER2 binding peptides " used herein, " HER2 mhc class iis binding peptide ", " binding peptide ", " HER2 peptides ", " immunogene Property mhc class ii binding peptide ", " hla binding peptide ", " HER2 antigenic determinants ", " reaction peptide " etc. are referred to from HER2/neu albumen The sequence of matter obtains or the mhc class ii peptide based on it, and it is in about 20%-25% all human breast cancers and its equivalent It was found that a kind of targeting thing.HER2 ectodomains " ECD " refer to the domain in the extracellular HER2 including its fragment, its Either it is anchored on cell membrane or in the circulating cycle.HER2 intracellular domain " ICD " is referred in intracytoplasmic HER2/neu eggs White domain.According to preferred embodiment, HER2 antigenic determinants or other binding peptides include 6 kinds of HER2 binding peptides, described HER2 binding peptides include 3 kinds of HER2 ECD peptides and 3 kinds of HER2 ICD peptides.

Preferable HER2 ECD peptides include：

Peptide 42-56：HLDMLRHLYQGCQVV(SEQ ID NO：1)；

Peptide 98-114：RLRIVRGTQLFEDNYAL(SEQ ID NO：2)；With

Peptide 328-345：TQRCEKCSKPCARVCYGL(SEQ ID NO：3)；

Preferable HER2 ICD peptides include：

Peptide 776-790：GVGSPYVSRLLGICL(SEQ ID NO：4)；

Peptide 927-941：PAREIPDLLEKGERL(SEQ ID NO：5)；With

Peptide 1166-1180：TLERPKTLSPGKNGV(SEQ ID NO：6).

In embodiment, its donor has HLA A2.1 blood groups (HLA-A2^pos)

HER2 I class MHC peptides or antigenic determinant include：

Peptide 369-377：KIFGSLAFL(SEQ ID NO：7)；With

Peptide 689-697：RLLQETELV(SEQ ID NO：8).

“HER2^pos" be breast cancer type and a lot of other types of cancer classification or molecular isoform.At present, HER2 It is positive to be defined by FISH (FISH) experiments, by the intensity 2+ or 3+ of gene magnification and cytological stains.

“HER2^neg" defined by FISH, by lacking gene magnification, and it can include from 0 in most cases To 2+ cytological stains.

" separation ", which is meant, to be changed or removes from nature.For example, the nucleic acid that is naturally occurring in animal living or Peptide is not " separation ", but partly or completely same nucleic acid or peptide are divided from the coexisting substances in its nature Open is " separation ".The nucleic acid or protein of separation can exist in the form of substantially purifying, or can be in ex situ environment In the presence of for example, host cell.

Term " major histocompatibility complex " used herein or " MHC " are defined as specific gene cluster, it is many this The surface protein related to evolution being related in a little gene cluster coding for antigens submissions, these surface proteins be in histocompatbility most Important determinant.I class MHC, or MHCI classes mainly play a major role in the antigen submission to CD8T lymphocytes.II classes MHC or MHC II classes are mainly to CD4⁺Played a major role in the antigen submission of T lymphocytes (T- auxiliary cells).

It is used herein that " ripe DC " means that expression includes high-level II classes MHC, CD80 (B 7.1) and CD86 (B7.2) The BMDC of molecule.On the contrary, immature BMDC (" iDCs " or " IDCs ") expresses low-level II classes MHC, CD80 (B7.1) and CD86 (B7.2) molecule, but still can absorb antigen." ripe DC ", which also refers to, to be present in vivo, body Outside, in vitro, or in the DC 1 of antigen presenting cell of the host either in subject or polarization (that is, it is fully able to Promote cell-mediated immune).

CD4⁺" measurement " of Th1 responses (or Th1 responses) for each test component by analysing anti-HER2 CD4⁺Th1 is immune should Answer to define：(1) overall anti-HER2 response rates (percentage that the subject of peptide is reacted with response >=a kind represents)；(2) response group Storehouse (to be represented by the par (n) of the reaction peptide of each tested group of identification)；Cumulative acknowledgements (with from each tested (c) Group 6 kinds of II class MHC binding peptides total overall reaction spot entire quantity come represent (from IFN-γ ELISPOT analysis it is every 10⁶The spot formation cell " SFC " of cell).

" non-suspicious HER2^neg" it is defined as non-gene magnification and 0 or 1+ cytological stains." suspicious HER2^neg" be defined For non-gene magnification, but it is 2+ in cytological stains.

" response rate " or " anti-HER2 response rates " is used interchangeably herein, means at least 1 in 6 kinds of binding peptides Subject's percentage of kind response.

" response group storehouse " is defined as by the par (" n ") of the reaction peptide of each tested group of identification.

" sample " or " biological specimen " used herein mean the biomaterial from subject, including but not limited to blood Liquid, organ, tissue, allochthon, blood plasma, saliva, urine and other body fluid.Sample can be any source from subject Sample of material.

Term " subject ", " patient ", " individual " etc. are used interchangeably herein, refer to suitable for described here Any animal of method, or its no matter cell in vitro or in the original location.In certain non-limiting embodiments, patient, subject Or individual is the mankind.

Term " targeted therapy " used herein refers to the treatment of cancer using medicine or other materials, the medicine or Other materials disturb the specific target molecules for being usually directed to cancer cell growth, and almost anti-to realize without damaging to normal cell The effect of tumour.On the contrary, conventional cytotoxic chemotherapy medicine, for all cells divided.In breast cancer Treat monoclonal antibody in, particularly trastuzumab/It is used as target using HER2/neu acceptors.

" T/C " is defined as trastuzumab and chemotherapy.What this referred to receives bent appropriate list before/after mammary cancer surgery Anti- and two kinds of treatments of chemotherapy patient.

Term " T- cells " used herein or " T cell " are defined as participating in many cell-mediated immunes react Thymus gland-source cell.

The reference cells such as term " T- auxiliary cells " used herein, " helper cell ", " Th cells " represent can be by ability The lymphocyte subgroup (a kind of white blood corpuscle or white blood cell) including different cell types of field technique personal identification.Especially, T- auxiliary cells are that major function is imitated for the activation of other B and T lymphocytes of promotion and/or macrophage and the T- to play a role Answer cell.Helper cell is divided into the two maxicell hypotypes of " Th1 " or " 1 type " and " Th2 " or " 2 type " phenotype.These Th cells Secrete cytokines, protein, or peptide, the cell factor, protein, or peptide stimulate other white blood cells or with other white blood cells Interaction." Th1 cells " used herein, " CD4⁺Th1 cells ", " CD4⁺1 type T- auxiliary cells ", " CD4⁺T cell " etc. refers to Be the ripe T- cells for having expressed surface glycoprotein CD4.When with peptide antigen by CD4⁺T- auxiliary cells pass through in Ru Shu When the II classes MHC molecule of antigen-submission peptide (" APCs ") surface expression of prominent shape cell carries out submission, CD4⁺T- auxiliary cell quilts Activation.Once by MHC- antigenic compounds by CD4⁺T helper cell activates, CD4⁺T helper cell secreting high levels as interference Element-γ (" IFN-γ ") cell factor.This cell be considered as efficiently for it is some survived in host cell cause disease The bacterium of disease, and it is most important in the anticancer response of human cancer.

" Treg " " T used herein_reg" and " regulatory T cells " refer to the police of immune system, it is immune for adjusting The anti-cancer activity of system.It can increase in some cancers, and it is that immunotherapy is produced in resistance in these cancer types Jie's thing.

" therapeutically effective amount " or " effective dose " used herein are used interchangeably, and refer to compound described herein, The quantity of preparation, material or composition, when administered patient, it can effectively realize special biological effect.Structure described herein Into the compound of " therapeutically effective amount ", preparation, the quantity of material or composition can according to compound, preparation, material or composition, Morbid state and its seriousness, the age of patient under consideration etc. and change.Therapeutically effective amount is by a people in the art Member refers to his/her knowledge and the displosure routinely to determine.

Term " treatment " used herein refers to treatment or prevention measure described herein." treatment " method is used to needing The mode for wanting the subject of the treatment in the present embodiment, composition or method to take measures, for example, tormented by disease or imbalance Subject, or the subject of this disease or imbalance may finally be obtained, in order to prevent, treating, delaying, reduce seriousness or improvement The symptom of one kind of multiple imbalance or recurrence imbalances, or the survival of extension subject make it exceed expection when not applying this treatment Survival.

Term " vaccine " used herein is defined as being used to animal, preferably mammal, more preferably human administration Cause the material of immune response afterwards.Once introduce subject's body in, vaccine can cause immune response, the immune response include but It is not limited to, antibody producing, cell factor and/or other cell responses.

Scope：Through the disclosure, the different aspect of embodiment can be represented by a range format.It should be appreciated that Be range format description just for the sake of convenient and succinct, should not be construed as the mechanical limit to scope of embodiments It is fixed.Therefore, the description of scope is considered as clearly disclosing all possible subrange and the institute in the scope It is possible to individual numerical value.For example, the description of the scope from 1 to 6 is considered as having specifically disclosed for example from 1 to 3, from 1 To 4, from 1 to 5, from 2 to 4, from 2 to 6, the subrange of grade from 3 to 6, and the individual numerical value in the scope, such as 1,2, 2.7,3,4,5,5.3 and 6.Width regardless of the scope, this explanation are all suitable for.

Now with detailed reference to different embodiments, the example in these embodiments is also with accompanying drawing, photo, and/or illustration To illustrate.

Specifically describe

The lifetime risk of mammary gland carcinogenesis is nearly 1/8th.Erb-B2 oncogene (HEK-2/neu) is in phase When the molecular drive that the mammary gland of quantity, ovary, stomach oesophagus, lung, pancreas, prostate and other entity tumors are overexpressed.HER2 mistakes Express (" HER2^pos"), the carcinogenic driving of molecule of some tumor types including about 20%-25% breast cancer, its with more More clinical affecting conditions, to chemotherapeutic resistance, the ratio of height recurrence and transfer, and poor overall prognosis phase Close.Breast cancer in the early stage, HER2 overexpression are the invasions with enhancing, tumor cell migration and angiogenic factors Expression is related, and it shows that HER2 is promoting tumour to occur to play key effect in environment.In DCIS (" DCIS ") In retrospective analysis, the DCIS lesions possibility related to infiltrative breast carcinoma for being overexpressed HER2 is that no HER2 is overexpressed 6 times of DCIS lesions.

Although molecular targeted therapy is used as target using HER2, i.e. trastuzumab, is generated in this breast cancer type Huge positive clinical effect, but resistance, Yi Jixiang are produced to existing HER2 treatment almost all in morbid state late The women's diseases recurrence for receiving targeted therapy when large scale all demonstrates the extra strategy using HER2 as target of needs.Although pin Carry out activating immune system to HER2 to alleviate metastases and prevent that the vaccine prospect of recurrence from being encouraging, but without complete It is complete to realize.Therefore, still need extra test and treat to diagnose and treat HER2 breast cancer.Embodiment described here solves These problems.

However, the anti-HER2 CD4 of whole body in the tumor of breast of HER2- drivings occurs⁺The effect of Th1 responses remains unchanged not It is clear.Embodiment described here is based in HER2^posAnti- HER2 CD4 in tumorigenic continuous process in-breast cancer⁺Th1 The identification that response is gradually lost, it is looked as HER2- specificity and independent of regulation T- cells (T_reg).Especially, Anti- HER2 CD4⁺Th1 responses continue to progress from negatively correlated with HER2 expression and disease.In addition, in HER2^pos- wellability mammary gland In cancer, the anti-HER2 Th1 responses of downward are had any different after the inoculation type of HER2- pulses 1 polarization BMDC (" DC1 ") vaccine Ground recovers, but with the HER2- targeted therapies of the trastuzumab and chemotherapy (" T/C ") that will be described in more detail herein it Do not recover afterwards or by other such as surgical resection arts or the standard treatments of radiation, the response of downward.It is extensive Multiple anti-HER2 Th1 responses seem to continue within least six moon or longer time.

Preferred embodiment described here provides the anti-cancer CD4 that circulation is produced and detected in mammalian subject⁺ The material and method of Th1 responses.Provide the anticancer CD4 for producing circulation⁺The blood of Th1 responses (that is, IFN-γ-secretion) is surveyed Test/test, and the production of caused IFN-γ is detected and measure.In another preferred embodiment, comprising CD4⁺Th1 is thin Subject's blood sample of born of the same parents and antigen-presenting cell or its precursor enters horizontal pulse with II class MHC immunogenic peptides, the II Class MHC immunogenic peptides are based on tormenting subject's and the cancer types of immune response can be induced in the subject.It is excellent The antigen presenting cell of choosing or its precursor are ripe or immature dendritic cells or its monocyte precursor.Especially excellent Select in embodiment, cancer, which is preferably, expresses HER2, and mammalian subject is preferably the mankind, and preferred cancer is HER2^posBreast cancer, human experimenter are women.

Here identified anti-HER2 CD4⁺The decay of Th1 responses realizes situations below, in blood test The caused immune response detected be only used as cancer diagnosis/response fallout predictor or by its with order to recover patient's immune response Specific vaccine is used in series.Diagnostic and therapeutic method with relying on tumour cell identification is completely contradicted, described herein preferable The emphasis of cancer diagnosis and treatment is transferred to the use of patient's immunity and blood test to determine and/or pre- chaining pin by embodiment To the immune response of cancer, including the patient with risk of recurrence.

Preferred embodiment is provided：By separating unexpanded PMBC (" PBMCs ") from subject, and By the PBMCs with including the group that immune response, HER2- sources II class MHC hla binding peptides can be produced in subject Compound enters horizontal pulse, and anti-HER2 CD4 are circulated to be produced in mammalian subject⁺The responses of Th 1.Do not expect by any specific Theoretical constraint, when binding peptide is by submission to the CD4 being present in PBMC samples⁺During Th1 cells, they activate CD4⁺Th1 cells And the CD4 of this activation⁺Th1 Cells Interferon Productions-γ (" IFN-γ ").From the precursor being included in subject's PBMC samples The DC 1s (1 type polarization BMDC) (" APCs ") obtained in multipotency monocyte are antigen presenting cell (" APCs "), its Once being changing to the antigen presenting cell of Antigen exposed to binding peptide, it is by II classes MHC hla binding peptides submission to sample Subject CD4 in this⁺Th1 cells, so as to activate CD4⁺Th1 cells are so as to producing/secretion of gamma-IFN.And caused IFN- γ is used subsequently to analysis measure.

In alternative preferred embodiment, by by the advance unprovoked purifying in subject's blood sample CD4⁺The BMDC of T- cells and autologous prematurity or maturation (" iDCs " or " mature DCs ", jointly, " DCs ") training altogether Support, produced in mammalian subject and circulate anti-HER2 CD4⁺The dendron shape of Th1 responses, the autologous prematurity or maturation Cell be by including can be produced in subject immune response, HER2- sources II class MHC hla binding peptides combination Thing pulse.It is undesirable to be bound by any theory, when binding peptide is by submission to the CD4 being present in T cell sample⁺Th1 is thin During born of the same parents, they activate CD4⁺Th1 cells and the CD4 of this activation⁺Th1 cells produce/secrete interferon-γ.Immature dendron Shape cell maturation is to DC1 ' s, and it is by the CD4 of II classes MHC binding peptides submission to the subject being present in sample⁺Th1 cells from And activate CD4⁺Th1 cells produce IFN-γ, and it is used subsequently to analysis measure.

In two alternative preferred embodiments that anti-HER2 immune responses are produced in subject, anti-HER2 CD4⁺Th1 The IFN-γ of cell secretion is detected and determined by IFN-γ enzyme-linked immunospot assay (" ELISPOT "), art technology Personnel, which will be appreciated that, to be detected using other method.It is, for example, possible to use flow cytometry, enzyme linked immunosorbent assay (ELISA) (" ELISA ") and immunofluorescence (" IF ") monitor immune response.Alternatively, detected with direct IFN-γ, such as whole is shown CD4⁺The ELISPOT of cell blots, which is formed, to be compareed or in addition, in the example of patient's immunologic surveillance, determines IFN-γ pair IL-10 ratio (showing what is completed in reference example and Fig. 8 E) is favourable.These detections are particularly with the trouble with risk Person is favourable.Further, the application of immunofluorescence is provided by using the ELISPOT readers for carrying out reading result with fluorescence Determine and show the other method of immune response.In such example, it as a result can be arranged to show 2,3 kinds or more thin The immune molecule of intracellular cytokine/other secretions, each result is shown with different colors in identical clinical samples.

Those skilled in the art are not difficult to find out, except BMDC and monocyte, other can be used suitable APC, for example, macrophage and B cell.

By using IFN-γ ELISPOT experiments, to detect IFG- γ secretion, (positive peptide should in a preferred embodiment Answer：Minimum threshold 20SFC/2x 10⁵It is higher than unprovoked control group with 2x).As a result three kinds of Th1 responses are preferably expressed as Measurement：(a) overall anti-HER2 response rates (being represented with subject's percentage that right >=a kind of reaction peptide produces response)；(b) should Answer a group storehouse (to be represented by the par (n) of the reaction peptide of each tested group of identification)；Cumulative acknowledgements (with from each (c) The entire quantity (every 10 from IFN-γ ELISPOT analyses of the reaction spot of tested group of 6 kinds of II class MHC binding peptides⁶Cell Spot formation cell " SFC ") represent.

For HER2^posThe preferred embodiment of cancer is thin with the composition pulsed dendritic shape including 6 kinds of II class MHC binding peptides Born of the same parents, immature or I types polarization DC1s, the II classes MHC binding peptides from can produce in patients immune response HER2 it Middle acquisition is obtained based on it.HER2 II class MHC binding peptides or antigenic determinant include：

Peptide 42-56：HLDMLRHLYQGCQVV(SEQ ID NO：1)；

Peptide 98-114：RLRIVRGTQLFEDNYAL(SEQ ID NO：2)；

Peptide 328-345：TQRCEKCSKPCARVCYGL(SEQ ID NO：3)；

Peptide 776-790：GVGSPYVSRLLGICL(SEQ ID NO：4)；

Peptide 927-941：PAREIPDLLEKGERL(SEQ ID NO：5)；With

Peptide 1166-1180：TLERPKTLSPGKNGV(SEQ ID NO：6).

In the embodiment that donor has A2.1 blood groups, the class MHC peptides of HER2 1 or antigenic determinant include：

Peptide 369-377：KIFGSLAFL (SEQ ID NO：7)；With

Peptide 689-697：RLLQETELV(SEQ ID NO：8).

As described further below, HER2 binding peptides/antigenic determinant in preferred embodiment is not limited to as above carry The six kinds of peptides arrived, it will also be carried out including functionally equivalent or herein discussed in detail by SEQ ID NOS：1-6 differentiates Binding peptide substitute, also other I class peptides can be used for subject, the subject have A2.1 and A3.1 blood groups with And other include the blood group (for example, A5, A6) of the I class peptides combined with any phenotype.

Except breast cancer, also many other HER2^posSolid carcinoma, for example, the cancer of the brain, carcinoma of urinary bladder, the cancer of the esophagus, lung cancer, pancreas Gland cancer, liver cancer, prostate cancer, oophoroma, colorectal cancer, and stomach cancer, and other cancers, the thing in embodiment described here Matter and method can be used for diagnosing and treat above-mentioned cancer.Therefore, six kinds of foregoing description anti-HER2 binding peptides can be according to this In embodiment use so as to producing the immune response that can be detected, and six kinds of anti-HER2 binding peptides of foregoing description are to these Diagnosis with other expression HER2 cancers is useful.

Can be by using the same anti-HER2 binding peptides of foregoing description or using any HER2 with immunogenicity The component of composition, such as DNA, RNA, peptide, or protein or such as ICD and ECD domains carrys out vaccine development, the vaccine with The tumour for expressing HER2 is targeting thing.For example, subject can be with transfer needle to can be used to detect the full HER2 of patient's immune response Albumen and six kinds of above-mentioned vaccines with reference to binding peptide.Can also be the similar vaccine of other kinds of cancer exploitation, such as including Other members of HER1, HER3 and c-MET HER2 families.

Although existing preferred embodiment is intended to the HER2 for the treatment of and Diagnosis of Female^posBreast cancer, those skilled in the art should This is easily it is understood that the present embodiment is not limited to women.Current preferred embodiment includes male, for example, expression HER2 Prostate cancer, in addition to other mammalian subjects.

Composition

Preferred embodiment includes the application of the peptide based on HER2 albumen or the separation obtained from HER2 albumen.Preferred embodiment Binding peptide represent the antigenic determinant of corresponding HER2 albumen.Although presently preferred embodiment is with six kinds of HER2II classes MHC Binding peptide/antigenic determinant is characterized, and other possible HER2II classes MHC peptides can be used in current embodiment, because whole As long as any component of individual HER2 molecules its in patient's body have the source that sufficient immunocompetence can serve as other binding peptides.

In a preferred embodiment, HER2 binding peptides include

Six kinds of HER2 II class MHC binding peptides, have sequence：

Peptide 42-56：HLDMLRHLYQGCQVV(SEQ ID NO：1)；

Peptide 98-114：RLRIVRGTQLFEDNYAL(SEQ ID NO：2)；

Peptide 328-345：TQRCEKCSKPCARVCYGL(SEQ ID NO：3)；

Peptide 776-790：GVGSPYVSRLLGICL(SEQ ID NO：4)；

Peptide 927-941：PAREIPDLLEKGERL(SEQ ID NO：5)；With

Peptide 1166-1180：TLERPKTLSPGKNGV(SEQ ID NO：6).

Pass through SEQ ID NO：The HER2 antigenic determinants of 1 identification represent the position 42-56 of HER2 protein.Pass through SEQ ID NO：The HER2 antigenic determinants of 2 identifications represent the position 98-114 of HER2 protein.Pass through SEQ ID NO：3 identifications HER2 antigenic determinants represent the position 328-345 of HER2 protein.Pass through SEQ ID NO：The HER2 antigenic determinants of 4 identifications Represent the position 776-790 of HER2 protein.Pass through SEQ ID NO：The HER2 antigenic determinants of 5 identifications represent HER2 protein Position 927-941.Pass through SEQ ID NO：The HER2 antigenic determinants of 6 identifications represent the position 1166- of HER2 protein 1180。

Further, technical staff can further perceive embodiment described here be not limited to use with it is described herein The related all six kinds of binding peptides of preferred embodiment.Any quantity of described binding peptide can be used for blood samples of patients survey Test, with relatively low scope about 2 or 3, it is necessary to which that alert is the CD4 of patient⁺T- cells must have enough immunocompetences to cause IFN-γ produces.Therefore, HER- sources II classes binding peptide with less than/be different from it is described herein related to preferred embodiment Six kinds of II class binding peptides the example that is used of form in, according to caused immune response in subject, the quantity of binding peptide It can be substantially below or higher than six kinds.

As stated here, the HER2 binding peptides of preferred embodiment also include with by SEQ ID NOs：1-6 identification peptide be The peptide of functional equivalent.Such functional equivalent may have the sequence changed, in the sequence of change, in corresponding HER2 One or more of peptide sequence amino acid is substituted, or deletes or add one or more ammonia from corresponding reference sequences Base acid.For example, 1-3 amino acid can be added to aminoterminal, c-terminus or both ends.In some instances, HER2 peptides can To be glycosylated.

HER2 binding peptides either can be cyclized or linearize according to any peptide of the present embodiment.When cyclisation, antigen is determined Determining cluster can be cyclized in any suitable manner.For example, disulfide bond can be in selected cysteine (" Cys ") shape between Into to provide desired conformation.It is believed that the formation of cyclisation antigenic determinant can provide the conformation for improving immune response.

In other examples, HER2 binding peptides can be reverse-reversion isomers of HER2 binding peptides.Inversely-reversion Change be included in all amido links inverted in peptide backbone.Such reversion can replace L- amino by using D- amino acid Acid is to invert the chirality in the direction of sequence and anti-phase each amino acid residue to realize.Such reverse-reversion isomeric forms It may keep planarizing and at least some peptide bonds having conformation limitation.

Non-conservative amino acid substitution and/or conservative amino acid substitution can also be carried out.When substituted amino acid and ginseng It is conserved amino acid substitution when examining the structure or similar chemical property of corresponding amino acid in sequence.As an example, conservative amino Acid substitution is related to an aliphatic or hydrophobic amino acid, for example, alanine, valine, leucine and isoleucine, by with another Individual substitution；One amino acid containing hydroxyl, for example, serine and threonine, are substituted with another；One acidic residues, For example, glutamic acid or aspartic acid, are substituted with another；One residue for including acid amides, for example, asparagine and glutamy Amine, replaced with another kind；One aromatic residue, such as phenylalanine and tyrosine, replaced with another；One alkalescence Residue, such as lysine, arginine and histidine, replaced with another；One p1 amino acid, for example, alanine, silk ammonia Acid, threonine, methionine and glycine, replaced with another.

In some instances, delete and add be positioned at preferred embodiment peptide binding sequence aminoterminal, c-terminus or Person both ends.For example, the amino acid sequence that has of HER2 binding peptide equivalents compared with corresponding HER2 peptide binding sequences at least 70% is identical, and at least 80% is identical, and at least 85% is identical, and at least 90% is identical, 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% be identical 's.At least 90% identical sequence has the change of not more than 1 in every 10 amino acid of reference sequences, i.e. deletes, increases Any combinations for adding or substituting.By using it is known or in this area program leaved for development by the amino acid sequence and ginseng of change Examine sequence to be contrasted, to determine percent homology.

For the functional equivalent longer than corresponding HER2 peptide binding sequences, functional equivalent can be with least 90% Sequence is identical with the sequence for being located at HER2 peptide sequences side in HER2 peptide sequences and wild type HER2 albumen.

The functional equivalent of HER2 binding peptides can by the sequences of the modified peptides and afterwards ability of polypeptide caused by detection It is identified, the ability refer to stimulate the monocyte of subject, BMDC or other binding peptide/antigenic determinant is passed In to CD4⁺The ability of the antigen presenting cell of Th1 cells.

According to other embodiment, there is provided chimeric peptide and composition including one or more chimeric peptides.According to different Embodiment, chimeric peptide include HER2 peptides, peptide in addition and the joint that the HER2 peptides are connected to other peptide, should be further Understand that any suitable joint is likely to be used.For example, according to used peptide, HER2 binding peptides may be connected to other The aminoterminal or c-terminus of binding peptide.Design feature of the positioning and selection of other peptide depending on HER2 peptides, if be α spirals Or β-bend or chain.

In another embodiment, joint can be from about 2 to about 15 amino acid, about 2 to about 10 aminoterminals or Person is from about 2 to the peptide of about 6 aminoterminal length.Chimeric peptide can be linear or cyclisation.In addition, HER2 peptides, in addition Peptide, and/or joint can be the forms of reverse-reversion.Therefore, HER2 peptides can be the form of reverse-reversion together.It is optional Ground, HER2 peptides and other peptide can be the forms of reverse-reversion.In another example, HER2 peptides, other antigenic determinants Can be the form of reverse-reversion with joint.

Peptide including chimeric peptide can be prepared using known technology.For example, peptide can be by using recombinant DNA technology Or the preparation that the method for chemical synthesis is synthesized.The peptide of the present embodiment can be with separately synthesized or be synthesized into by two or more The longer polypeptide of peptide composition.The peptide of presently preferred embodiment is preferably separated, i.e. substantially not by the place of other Lock-ins The constraint of host cell proteins and its fragment.

The peptide and chimeric peptide of the present embodiment can be synthesized with commercially available Peptide synthesizer.For example, it can make With description in Kaumaya, P.T.P., etc. " DeNovo " is in peptide, design, in synthesis and bioactivity, as exempting from for potential vaccine Epidemic focus peptide and antigen determinant engineering science, the content in pp 133-164 (1994).For example, HER2 binding peptides can with it is another Outer binding peptide is collinearly synthesized to form chimeric peptide.Can be with Fmoc/t-But chemical methods come synthetic peptide.Peptide and chimeric peptide can be with It is cyclized in any suitable manner.For example, can by using the discrepant cysteine residues being protected, iodine oxidation, Water is added to promote the associated formation of the removal of Acm groups and disulfide bond, and/or silyl chloride-sulfoxide to obtain Disulfide bond.

Peptide and chimeric peptide can also be by using cell free translation system and the DNA of encoding antigenic determinants or binding peptide Obtained in conformation and RNA molecule produces.Alternatively, antigenic determinant or chimeric peptide can be by using including codings point Other antigenic determinant or the expression vector transfection host cell of chimeric peptide, then expression of the inducing polypeptide in host cell. For recombinant production, including encoded peptide antigenic determinant, the recombinant precursor of one or more sequences of chimeric peptide or its variant Host cell is introduced by conventional method, these conventional methods such as calcium phosphate method transfects, the transfection of DEAE-dextran mediations, Microinjection, cationic-liposome-mediated transfection, electroporation, transduction, scrape and carry (scrape lading), trajectory introduces (ballistic introduction) or infection.

As long as modification does not destroy the bioactivity of binding peptide, the binding peptide of the present embodiment can include modification, for example, glycosyl Change, oxide side chain or phosphorylation.Other modifications include introducing D- amino acid or other amino acid simulants.

The binding peptide of the present embodiment can be prepared as the combination for including two or more peptides.Peptide can be mixture or Make to be conjugated each other by using standard method.For example, peptide can be expressed with single peptide sequence.Peptide in combination can be phase It is same or different.

The present embodiment should also be interpreted as including " mutant " of the peptide (or encoding its DNA) of embodiment, " derivative Thing ", and " variant ", mutant, derivative and variant are the peptides of one or more amino acid changes (or when referring in embodiment It is when encoding its nucleotide sequence, is one or more base-pairs changed), the peptide (or DNA) so obtained with arranging here The sequence of act is different, but has identical bioactivity with peptide disclosed herein.

Some embodiments also provide the polynucleotides for encoding at least one peptide, and the peptide is selected from any one or more SEQ ID NOS：The peptide of 1-6 sequences.Nucleotide sequence includes being transcribed into RNA DNA sequence dna and is translated into the RNA of peptide Sequence.According to other embodiment, polynucleotides are to deduce what is come from the amino acid sequence of the peptide of preferred embodiment.Such as this area Known, when retaining the bioactivity of translated polypeptide, due to redundant codon, some optional polynucleotides are possible.

Further, preferred embodiment includes the nucleic acid of separation, and the nucleic acid coding has with binding peptide disclosed herein The peptide of substantial homologous.Preferably, the nucleotide sequence of the seperated nuclear acid of peptide of the present invention is encoded with encoding in preferred embodiment The nucleotide sequence of the seperated nuclear acid of binding peptide is " substantial homologous ", i.e. about 60% is homologous, and preferably about 70% is homologous, It is even more preferably still about 80% homologous, more preferably about 90% is homologous, is even more preferably about 95% homologous, even more Preferably about 99% is homologous.

The scope of preferred embodiment, which should clearly be understood, includes homologue, analog, variant, derivative and salt, Including shorter and longer peptide and polynucleotides, and the peptide and polynucleotides class substituted with one or more amino acid or nucleic acid Like thing, and amino acid known in the art or nucleic acid derivative, alpha-non-natural amino acid or nucleic acid and synthesizing amino acid or core Acid, it provides it is that these modify the immunologic competence that must protect original binding peptide.According to principle described here, especially, Any active fragment and extension, conjugate and mixture including active binding peptide.

Homologous any of nucleic acid that preferred embodiment should be interpreted as including and be described herein and refer to and The nucleic acid of all separation.Assuming that these homologous DNA have the bioactivity of binding peptide disclosed herein.

It will be understood to those of skill in the art that the nucleic acid of preferred embodiment include coding preferred embodiment peptide RNA or DNA sequence dna, and the form of its any modification, including cause more stable when nucleotides sequence is listed in acellular or related to cell DNA or RNA chemical modification.The chemical modification of nucleotides can also be used to improve nucleotide sequence by the efficiency of cellular uptake Or its efficiency for expressing in the cell.The combination of any and all nucleotide sequence modification is pre- in a preferred embodiment Measure.

Further, using recombinant DNA method known in the art, such as in Sambrook and Russell, supra and Ausubel etc., described by supra, any number of step can be used for the mutant of the peptide of preferred embodiment, derivative or The generation of variant form.The step of DNA sequence dna by changing coded polypeptide introduces amino acid change in peptide or polypeptide is at this Field is known, also at these, and other, paper is described.

The nucleic acid of binding peptide is encoded in preferred embodiment can be incorporated into suitable carrier, for example, retrovirus vector Body.These carriers are well known in the art.Nucleic acid effectively can generally be imported into expectation including the carriers of these nucleic acid Cell, this cell is preferably selected from patient.

Cryopreservation

After subject obtains PMBC and separated, carry out any blood test described here/ It is stored refrigerated using being carried out for method known to those skilled in the art before experiment.

Use diagnosis/prognosis/Treatment monitoring instrument

As described herein, it has been found that, circulate HER2- reactivity IFN-γs^pos CD4⁺The loss of Th1 cells is opened earliest Start from DCIS breast cancer and in early stage invasive stages I HER2^posSubstantially decline in tumour.More specifically, from healthy donors to HER2^posDCIS (DCIS) arrives HER2^posIn the continuous process that IBC (infiltrative type breast cancer) occurs across tumor of breast Anti- HER² CD4⁺Th1 responses progressively decay.The reason for immune response is lost, be not because, i.e. be not due to and cancer Relevant immunosupress, it does not seem relevant with from the increase for moving to infiltration lesion, and it is independently of regulatory T cells.

Based on this discovery-HER2 CD4⁺The embodiment of the loss of Th1 immune responses is provided as individual healthy on surface Breast cancer and the method for other cancers are screened, these breast cancer and other cancers can by mammography or other screening techniques It will not can be detected, this method includes carrying out tachysynthesis detection/experiment to detect anti-HER2 CD4⁺Th1 response rates, it is excellent Embodiment is selected to carry out blood testing.These individual test results are lower than those healthy individuals, and this causes the survey more determined The implementation tested and faster treat option can be realized.For example, blood test here can be advantageously used for differentiating and be in risk In, vaccine inoculation may be believed to reduce the patients of its HER-2 expression mammary cancer risks.For example, in risk Patient can be following completion lactation, pregnancy and other may reduce response life stress event patient.

Due to the factor such as genetic predisposition or life style, such screening technique is for there is excessive risk to develop into mammary gland The patient of cancer is beneficial, and from the angle of diagnosis, immune biomarker can obtain development with for screening such height-wind Fluctuation immune anti-HER2 Th1 in the patient of danger.When IHC dyeing or fish analysis breast biopsy sample merely provide it is swollen When the snapshot for the separation that knurl is evolved, immunoassay (such as with such potential biomarker) can provide tumour from Right history and the information of immune response.It can be used for the patient that prediction is diagnosed as any breast cancer, no matter whether it has HER-2 The new mammary gland event of expression or the risk of recurrence.

In another embodiment, diagnosis or monitoring test based on anti-HER2 Th1 responses loss can be used for pre- measuring tool There is HER2^posWhether the patient of breast cancer nonimmune treats the response that has had for the standard of the additional trastuzumab of such as chemotherapy.

According to the further embodiment that will be described in detail herein, with targeting (for example, trastuzumab) or often The breast cancer treatment (that is, chemotherapy) of rule is compared, and is connect by the II classes peptide (DC1 is immunized) and autologous DC1 vaccines in HER2- sources Kind, CD4⁺Th1 responses can preferentially recover.So, in HER2^posIn-IBC patients, after HER2- pulse DC vaccine inoculations, and It is not the CD4 after trastuzumab/cytotoxic chemotherapy (" T/C ") treatment⁺Th1 responses are effectively recovered.Cause This, in preferred embodiment before vaccine inoculation or the test of vaccine inoculation laggard promoting circulation of blood liquid with determine the recovery of Th1 immune responses or The degree of non-recovery.So as to which patient can be test by blood here and easily reevaluate theirs after breast cancer treatment CD4⁺Th1 responses are to determine any CD4 previously found⁺Whether the immune losses of Th1 have been recovered by vaccine inoculation.

In another embodiment, dependent on anti-HER2 CD4⁺The blood test of Th1 responses decay is determined for DC1 Vaccine inoculation whether sufficiently recovered or strengthened anti-HER2 be immunized to can be to cancer further invasion and attack provide guarantor The level of shield.The vaccine inoculations of Post-DC 1, the blood test in preferred embodiment can carry out repeatedly, preferably curing according to patient The timetable that teacher recommends, so as to follow the trail of the CD4 of patient⁺Th1 immune states.After vaccine inoculation, due to vaccine-induced pair The continuation of HER2 prodrug sensitizations and the possibility protected for a long time, these additional tests can be carried out The a lot of moon, for example, at least to 60 months or more after vaccine inoculation.

The infiltrative breast carcinoma patient that blood test used herein can be used for showing HER2 expression is in chemotherapy/song The degree of HER2- response rates after appropriate monoclonal antibody treatment.There is illustrated correlation, the correlation is that such patient will be to controlling Reaction how, and the therefore correlation between the final result prediction quality of such patient are made in treatment.For example, that lowers is anti- The risk for the enhancing subsequently recurred is predicted in HER2th1 responses in the patient that adjuvant-T/C is treated.

The method that other embodiment provides immunization strategy, i.e. DC vaccine inoculations, to improve or recover in HER2^posInfiltration Anti- HER2 CD4 in property patient with breast cancer⁺Th1 loses.The ability of " immune restoration " can be utilized to combine current bent appropriate list Treatment-resistant is used to treat.Also provide and combine vaccine inoculation with the standard care including the additional trastuzumab of chemotherapy General principle.

Another embodiment shows to predict the immune-related of new mammary gland event risk.Controlled with chemotherapy/trastuzumab The HER2 for the treatment of^posIn-IBC patients, responding control is shown, specifically, the anti-HER2 CD4 of downward⁺Th1 responses and new breast The increased risk of gland cancer event institute is relevant.Therefore the response so lowered can be used for whether prediction result such as patient is possibly subjected to New mammary gland event, and if it happens, whether patient needs additional treatment.So as to based on this exploitation biomarker.

Further, observe here, express the HER2 of IFN-γ and TFN- α acceptors^posBreast cancer cell, once it is exposed to Th1 sources-cell factor (including IFN-γ and TNF-α, the typical cell factor secreted by Th1 cells) will undergo cell Apoptosis, show in the physiology course that such as breast is degenerated, anti-HER2 Th1 cells are to controlling or eliminating HER2 expression cells Work.All HER2 described here by detection^posBreast cancer cell line is found that IFN-γ and TFN- α The expression of acceptor, it is seen that these anti-HER2 CD4⁺The generation of Th1 cells makes the breast cancer cell line Apoptosis of HER2 expression The solvable factor.Show in the physiology course that such as breast is degenerated, anti-HER2 Th1 may be to controlling or eliminating HER2 expression Cell have effect, CD4 can also be explained⁺Th1 cells are how to accomplish that II classes can not be identified^negI classes^posTumour cell, but The destruction of tumour cell can be adjusted.

It is such as specifically described herein, it further embodiment provides prediction HER^2posAuxiliary new to standard is controlled in breast cancer Pathology response rate is immune-related caused by treatment.Design experiment with study expression HER2 infiltrative breast carcinoma patient in chemistry The degree of HER2 response rates after therapy/trastuzumab treatment will to what extent reply the correlation for the treatment of with them.This The experiment of sample discloses prediction and produces the immune-related of pathology response rate to standard lower rectal cancer.Completely should with no pathology The patient answered compares, and finds lower rectal cancer complete response and significantly higher anti-HER2 CD4⁺There is correlation between Th1 responses.

The HER2 of T/C- treatments^posThe amplitude that the HER2- specificity Ts h1 of-IBC patients suppresses is follow-up with new mammary gland event The increased risk of recurrence is relevant, on the contrary, the anti-HER2 CD4 of foregoing description⁺Holding immune Th1 to new assistant chemical with treating The complete pathology response of method is relevant.Altogether, the anti-HER2 Th1 immune response activities of these as shown by data are used as giving birth to Substance markers with help to identify it is vulnerable, there are clinical or pathology risk of failure patient subgroups.

Although the present embodiment described herein may include the special reference of the tumor of breast to expressing HER2, this area skill Art personnel should be appreciated that other kinds of expression HER2 tumour, only for example, oophoroma, stomach oesophagus cancer, lung cancer, pancreas Gland cancer, liver cancer, prostate cancer and other solid tumors, may have benefited from the teaching of the present embodiment.Similarly, people in the art Member can experience the breast cancer that teachings herein extends to non-expression HER2, including three times negative tumours and the ER- positives Tumour and other tumours.

In addition, can be according to preferred embodiment also from other HER families targeting thing of acceptor TYR kinase families Use.HER families are related to by four kinds in many cancers, related signaling molecules form：HER1, HER2, HER3 and HER4.To the greatest extent Pipe, it is known that HER-2 overexpression is found in about 20% to 25% breast cancer, and other HER family members are also found Related in early stage and infiltrative breast carcinoma and other cancers.For example, breast carcinomas of the HER1 in minority, is typically Three times feminine gender.C-Met is that have involved growth factor receptors in the recurrence of activation HER3 many cancers.HER3 is being tied It is overexpressed in intestines, prostate, mammary gland and melanoma.HER3 is in substantial amounts of DCIS lesions and breast carcinoma.Receiving DC1 In some patients of HER2 vaccine inoculations, detected in the DCIS that HER3 can remain in surgical operation.Therefore, other HER Family targets thing, such as causes other of breast cancer and other solid carcinomas HER families to target thing, such as：HER3, HER1 and c- It Met, may be useful as targetting thing, and be also developed for the peptide vaccine of these other targeting things, as retouching here The HER2 stated exploitation.Therefore, for identifying driving/quilt which molecule is expressed in the tumor of breast of patient including carcinogenic The breast cancer driving (panel) for the carcinogenic driving (oncodrivers/proposed oncodrivers) proposed, such as HER2, HER3, HER1 and C-Met, complementary therapy can be developed into and for vaccine target molecule.Therefore, it is contemplated that except described herein For HER2 DC1 vaccines, similar vaccine can also develop for non-express HER2 breast cancer type.

Example

Preferred embodiment is described in detail with further reference to following test examples.These examples are only with the purpose of explanation There is provided, it is outer unless otherwise prescribed, it is therefore intended that to be limited.It is therefore preferable that embodiment should be in no way to be construed as being limited to it is following Example, more specifically, it should be interpreted as including any and all change, its result as teaching provided herein Become apparent.

There is no further description, those skilled in the art are believed to, illustration using previous description and afterwards, system Make and the method using the present embodiment and practice calls.So as to which following working example particularly points out preferred embodiment, not Any mode of disclosed remainder can be construed as limited to.

Following reference example includes method, as a result with discuss part.

Reference example

Method

Patient selects and research and design

By the approval of University of Pennsylvania's institutional review board, 143 patients are recruited to participation at present by successive The research of description is simultaneously agreed to.At healthy donors (" HD ") (n=21), there is benign breast disease (" BD ") (n=10), HER2^neg- DCIS (n=11), HER2^neg(0/1+) IBC (n=11), HER2^pos- DCIS (n=31), and HER2^neg- IBC (n= 22) anti-HER2 CD4 are checked in patient⁺Th1 (" Th1 ") response.It is incorporated into HER2^pos- DCIS new auxiliary DC1 immunization methods And in operation find there is stage I HER2^posThe Th1 responses of-IBC (n=11) patient, before and after immune Analyzed (immediately and after >=6 months).By the HER2 of untreated before^posTh1 responses and the T/C of-IBC patients is controlled Treatment stage I-III HER2^pos- IBC patients (n=37) are contrasted.Fig. 1 shows the patient for meeting study condition and donor Group.Monitor T/C- treatments HER2^posThe development of-IBC patient's mammary gland events, mammary gland event are defined as any local or distal end Recurrence.Table 1 below show this study population demographics and to tumour (year of individual patient subgroup the characteristics of related Age, race, AJCC pathologic stages, hormone receptor situation, chemotherapy time and the time (applicatory) for completing trastuzumab) (“IBC”：Infiltrative breast carcinoma；“DCIS”：DCIS；“T/C”：Trastuzumab/chemotherapy).After T/C treatments, Observe HER2^posThe development of the follow-up mammary gland event (" BEs ") of-IBC patients, mammary gland event is defined as any local or remote End recurrence.The Th1 immune responses of all subjects generate and by perspective analyses.

Vaccine test designs and immunization program

Two kinds are directed to HER2^pos1 type of HER2- pulses of-DCIS patients-polarization dendritic cell vaccine inoculation, " DC1 vaccines The lower rectal cancer of inoculation " is developed.Dendritic cell vaccine is prepared according to preceding method.Koski, G.K. etc. are referred to, J.Immonother.35(1)：54 (2012) (" Koski etc. ")；Sharma, A. etc., cancer 118 (17)：4354(2012) (" Sharma etc. ")；Fracol, M. etc., Ann.Surg.Oncol.20 (10)：3233(2013)；Lee, M.K.4th etc., Expert Rev.8(11)：e74698(2013)；Czemiecki, B.J. etc., Cancer Res.67 (4)：1842(2007)； Czerniecki, B.J. etc., cancer research 67 (14)：6531(2007)；With US publication application US 2013/0183343 A1。

Fig. 2 shows the dendritic cell vaccine vaccination strategies in this research.As there is shown with as, passing through series connection Leukopheresis and counterflow centrifugal elution obtain monocytic dendritic shape cell precursors (CD14 from subject⁺Peripheral blood mononuclear is thin Born of the same parents).BMDC is thin with grain in macrophage serum free media (Cellgro/Mediatech, Manassas, VA) Born of the same parents-macrophage colony stimulatory factor (" GM-CSF ") (250IU/mL；Berlex, Wayne, NJ) and IL-4 (1000 μ/mL； R＆D Systems, Minneapolis, MN) overnight incubation-they are considered as immature BMDC (" iDC ").Connect Several days to get off, 6 kinds of HER2II class MHC binding peptides (42-56 (SEQ ID NO of these immature BMDCs：1)； 98-114(SEQ ID NO：2)；With 328-345 (SEQ ID NO：3) (HER2 ectodomain), and 776-790 (SEQ ID NO：4)；927-941(SEQ ID NO：5)；With 1166-1180 (SEQ ID NO：6) (HER2 intracellular domain) (see, Disis, M.L. etc., Clin.Can.Res.5 (6)：1289 (1999) (" Disis, etc. ")) (United States Biochemical is studied, western refined Figure, the State of Washington；The lyophilized storage of peptide is simultaneously restored for using in sterile PBS) enter cultures of the horizontal pulse at 8~12 hours Afterwards, interferon gamma (" IFN-γ is added ") (1000U/mL).Second day, add NIH normative reference product lipopolysaccharides (" LPS ") (10ng/mL) is the polarization phenotype (" DC1 ") of 1 type to cause BMDC 6 hours whole activation before harvest.For HLA-A2.1^posPatient, with 2 kinds of extra 1 class MHC binding peptides (peptide 369-377 (SEQ ID NO：And peptide 689-697 (SEQ 7) ID NO：8)) pulse DC1s.Wash the cell of harvest, it is thus identified that the batch release standard of the survival abilities of ＞ 70%, gram-negative Property, endotoxin ＜ ＜ 5EU/kg.

Internal node vaccine injection and internal lesion vaccine injection have been carried out as being described according to Koski etc..In short, Immune is the complex clinical research center administration of the hospital of the University of Pennsylvania specified by NIH (NIH) 's.Injection includes the DC 1s for 1000~20,000,000 HER2 pulses being suspended in 1ml Sterile Salines, and passes through ultrasonic guidance Inguinal lymph node is fed, mammary gland, or both has.Immune administration, weekly, altogether to 6 weeks, all patients are complete It is immune into 6 times.Ia safety and toxicity data have been reported that in former Sharma etc..

The detection of immune response

The anti-HER2 CD4 of circulation⁺Response is unexpanded by using 6 kinds of foregoing HER2II class binding peptide pulse patients Caused by PMBC, interferon gamma (" IFN-γ ") is determined by enzyme-linked immunospot assay (" ELISPOT ") Generation.Enzyme-linked immunospot assay (" ELISPOT ") is carried out according to Koski et al. methods described.In short, PVDF is thin Diaphragm plate (Mabtech Inc., Cincinnati, OH) captures antibody (1-D1K (Mabtech)) coating overnight with anti-IFN-γ. Peripheral blood by the isolated refrigeration of density-gradient centrifugation method is unicellular, is put to and preheated supplements 5% people's blood In clear DMEM culture mediums.It is washed and after closing in culture plate, peripheral blood it is unicellular by it is triplicate be placed in plate (2 × 10⁵Cells/well), then culture plate selects any one (peptide of the II classes binding peptide (4 μ g) with HER2- sources at 37 DEG C 42-56(SEQ ID NO：1)；Peptide 98-114 (SEQ ID NO：2)；Peptide 328-345 (SEQ ID NO：3)；Peptide 776-790 (SEQ ID NO：4；Peptide 927-941 (SEQ ID NO：5)；With peptide 1166-1180 (SEQ ID NO：6), only culture medium is (unprovoked Control) or positive control (Anti-Human's class CD3 and anti-CD28 antibody (each 0.5 μ g/mL)) be incubated 24~36 hours (two anti- Body is BD Pharmingen, San Diego, California).After washing, antibody (7B6-1- biotins (Mabtech) will be detected； 100μg/mL；) be added in each hole, 2 hours of culture plate culture at 37 DEG C.Then, the ratio according to 1: 1000 is added Streptavidin-horseradish peroxidase after being diluted in PBS+0.5%FCS, is then further cultured for 1 at 37 DEG C Hour.Tmb substrate liquid (Kirkegaard＆Perry laboratories, Th1Gaithersburg, MD) is next added into, with display Spot formation.After color change, with running water washing hole.Spot formation cell is counted with automatic ELISPOT readers The quantity (ImmunoSpot CTL, Cleveland, Ohio) of (" SFC ").

In addition, it is by stimulating the PBMCs for being available for studying obtained from specific patient subgroups to detect to recall Th1 responses , described stimulate stimulates Candida albicans (Allermed using according to the memory after 1: 100 dilution proportion Laboratories, San Diego, California) and tetanus toxoid (Santa Cruz Biotechnology, Dallas, De Ke Sa Si).In order to determine T_regAnd/or the correlation function activity of Th2 phenotypes, existed according to Guerkov, R.E. etc. J.Immunol.Meth.279：The report of 11 1 (2003), using 2.5 μ g/mL anti-CD 3 antibodies as positive controls, pass through ELISPOT determines IL-10 production.

Because ELISPOT inside repetition otherness is relatively low (data are not shown), implements and determine antigentic specificity response Empirical method.It is defined as the positive response of the single class peptides of HER2 2：(1) after the background not stimulated is subtracted, examination Minimum threshold in verifying is 20SFC/2 × 10⁵Cell (2) antigentic specificity SFC at least twice of growths in background.Often Individual patient's group both defines 3 single CD4⁺Th1 responses are measured.(a) the overall response rates of anti-HER 2 (that is, right >=1 peptide Carry out corresponding Proportion of patients (" response rate ")) (b) reaction peptide average (" response group storehouse ") (c) 6 kinds of peptides cumulative acknowledgements (it is reported as SFC/10⁶Cell) (" cumulative acknowledgements ").

ELISPOT test bay precision

Maecker, HT. et al. is in BMC Immunology 9 according to before：The report of 9 (2008) (" Maecker etc. "), The measure of ELISPOT test bay precision is carried out.When flat for accumulating Th1 responses drafting caused by five stimulated donors After the equal coefficient of variation (CV) (3 parallel repetitions are carried out in 3 days), it is observed that the non-linear relation of a characteristic, wherein Stimulation is to use the ectodomains of HER 2 (ECD) peptide mixer (peptide 42-56 (SEQ ID NO：1)；Peptide 98-114 (SEQ ID NO：2)；With peptide 328-345 (SEQ ID NO：3)) stimulated in vitro.As can be seen from Figure 3A, when cumulative acknowledgements are close to 0 When, average coefficient of variation can steeply rise.Because average coefficient of variation (CV) and cumulative acknowledgements level are nonlinear relations, 3 Individual experiment standard deviation (" SD ") individually daily in 3 days is directed to accumulative Th1 responses and is drawn out, as testing experiment Between otherness means.As shown in Figure 3 B, it is found that standard deviation and cumulative acknowledgements are linearly related.(connecting line represents to produce SD linear regression, with parallel dotted lines show return 95% confidential interval.)(R²=0.96, P ＜ 0.0001).

Linear research is carried out, wherein the triplicate periphery obtained from two height-response HER2- reaction response bodies Blood monocyte sample oneself is known that PMBC is serially diluted in the non-response bodies of HER 2 of allosome, and in body The ECD peptide mixers of external application HER 2 (peptide 42-56 (SEQ ID NO：1)；Peptide 98-114 (SEQ ID NO：2)；With peptide 328-345 (SEQ ID NO：3)) stimulate.Same non-response donor is also used in all detections.Being subtracted in each dilution point does not stimulate Background.In two groups of donors (No. 1 triangle；No. 2 circles) all observed between Th1 responses and diluted concentration it is significant linear Relation.Generally speaking, these as shown by data ELISPOT is accurate, reliably, and repeatable.

HER2 antibody tests

The anti-HER2 antibody of the IgG1 and IgG4 of interior life in patients serum is tested using ELISA.EIA/RIA plates are by carbonic acid The ECD peptides of HER 2 coating (5 μ g/ml in hydrogen salt buffer；Speed Biosystems, Rockville, MD), at room temperature (RT) it is incubated a night.Ensuing that day, culture plate is by the middle closing of 1% casein in PBS, by quadruplicate serum (1: 100 dilution) is added in Block buffer, is incubated 2 hours, is being added for IgG1 or IgG4 (Life Technologies, Grand Island, NY) there is the HRP after specific, 1: 500- dilution to be conjugated before anti-human secondary antibody Washing is three times.After 1 hour is incubated, culture plate is washed, continues (Kirkegaard＆Perry with tmb substrate solution Laboratories laboratories).

Flow cytometry

PBMC suspension in FACS buffer solution (PBS+1%FCS+0.01% nitrine) and anti-human-CD3 ,-CD4 ,-CD8 ,- CD83 ,-HLA-DR ,-CD1 1b ,-CD33 ,-CD19, in-CD56 ,-CD16 (allBD Biosciences, San Jose, California) Prepare ,-CD4 and-CD25 (both Biolegend, San Diego, California) is used for determining the PBMC of correlation immunophenotype. After washing, cell and mixtures of antibodies are cultivated 30 minutes at room temperature.After culture, cell is washed three times with FACS buffer solution, And cell is fixed with 2% paraformaldehyde.The sample of dyeing was analyzed in 24 hours.One is used according to the explanation of manufacturer FoxP3 fixes/and permeabilization kit (Biolegend) carries out the peripheral blood containing anti-FoxP3 (eBioscience, San Diego, California) The cell inner dyeing of monocyte.Flow cytometry is carried out using BD LSR-II cell instruments, uses CellQuest Pro^TM (BD Biosciences) software enters the analysis of line data set.

Case dyes

Will be from HER2^posWhat is obtained in-DCIS and IBC tumours fixes through formalin, the tissue block section of FFPE, And after haematine and eosin dyeing, go to assess cancer week lymphocytic infiltration degree.Compound token IF (PerkinElmer, Wal Se Mu, Massachusetts) it is used for measure from HER 2^posLymphocyte subgroup in the sample that-DCIS and IBC tumours obtain. (see Wang, C etc., Journal for Immunotherapy of Cancer 118：1(Suppl.1)54(2013)) (" Wang, C., et al. ").The use of tyramide signal amplifying technique is the CD4 with fluorescence labeling of the same race, CD8, CD20 and 4 ', The tumor staining of 6 '-diaminourea -2-phenylindone.Image is analyzed using the mode identificating software of Vectra Multispectral microscopes, And identify tumour, matrix and T/B lymphocytes.

Apoptosis is analyzed

BC cell lines (Ithimakin, S. etc., Can.Res.73 with HER2 expression spectrum：1635-46(2013))- HER2^highSK-BR-3, HER2^intermediateMCF-7, HER2^lowMDA-MB-231(American Type Culture Collection) cultivated in RPMI-1640+10%FBS (Cellgro/Mediatech, Manassas, Virginia).50 ×10³Cell is laid down in transwell systems (BD Biosciences), with 10⁶CD4⁺T cell and 10⁵DC Is are (ripe BMDC) or iDC cells (immature dendritic cell) co-cultivation.Foundation Sharma et al. report, DC Is, no Ripe DC cells and CD 4⁺T cell is obtained from the patient after the inoculation chosen.DC Is/iDCs, with 2 class HER 2 Or irrelevant control BRAF peptides (20 μ g/ml) pulse 24 hours at 37 DEG C.Particularly, as being shown in figure I0A, 50×10³SK-BR-3 and only culture medium (complete medium), 10⁶People CD 4⁺T cell (only CD 4⁺), 10⁶CD4⁺T cell+ 10⁵The peptides of 2 class HER 2 (" iDCH ")-or the iDCs of 2 irrelevant class BRAF peptides (" iDCB ")-pulses, and 10⁶CD4⁺T cell +10⁵Each HER2 (" DC1H ")-or DC Is of BRAF (" DC1B ")-pulse.DC 1s/iDCs 2 class HER2 or non-right According to BRAF peptides (20 μ g/ml) pulse 24 hours at 37 DEG C.Control wells only include culture medium or CD4+T cells.

The anti-human TNF-αs of IgG (0. (0.06ug/mL is per 0.75ng/mL TNF-αs) and the IFN-γ of polyclonal goat (0.3ug/mL is per 5ng/mL IFN-γs) antibody (R＆D Systems, Minneapolis, the Minnesota State) be used to neutralize Th1 cell factors, the IgG homotypes of goat are as control.Follow-up processing, BC cells are cleaved, and carry out Western blotting point Analyse to detect the caspase-3 mRNA of cracking.The degree of karyorrhexis is assessed by DAPI dyeing.In addition, half Guang for passing through cracking The detection of aspartase -3 uses (i) from iDC：CD 4⁺Or DC 1：CD 4⁺The supernatant that T cell co-incubation thing obtains, or (ii) The 50 of TNF-α (being shown to be 10-200ng/mL)+IFN-γ (being shown to be 100-2000U/mL) (R＆D Systems) hatching × 10³The apoptosis of BC cells.

Express high-level rodent HER2/ErbB2 (HER2^highTUBO and MMC15, [the latter is Li-Xin Wang Reagent, Cleveland clinic]) and HER2^low/neg(4T1) transgenosis muroid cell line of mammary gland and single culture medium (RPMI- 1640+10%FCS), muroid rmTNF- α (ng/ml are individually recombinated；Send general Tyke), single rmIFN- γ (12.5ng/ml, Send general Tyke), or cultivated 72 hours at 37 DEG C in rmTNF- α+rmIFN- γ mixed culture medium.Ensuing tryptose In enzyme, the cell of harvest is washed, and is resuspended in FACS buffer solution, adds FITC-Annexin v (4 μ l) and PI (2μl).Cell cultivates 20min at 4 DEG C, washes twice, and then carries out flow cytometry.The cell of apoptosis is defined as those The positive is all rendered as to two kinds of mark dyeing.Vinculin, which is used as loading, to be compareed.Be averaged caspase-3 mRNA/film accordingly Join protein ratio ± SEM, show the multiple of apoptosis, ImageJ software quantifications can be used.

ELISA

It is IFN-γ and TNF-α (BD to detect antibody and standard using capture and biotinylation according to manufacturer's recommendation Pharmingen)。

Statistical analysis

The distribution of patient characteristic and immune response variable are summarized using descriptive statistics.By average, SEM and model Enclose and continuous variable is summarized, and classified variable is according to frequency and percentage.When being necessary, using data conversion (natural logrithm or square root), to meet the hypothesis situation of parameter testing.In the case of 3 groups of ＞, multiple ratio afterwards is used Compared with matching test ANOVA or rank test (Kruskal-Wallis) (non-parametric) contrast lasting changeability. Student is used for 2 groups of contrasts.Fisher precision test be used to compare changeability in multi-level form.Student's Pairing t- examine and McNemar accurate test be all used in Th1 response variables assess patient in match change (such as Before vaccine inoculation and after inoculation).P value≤0.05 is statistically regarded as significantly.All tests are all two-sided.Make Statistical analysis has been carried out with SPSS (IBM Corporation) or StatXact (Cytel companies, San Diego, California).

As a result

Patient characteristic

After random continuous registration, the standard that 143 subjects meet to participate in research is shared.The average age of participant Be 53.1 ± 1.4 (scope, 21~88) year, and most of (79.0%) is white people.Patient/donor group, and they take out The time of blood, show in Fig. 1 and above-mentioned method part.The feature that the donor demographics of research participant is related to tumour exists Listed in detail in upper table 1.It is HER2 respectively^pos- DCIS is organized and 26 (83.9%) and 11 (50.0%) of-IBC groups participate in Person, participate in before and be directed to HER2^pos- DCIS the type of lower rectal cancer 1 polarization (DC1) vaccination tests.Their patient/ Tumoral character had obtained Sharma et al. report.

The anti-HER2-Th1 immunitys loss of synthesis is relevant with the development of tumor of breast

IFN-γ ELISPOT testing inspections unicellular (" PBMCs ") using peripheral blood, being stimulated by the external peptides of HER 2 The anti-CD4 of HER 2 of whole body⁺Change of the Th1 responses in tumorigenic continuous process.3 Th1 responses are compared between group to refer to Mark：(a) average of the overall responses of anti-HER 2 (right >=1 peptide have the Proportion of patients of response) (b) reaction peptide, and (c) above-mentioned 6 The cumulative acknowledgements of 2 class peptides of kind.When the donor (" HD ") or benign breast disease patient with health contrast, (Fig. 1, A group), observation To in HER 2^posThere is one significantly progressively to decline for Th1 responses in patient with breast cancer.From the HER of untreated before 2^pos- DCIS (Fig. 1, C group) starts, in the stage I/II HER2 of untreated^pos- IBC reaches low spot (Fig. 1, F group), it was observed that Th1, which is immunized in all Th1 responses indexs, consistent gradual loss.For example the overall response rates of anti-HER 2 are from HD/BD 100% drop to HER 2^pos- DCIS 84% and HER 2^pos- IBC patients drop to 32% (P ＜ 0.0001).Equally , as shown in Figure 5A, in HD, BD, HER2 pos-DCIS, and stage I/II HER 2^posAlso observed in-IBC patients should Answer the similar of group storehouse and be remarkably decreased that (5.2 ± 0.2 compare 0.4 ± 02 compared to 4.5 ± 0.4 compared to 2.0 ± 0.3；P ＜ .0001) It is remarkably decreased that (259.9 ± 23.5 compare 225.1 ± 25.5 compares 32.3 ± 5.4 spots compared to 126.1 ± 24.4 with cumulative acknowledgements Point forms cell (SFC)/10⁶Cell, p ＜ 0.0001).When with response group storehouse (P ＜ 0.001) and cumulative acknowledgements (p= 0.001) when, rather than overall response rate (p=0.07) is assessed, for comparing (post-hoc), HER2 afterwards^pos- DCIS patients Th1 responses be substantially less than healthy donors.HER2^posThe Th1 responses of-IBC patients are further suppressed, because these patients With HER2^pos- DCIS patients compare with significantly lower overall response rate (p=0.0003), group storehouse (p-0.001) and accumulated Response (p ＜ 0.001).Every 10⁶PBMC reacting cells percentage arrives HER2 the 0.03% of HD^posThe 0.003% of-IBC patients Between.

It may be noted that HER2 of the Th1 responses in untreated^neg- DCIS (Fig. 1, B group), or HER2^neg- IBC (Fig. 1, D Group) there were significant differences between patient and HD/BD patient.However, and HER2^neg- DCIS patients compare, HER2^pos- DCIS patients Anti- HER2 Th1 groups storehouse (p ＜ 0.001) and cumulative acknowledgements (p=0.002) it is significantly less.Likewise, and HER2^neg- IBC suffers from Person compares, HER2^pos- IBC patients have lower response rate (P=0.0003), organize storehouse (p ＜ 0.001) and cumulative acknowledgements (p= 0.001), as shown in Figure 5A.

Just as shown in Figure 6 in the anti-of all HER2 ectodomains (" ECD ") and intracellular domain (" ICD ") peptide In implementations of taking on service jobs (p ＜ 0.0050), the special contribution for the accumulation Th1 responses that single HER2 peptides are organized to patient is demonstrated from HD/ BD to HER2^posTh1 responses between-IBC patients have it is similar successively decrease,.Th1 immune responses are to having in DCIS/IBC patient The focusing (focus) that the HER2 antigenic determinants of selectivity are out of proportion can not explain HER 2^posTh1's is gradual during tumour occurs Loss.

In order to investigate the Th1 responses of HD/BD donors it is whether out of proportion be higher than some subgroup, compare all ages and classes (＜ 50 years old (n=16), >=50 years old (n=15)), different menopausal states (premenopausal (n=16), post menopausal (n=15)) are different Ethnic (white people (n=23), other (Black people/Asian etc., n=8)) or different gestation (0 time (n=12), 1 (n of ＞ =19) response).In based on age, race, the sorted HD subgroups of menopausal state, it is not observed in anti-HER2 Th1 There is obvious difference in group storehouse or cumulative acknowledgements.However, the donor (for example, >=1 gestation) of gestation has than nogestational The considerably higher Th1 groups storehouses of anti-HER 2 (5.3 ± 0.2 compare 4.6 ± 0.2, p=0.01) of donor and cumulative acknowledgements (293.1 ± 21.2 compare 178.2 ± 19.0, p=0.0008) (Fig. 5 C).HD/BD and HER 2^posThe Th1 of-IBC donors (each n=4) should The otherness answered in time is detected.From the point of view of the blood drawing at same patient >=6 month interval, Th1 groups storehouse detected It is relatively constant for the time with cumulative acknowledgements, as shown in Figure 7.

HER2^posThe forfeiture of-IBC moderate resistance HER2 IgG1 and IgG4 antibody responses

Existing anti-HER2Th1 responses are in HER2 before noticing in HD^posAfter decaying in tumor of breast, using from health Donor, HER2^pos- DCIS and HER2^posHER2 ectodomain of the Virus monitory serum of-IBC patients extraction for restructuring (ECD) reactivity of peptide.It has evaluated IgG1 Ia to Th1 and the IgG4 related with chronic antigen exposure.Such as Fig. 5 D It is shown, with healthy donors (n=12) and the before HER2 of untreated^pos- IBC patients (n=7) compare, in HER2^pos-DCIS By ELISA observed anti-HER2 IgG1 and IgG4 in patient (n=10 IgG1, n=11 IgG4), (p is ＜ 0.0001) level has relative raising.HER2^posThe relatively low Anti-HER 2 level of-IBC patients shows in disease In progress, the loss of the anti-HER2 responses of endogenous.

CD4 in suspicious expression HER2 IBC⁺Th1 responses are with it in non-suspicious HER2^negIt is visibly different in-IBC

It has detected HER2^negThe Th1 situations of-IBC patients with determine anti-HER2 Th1 be immunized relative reduction subgroup.When with Non- suspicious HER2^negWhen-IBC patients (IHC 0/1+) (n=11) compare, suspicious expression HER2 IBC patient (IHC 2+/FISH It is negative) (n=7) show obvious lower overall response rate (28.6% [IHC 2+] compare 100% [IHC 0/1+], p= 0.002), organize storehouse (0.3 ± 0.2 compares 3.9 ± 0.3, p=0.0001) and cumulative acknowledgements (21.4 ± 6.5 compare 191.2 ± 11.7SFC/10⁶Cell, p=0.002).As shown in Figure 5A, the Th1 responses of suspicious expression HER2 IBC patient and HER2^pos- The response of IBC patient is similar.IL-10 productions are determined by ELISPOT and pass through Flow Cytometry Assay T_reg(CD4⁺CD25⁺ FoxP3⁺) cell relative scale when, suspicious and non-suspicious HER2^negDo not have obvious difference between-IBC patients (not Provide data).

Th1 responses loss T cell tolerance horizontal with host or increased immunosupress circulation immunity phenotype are unrelated

The immunocompetence of evaluable donor subgroup is to determine CD28 anti-to anti-CD3/ by using IFN-γ ELISPOT Th1 responses assess；These responses also serve as donor specific positive control in all ELISPOT experiments.Anti- CD3/ The intermediate value of CD28 responses is in HD/BD (n=31), HER2^neg- DCIS (n=11), HER2^neg- IBC (n=11), HER2^pos-DCIS (n=5), HER2^posThe HER2 of-IBC (n=11) and T/C treatments^posIt is not different between-IBC (n=37) that (1098 compare 1104 compare 1032 SFC/2x10 compared to 1099 compared to 1032 compared to 1318⁵Cell, p=0.22), respectively such as Fig. 5 B institutes Show.In addition, respectively as shown in Figure 8 A, exciting stimulant (tetanus toxoid), (105 ± 17.0 compare 101 compared to 96 ± 15.6 ±1 1.3SFC/2x10⁵) (185 ± 10.2 compare 199 ± 15.3 to 181 ± 14.6SFC/2x10 with candida albicans bacterium⁵) Th1 responses are in evaluated HD (n=10), HER2^posIt is phase between-IBC (n=11), and IBC (n=10) group of T/C treatments As.Jointly, the gradual loss of the anti-HER2 Th1 responses of the breast cancer of these as shown by data HER2 drivings is not attributed to The tolerance of host-level T cells or the ability of impaired antigen submission in IBC patient peripheral's blood monocytes.

Respectively as shown in Figure 8 B, using flow cytometry, from healthy donors group, HER2^pos- IBC groups, T/C treatments HER2^posCD3 in the PMBC of-IBC groups⁺CD4⁺(72.8 ± 2.3% compared to 62.6 ± 3.2% compared to 63.3 ± 6.9%, p=0.26) and CD3⁺CD8⁺(25.1 ± 2.9% compare 38.2 ± 6.6%, p=0.15 compared to 37.9 ± 4.7%) is thin The average proportions of born of the same parents have no significantly different.Not it was observed that B cell (CD 19+) or NKT (NK) cell (CD3- between group CD16⁺) ratio difference (data are not shown).General immunity is compared in following group and suppresses phenotype.As shown in Figure 8 C, HD, HER2^pos- IBC, and T/C treatments HER2^posBetween-IBC subgroups CD4+CD25+FoxP3+ cells (Treg) (1.8 ± 0.3% compares 1.7 ± 0.3%, p=0.78 compared to 1.5 ± 0.2%) and CD11b+CD33+HLA-DR-CD83- cells (marrow source Property suppress cell " MDSC ") average proportions of (0.6 ± 0.1% compared to 1.0 ± 0.3% compares 0.9 ± 0.1%, p=0.33) are simultaneously It is not significantly different.

HER2- specificity IL-10 production detects also by ELISPOT in patient subgroups, and the IL-10 is 2 type T- Aid in (" Th2 ") or T_regThe replacement of function.As in fig. 8d, in HD, HER2^posBetween-IBC, and T/C treatment IBC groups, he Anti- HER2 responsiveness (all 100%), group storehouse (1.8 ± 0.4 compared to 1.8 ± 0.2 compare 2.0 ± 0.3) and cumulative acknowledgements (77.4 ± 15.2 compare 92.8 ± 4.7 compared to 66.6 ± 8.2) is not obvious different.As illustrated in fig. 8e, IL-10 is produced Anti- CD3 stimulants are all similar in all evaluation groups.However, the overall IL-10 productions between subgroup do not have difference, Donor matching HER2 specificity IFN-γs: IL-10 yield than from 6.6: 1 (phenotypes favourable with respect to Th1) in HD groups drastically Change to and do not treat HER2^posThe HER2 of 0.74: the 1 and T/C treatments of-IBC patients group^posThe 0.97: 1 of-IBC patients group is (relative Treg/Th1 advantageous phenotypes) (p=0.009) (top side).

Systematic Th1 responses loss and transport to HER2^posThe T lymphocytes out of proportion of-IBC lesions are unrelated

From pathological angle, to 14 HER2^pos- DCIS and 8 HER2^pos- IBC lesions have carried out immunohistochemistry Analysis, to determine systematic IFN-γ^posCD4⁺Whether response loss is thin with the lymph out of proportion of transport to IBC lesions Born of the same parents are relevant.As a result as shown in Figure 9 A.However, in the assessment patient of most of (12/14,85.7%), it was observed that medium (>= 15% matrix shifts) it is collected at matrix area (top) (arrow outside conduit containing DCIS to the lymphocyte level of higher (>=25%) Head is shown), (8/8,100%) (bottom) observes the relative shortage lymph around intrusion focus in all 8 IBC patients Cell (arrow).

According to Wang, C et al. description, the lymphocyte by new multiple labeling immunofluorescence image technical Analysis Phenotype, this method can distinguish tumour and matrix area, can reliably detect relative CD4⁺(green), CD8⁺(yellow) and CD20+ (red) subgroup.As a result as shown in Figure 9 B.HER2^posMost of matrix (STL) of-IBC tumours and tumor-infiltrated leaching Bar cell (TIL) is by CD8⁺Cell forms (upper right side).In addition, compared with DCIS focuses (upper left side), in HER2^pos- IBC swells Relatively small number of CD4 is observed in knurl⁺TIL/StL.Cancer week CD4 out of proportion⁺T cell is transported to HER2^pos- IBC lesions are simultaneously FN- γ can not be explained^pos CD4⁺The systematicness consumption of T- cell subsets.

The BC cells of higher/medium HER2 expression rather than relatively low HER2 expression are to CD 4⁺The Apoptosis of Th1 mediations is quick Sense

It has evaluated Th1 and mediate effect to external HER2^highSK-BR-3, HER2^intermediateMCF-7 and HER2^lowMDA- The influence of MB-231 BC cell lines.Using transwell culture systems by the ever-increasing class peptides of the HER2 2-specificity of ratio CD4⁺The expression HER of Th1 cells and mentioned kind BC cells co-culture, and the Th1 cells are stimulated by HER2- pulses DC1, make The dose-dependent apoptosis of SK-BR-3 of impact is result in transwell culture systems, it is examined by western blots method Survey increased caspase-3 mRNA prove, as shown in Figure 10 A.The visible MCF-7 rather than MDA-MB-231BC cells of Figure 11 A.Phase Instead, as shown in Figure 10 A and Figure 11 A, with CD4⁺Apoptosis is not very notable relatively in the BC cells that cell co-cultures, described CD4⁺T cell immature DCs (iDC H and iDC B) or the DC Is (DC1B ' s) of 2 class peptides (BRAF)-pulse of control Stimulate.The quantity of Th1 cell factors in the supernatant that these are co-cultured by elisa assay, it can be seen that show compared to Figure 10 A The CD4 gone out⁺：BRAF compares DC1, CD4⁺T cell：IFN-γ and TNF-α dramatically increase caused by the DC1 of HER2 pulses, corresponding In the apoptosis degree observed.

As shown in Figure 1B, when with CD 4⁺T cell：The DC1 cocultures rather than and CD4 of HER2 pulses⁺：HER2-iDC or Person CD4⁺：When the supernatant of the cocultures of BRAF controls-DC 1 is cultivated together, it can be observed in SK-BR-3 cells similar Specificity antiapoptotic.Compared with control group, the specific Th1 cells of HER2- to causing 25 times of increase of SK-BR-3 apoptosis, This can be dyed from Figure 10 B DAPI find out, right figure and column diagram.Consider, the anti-HER2 CD4 of these as shown by data⁺Th1 Cell generates the HER2 that solvable sexual factor has regulated and controled high/middle expression, rather than the HER2 of low expression, breast cancer cell.

Importantly, as shown in Figure 10 A, HER2^highSK-BR-3 apoptosis can be shown by neutralizing IFN-γ and TNF-α Rescue is write, show the Th1 cell factors of pleiotropism plays the role of key in the specific Apoptosis of HER2 is regulated and controled.In order to enter One step studies these phenomenons, have detected the influence of IFN-γ and TNF-α to BC cells.No matter HER2 is expressed, human body BC is thin Born of the same parents can equably maintain IFN-γ and TNF-α expression of receptor, as illustrated in figure 10 c.IFN-γ and TNF-α processing cause HER2^highSK-BR-3 and HER2^intermediateM F-7 notable apoptosis, not including HER2^lowMDA-MB-231, such as Figure 11 C institutes Show.Next, remained to assess the HER2 expression of MDA-MB-231 cells recovery to the quick of Th1 cytokine modulating apoptosis Perception, the HER2 plasmids (pcDNA-HER2) of MDA-MB-231 cell wild types or the empty vectors (pcDNA3 of control group； Kind gifts of Mark I.Greene, the University of Pennsylvania) transfection, and with IFN-γ and TNF-α (2000U/ml and 200ng/ml；It is different from the metering to MDA-MB-231 processing shown in Figure 11 C).It was observed that HER2 transfection after there occurs Notable apoptosis under IFN-γ/TNF-α induction, does not find this phenomenon (not providing data) in the MDA-MB-231 of blank transfection.

Finally, the HER2 specificity antiapoptotics of these Th1 cytokine modulatings obtain in the Mouse mammary cells of transgenosis Confirm.Handled, can be caused twice with the mouse IFN-γ and TNF-α of restructuring, rather than single cell factor HER2^highTUBO and MMC15 rather than HER2^low/neg4T1 notable apoptosis, is shown in Figure 10 D.This HER2^pos- IBC response damage The can after HER2 pulses DC inoculations is lost to recover, rather than after HER2 targetings or conventional therapy.

After HER2- pulse DC vaccine inoculations, rather than after HER2- targetings or traditional treatment, HER2^posIn-IBC Th1 responses loss be resumed

Analyze and give T/C treatments and HER2 pulses DC1 in Figure 12 A top layers and be immunized to HER2^pos- IBC patients Th1 The Different Effects of response.The stage I/II HER2 of untreated before^pos- IBC patients (n=22, Fig. 1 F groups) and T/C treatments Stage I-III HER2^posIn anti-HER2 responsiveness, (31.6% untreated compares 45.9＆T/ to-IBC patients (n=37, Fig. 1 G groups) C is treated, p=0.39), ((32.3 ± 5.4 compare for (0.4 ± 0.2 compares 0.8 ± 0.2, p=0.24) or cumulative acknowledgements in group storehouse 54.5±12.0 SFC/10⁶, p=0.97) on do not distinguish significantly.As shown in Figure 12 B top layers, and then to 11 stages 1 HER2^pos- IBC patients carry out the DC1 inoculations (Fig. 1 H groups) of HER2 pulses, however, it was observed that in anti-HER2 response rates (18.2% After being inoculated with before inoculation compared to 90.9%, p=0.0035), organize storehouse (0.3 ± 0.2 compares 3.7 ± 0.5, p=0.0001) and accumulate (29.7 ± 7.9 compare 162.8 ± 33.7SFC/10 for response⁶, p ＜ 0.0001) on increase significantly.After DC1 vaccine inoculations, Rather than T/C receive after impact Th1 recover influence can continue to and the stage 1 before untreated (n=11), T/C- (n=11) HER2 after treating (n=8) and being inoculated with^posThe stage of-IBC patients matching, as indicated in fig. 12 c.

It is have detected after DC1 vaccine inoculations to treat in IFN-γ with T/C^pos∶IL-10^posReact in T cell relative scale Difference.In the donor matching contrast carried out at the same time, but HER2 specificity IFN-γ (is compared after 196.8 ± 56.8 inoculations SFC/10 before 32.1 ± 6.1 inoculations⁶, p=0.02) and (79.0 ± 7.4 compare 33.8 ± 5.1SFC/10 with IL-10⁶, p=0.001) Response adds after the DC1 vaccine inoculations of HER2 pulses, IFN-γ: the relative scale of IL-10 responses is (relative from 0.95: 1 T_reg/ Th2- is favourable)/renewed vaccination changed to after 2.5: 1 (Th1 is favourable) vaccine inoculations (p=0.008).However, IFN-γ: IL- The relative scale of 10 responses not can be shown that (0.74: 1, p=0.78) the HER2pos-IBC patient compared to untreated before For T/C treat (0.97: 1) after there is one to clearly tend to the favourable phenotypes of Th1.

Longitudinal direction >=vaccine inoculation of 6 months after Th1 Immunological evaluations be possible to 9 patients's (81.8%).Such as Figure 12 D Shown in Figure 12 E, although all patients have been complete postoperative chemotherapy after inoculation, it has been observed that relative to connecing The more lasting anti-HER2 Th1 reactivities of datum line after kind, its Median Time are 16 months (6~16 years old scope), anti-HER2 Response (before 6 months after 100% >=inoculation are inoculated with compared to 22.2%, p=0.008), (4.0 ± 0.4 compare 0.3 ± 0.2, p in group storehouse ＜ 0.0001), (255.1 ± 49.2 compare 33.8 ± 9.2SFC/10 to cumulative acknowledgements⁶, p=0.006).

The subgroup analysis organized after T/C treatments has been carried out, it is (new auxiliary according to chemotherapy to study the difference of Th1 reactivities Help treatment or adjuvant)；From the trastuzumab of description is completed to the time (＜ or >=6 month) of research registration；Estrogen by State (the ER of body^posOr ER^neg)；With pathologic state (I~III).Figure 13 A show chemotherapeutic order (new auxiliary Adjuvant [n=25] is compared in treatment [n=12])；Figure 13 B show after the completion of trastuzumab time (＜ 6, [n=16] compared to >= 6 months [n=21]；), or Figure 13 C illustrate ER state (ER^pos[n=21] compares ER^neg[n=16]), do not have an impact Anti- HER2 Th1 responsiveness, organize storehouse, or cumulative acknowledgements (all p=NS).Importantly, Figure 13 D illustrate the AJCC stages I (n=8), stage II (n=20) or the patient of stage III (n=9) T/C treatments do not have difference in Th1 indexs, show It was observed that HER2^pos- IBC anti-HER2 Th1 losses do not have dependence with Disease Spectrum.In addition, these data are on the whole The leading Th1 reactivities that can show that part subgroup are not to cause the HER2 in T/C treatments observed^pos- IBC patients In the immune restoration that occurs comprehensively the reason for reducing.

The anti-HER2 Th1 responses lowered are relevant with bad clinical effectiveness

In order to assess the horizontal correlation of these discoveries, an assessment is carried out, to determine the HER2 of T/C treatments^pos-IBC Patient occur Th1 responses difference whether with follow-up mammary gland event (" BE "；Any local/distal end is defined as to recur) Develop relevant.Follow-up intermediate value is 33.5 months (interquartile range IQR25.5-45.8).It is as shown in table 2 below, there is shown HER2^pos- IBC patients occur after trastuzumab and chemotherapy follow-up breast cancer event (be defined as it is any local or Person's whole body recur) population and Clinical symptoms.8 patients's (21.6%) after T/C treatments are received there occurs follow-up BE, its Middle bit duration is 29 months (interquartile range IQR 16.2-36).Figure 13 E, left side, there is shown the trouble with no BE Person compares, and BE patient occurs, and there is the anti-HER2 responsiveness (top side) after significantly inhibiting (to have BE patient 12.5% compared to not Have BE patient 55.2%, p=0.048) and cumulative acknowledgements (bottom) (9.4 ± 3.6,66.9 ± 14.5 SFC/106, p= 0.046), but not including response group storehouse (centre), (1.03 ± 0.3 compare 0.13 ± 0.1；P=0.11)).

Table 2

Receive the HER2 of lower rectal cancer T/C T/C treatments at 12 (32.4%)^posIn-IBC patients, pathology is compared Complete response (pCR, being defined as not finding the infiltration BC of residual in postoperative pathological) and non-pathology complete response patient are in anti-HER2 Difference in terms of Th1 responses.Result on the right side of Figure 13 E, represent the knot for the pathology complete response that 4 patients's (33.3%) obtain Fruit, with significantly higher anti-HER2 groups storehouse (3.3 ± 1.1,0.13 ± 0.13, p=0.002) (centre) and cumulative acknowledgements (193.1 ± 64.9,13.6 ± 4.6, p=0.002) it is relevant.Anti- HER2 responses (100%, 25%, p=0.06) (top) are without statistics Learn meaning.

Discuss

Appearance (Topalian, S.L. etc., the N.Eng.J.Med.366 of counter point inhibitor：2443-54 (2012)), exempt from Epidemic disease regulating strategy for example, inoculation (Kantoff, P.W. etc., N.Eng.J.Med.363：411-22 (2010)), Toll-like receptor Activator, or for particular organization's specific epitope T cell treatment (Kalos, M. etc., Sci.Transl.Med.3(95)：95ra 73 (2011) and Rosenberg, South Africa, naturally comment on Clinical Oncology, 8：577- 85 (2011)) laid the foundation for significantly more efficient immunotherapy for cancer.Most of in these therapies are towards wide General immunoregulation.With these discoveries simultaneously, genomics has identified tumorigenic oncogenic molecules driving, including V- Raf murine sarcoma virus oncogene homologue B1 (BRAF), EGF-R ELISA (" EGFR "), hepatocyte growth factor receptor (" c-MET ") and HER2.Targeted therapies, which such as " drive ", can obtain encouraging response rate, and their success is relatively short Temporarily, because most of tumours eventually recur or become it is resistant to therapy (genuinePohlmann etc. and Flaherty, K.T. etc., N.Eng.J.Med.363：809-19(2010)).Identify that carcinogenic driving is specific immune scarce during tumor development The therapy apparatus meeting treated by disease may be provided for specific cancer subclass by falling into.Described herein is considered as in tumour generation Identify CD4⁺The preliminary research of Th1 immune deficiencies, the immune deficiency have specificity to the molecular drive of specific BC phenotypes, That is HER2/neu.

Anti- HER2 CD4⁺Decay immune Th1 starts from the DCIS stages before cancer, and is gradually lost in and invades profit in early days Disease stage.Seem specifically can also lose in the phenotype of HER2 overexpressions in addition, Th1 is immune.Widely used tumour In the continuous process of generation, HER2 is had confirmed herein^neg- DCIS and HER2^negThe anti-HER2 of-IBC patients (IHC 0/1+) Th1 responses are very similar with HD/BD donors, and significantly higher than HER2^pos(IHC 3+ or 2+/FISH are positive) DCIS It is similar in addition, the Th1 responses in suspicious expression HER2 (IHC 2+/FISH negative) individual can lose with IBC patient In HER2^pos- IBC patients.Particularly, HER2^negThe HER2- specific Cs D4 of-IBC patients⁺The maintenance of immunity, can part Explain that they are received to activate CD8 in ground⁺The improved clinical effectiveness obtained after HER2 peptide vaccines inoculation for the purpose of T cell. Benavides, L.C. are seen, etc. Clin.Can.Res.15：2895-904(2009).

To a certain extent it is shocking that HD/BD maintains the anti-HER2 Th1 cell quantities of recognizable circulation.By It is the membrane component for being present in branch's breast ductal cells on paper in the HER2 of edge design, (Press, M, F. etc., Oncogene 5：953-62 (1990)), so CD4 pre-existing in HD/BD⁺T cell response is the antigen submission in mammary gland Result caused by cell (" APCs ") submission HER2- antigenic determinants is seemingly believable.In fact, although and the age, kind Race or menopausal state are unrelated, and the donor for being higher than non-pregnancy is immunized in existing anti-HER2 Th1 in advance in HD/BD, special Other, the latter is the higher colony of the danger of BC development.In addition, pass through HER2^higRather than HER2^lowVivoexpression IFN-γ/ The cell for the significant HER2- specificity Ts h1 that cell factor IFN-γ and TNF-α in the BC cell lines of TNF-α acceptor are realized Apoptotic effect, indicate anti-HER2 Th1 may such as mammary gland physiology course it is excessive to controlling or eliminating HER2 Expression cell is helpful.Thus, pre-existing anti-HER2 Th1 are immune in healthy donors may be directed to tumour Event is protected, but the forfeiture of anti-HER2 Th1 functions can represent a kind of mechanism of tumour driving, and it can be avoided HER2^posImmunosurveillance in tumour generation.It is interesting that the recent evidence show that antigen (ratio of preferential circulating tumor correlation Such as MAGE6, EphA2) specific C D4⁺Th1 death programs may cause to observe in the melanoma tumors patient for having active disease Dysimmunity (Wesa, A.K. etc., the Front.Oncol.4 arrived：266(2014)).Similar mechanism also appears in current research In observe anti-HER2 CD4⁺In loss immune Th1-decryption, and targeting, these mechanism are for being intended to prevent early stage BC It is important for the development of immunological intervention.The functional meaning of anti-HER2 Th1 cells in these mechanism, and mammary gland stable state, It ensure that further research.

It is present in although previous HER2-Th1 is immune in HD/BD, HER2 reactivity humoral responses are not.In health Mammary gland in, non-inflammatory set in pass through antigen presenting cell early start CD4⁺Th1 cells, passing through IFN-γ/TNF-α secretion When causing the stable state of HER2 expression cells, it is impossible to drive the generation of antibody.However, in HER2^posIt is anti-in HER2- in-DCIS The relative increase for answering IgG1/IgG4 is related to intermediate, but can not lack Th1 responses.The appearance of HER2 antigens is to development Tumour stimulation, and follow-up antigen presenting cell retains Th1 cells in inflammatory environment, enables the antibody point of transient state Secrete.Finally, in HER2^posIn-IBC, CD 4⁺The decrease of T cell may corrode the continuous secretion of antibody, cause them final Loss.Early prevention and the control that these dissipation for adapting to immune aspect will cause these patients to realize tumour.

In addition to discussed above, loss immune anti-HER2 Th1 also can other other functions for example, chronic T is thin Born of the same parents are lost or outer peripheral tolerance and its influence to synergistic signal (such as TIMs, PD-L1, CTLA-4 etc.), or HER2- reacts the change of immunophenotype.Although in fact, overall IL-10 responses tumour occur non-individual body in have maintenance, when Use antigentic specificity IFN-γ: when IL-10 ratios are assessed, the response of HER2- specificity from favourable strong Th1- (HD/BD) to With respect to Th2/T_rea- favourable (in HER2^pos- IBC) phenotype functional transformations.Completely, it is although soft, 7/22 (32%) HER2^posThe Th1 response rates of-IBC patients, thus, it is possible to which ongoing Th1 antitumor immunes protection and tumour can be influenceed The T of tolerance in generating process_reg/ Th2 contributes (Levings, M, K etc., Blood 105：1162-9 (2005)) between it is flat Weighing apparatus.

Even so, loss immune anti-HER2 Th1 is not but in HER2^posCirculation immunity is caused to suppress in-IBC patients The reason for quantity increase.Although before research it has been reported that the later stage BC (stage III/IV) (Liyanage, U.K. Deng J.Immunol.169：2756-61 (2002)) and other entity tumors (Zhang, B. etc., in PLOS ONE 8 (2)： E57114 (2013)), in this research, early stage (stage I/II) IBC patient seem relative to HD have comparable to exempt from Epidemic disease suppresses.Thus, drastically decline of these patients in anti-HER2 Th1 responses is even more enforceable.Further, This anti-HER2IFN- γ of peripheral blood^posSculpture (immune is immunized with following unrelated (i) in the reduction of CD4+T cell subsets Sculpting), it is partial to selective HER2 reactive polypeptides activity due to not observed in the generation of continuous tumour.Second discovery It should explain with caution, however, because these data do not illustrate HER2 specific Cs D4 in tumor microenvironment⁺TIL collect or Loss.Finally, anti-HER2 Th1 immunosupress can not be explained with the horizontal T cell anergy of general IBC patient host.So And existing research is also without the exclusion horizontal anergy of antigen-specific cellular completely as the possible explanation of this phenomenon.

Importantly, the HER2 of this anti-HER2 Th1 consumption and T/C treatments^pos- IBC patients increased local or The risk of distal end recurrence is related.On the contrary, anti-HER2 Th1 reservation is relevant with the lower rectal cancer T/C after pCR.Total comes Say, the anti-HER2 Th1 immune response activities that these data illustrate to monitor after HER2 active treatments can identify and be subject to clinic Or the subgroup of pathology failure.In addition, anti-HER2 Th1 shortage and the result of unfavorable clinicopathologia are demonstrated and ground Study carefully the correctness for the therapeutic strategy that may reverse these immune losses.

(such as pathology stage), HER even after Disease Spectrum is controlled^2posBeing suppressed for-IBC patients is anti- HER2 targeting of the HER2 Th1 responses under operation, radiation, chemotherapy or no trastuzumab is to be affected.It is some Research has been proven that trastuzumab is reducing HER2^posTumour increase and induce in terms of its extinction ability (Dogan, I. etc., Mol.Cell.Biochem.347：41-51 (2011) road), while also demonstrate it and make HER2^posCell by cell factor chemistry Tumoricidal sensitiveness (Henson, E.S. etc., the Clin.Cancer Res.12 of therapy：645-53(2006)).Although there is this A little advantages, but using trastuzumab fail to significantly recover the HER2 specificity T h1 epidemic disease abilities of Most patients, including which Patient in stage I.In addition, it observed in the advanced stage tumours stage almost universal for these HER2 targeted therapies Resistance.Pohlman etc..It is then desired to the extra strategy using HER2 as target.

As described here, it is tactful as one, exactly it is immunized together with the 2 class peptides in HER2 sources and autologous DC1. After receiving lower rectal cancer, in HER2^posHER2 pulse DC1 vaccine inoculations are carried out in-IBC patients (preoperative), after immunity inoculation Anti- HER2 Th1 sustained restoration can be observed up to 60 months.Generally speaking, these data illustrate, (i) this The specific CD4 of HER2⁺Th1 immune deficiencies are not " fixed " in immunology, because it can be by suitably immune dry The measure of relating to is corrected for；(ii) is by vaccine inoculation (or other immune modulatory strategies) and existing based on body fluid The combination of HER2 targeted therapies can improve the long-term final result of this disease.In fact, in mouse model, cell (IFN-γ- The CD 4 of generation⁺, but be not CD8⁺, T cell (Sakai, Y. etc., Cancer Res.64：8022-8 (2004))) and body fluid pin Cooperation to HER2 immunitys is for eliminating HER2^posTumour is important (Reilly, R.T. etc., Cancer Res.61：880- 3(2001))。

Generally speaking, current discovery is for HER2^posImmunologic surveillance and the therapy selection of-BC patients is meaningful.Just As discussed, they confirm in the BC of HER2 drivings people at highest risk that anti-HER2 is immune added to standardization HER2 Correctness in targeted therapies；In fact, still there is the HER2 of residual disease after lower rectal cancer T/C treatments are received^pos- IBC suffers from Experiment in person and the effect of those test these combinations in the patient in the tumour later stage after adjuvant treatment has begun to Carry out.Further, because the monitoring policy (radiography, the IHC/FISH analyses of breast biopsy sample) of routine can only provide To the single snapshot of tumour change, the immune real-time fluctuations of anti-HER2 Th1 for monitoring high-risk patient can be to understand nature disease Journey and the Immunological Effect offer information to tumour.By CD4⁺Th1 immunologic detection methods are incorporated into the BC clinical trial designs in future It is reasonable.

It is summarized as follows, as far as we know, herein it is considered that the first step that molecular weight tumor drives in tumor of breast generation It is CD4⁺Th1 it is immune progressively and specific loss.Understand those and the anti-HER2 Th1 of downward are Ia unfavorable faces Bed and pathological examination show that the immune restoration of inoculation acquisition or other immunoregulation strategy are worth in these high-risk patients Middle research, recurred with alleviating the development of tumour or prevention.Also extra research has been carried out to determine anti-HER2 CD 4⁺Should Whether answer in other HER2^posAlso incurred loss (such as oophoroma, stomach cancer etc.) in cancer, and determines to occur in tumour During driving carcinogenic to other molecules produce whether immune Th1 is immune universal loss.

Test examples 1

Anti- HER2 CD4⁺Th1 responses be it is a kind of for HER2 it is positive it is breast cancer, after lower rectal cancer, with The relevant novel immune therapy of pathology response

In the practice in the present age, have it is larger can tumor resection patient often from trastuzumab and chemotherapy (T/C) lower rectal cancer is benefited in applying, and there are about 40%~60% can obtain pathological complete response (pCR).See Gianni, L., etc. Lancet 375：377-84(2010)；Untch, M. etc., J.Clin.Oncol.28：2024-31 (2010)；Untch, M. etc., J.Clin.Oncol.29：3351-7(2011).(" the ＜ compared with the Residual Disease of operative treatment PCR "), the pCR after lower rectal cancer T/C treatments is the method for the determination for reducing recurrence and improving long term survival chance.

Reference example above confirms HER2^posThe continuous process moderate resistance HER2 CD 4 that breast cancer tumour occurs⁺1 type T The immune gradually loss of auxiliary cell (Th1).It is particularly interesting, will of this HER2 specificity T h1 response in health Hope person and contain HER2^negThe patient of (0-1+) infiltrative breast carcinoma cell (" IBC ") retains.In HER2^pos- IBC patients In, this primary antibody HER2 Th1 losses will not be by standard treatment --- the influence of surgery excision, radiotherapy or T/C treatments, on the contrary It can recover after 1 type polarization BMDC (DC1) vaccine inoculation of HER2 pulses.In addition, be also shown for lowering resists HER2 Th1 responses, which predict, receives the risk that the patient that adjuvant T/C is treated has higher follow-up recurrence.These observations are facilitated Whether the anti-HER2 Th1 responses for similar downward can also be observed in omen is recurred known to another kind and ground Study carefully, i.e. ＜ pCR states (Kim, M.M. etc., Ann.Oncol.24 after lower rectal cancer T/C：1999-2004 (2013)), phase Instead, it is assumed that the prevention and recovery of anti-HER2 Th1 responses are also assumed to relevant with pCR.Thus pCR and ＜ pCR patients are anti- Difference in HER2 Th1 responses is detected to determine the modifiable immune correlation with pathology response.

87 HER2 are analyzed perspectively^posThe anti-HER2 CD4 of-IBC patients⁺Th1 responses, and compare stage I/ IIHER2^pos- IBC patients (n=22) and the HER2 of stage I-II T/C treatments^posThe difference of the response of-IBC patients (n=65). Generate after the completion of adjuvant trastuzumab in the group-anti-HER2 Th1 responses of T/C treatments-response is according to chemotherapy time (ratio As lower rectal cancer compares adjuvant) classification, or according to pCR and ＜ pCR state point further in lower rectal cancer group Level.PCR is defined as remaining intrusion cancer in the pathological examination to cutting off the lymph node of mammary gland sample and sampling (i.e., YpT0/Tis ypN0) it has been not present.

4 patients in ＜ pCR groups are reassembled 1 type polarization DC (DC1) vaccine for participating in an adjuvant HER2 pulse Inoculation test (NCT02061423).Analyze anti-HER2 Th1 response of these patients after immune preceding and immune.

Method

As described in reference example, the unexpanded PBMC anti-HER2 CD4 of circulation are detected⁺Th1 responses, institute State PBMC with the 2 class peptides in 6 HER2 sources (peptide 42-56, peptide 98-114, peptide 328-345, peptide 776-790, peptide 927-941, and Peptide 1166-1180) (SEQ ID NOS：External pulse 1-6)) is carried out, is determined by enzyme-linked immunospot assay (ELISPOT) The generation of IFN-γ, the description that ELISPOT is pressed in reference example are carried out.From HLA-A2.1^posThe PBMC obtained in donor uses 2 1 class peptide (peptide 369-377 (the SEQ ID NO in kind HER2 sources：And peptide 689-697 (SEQ ID NO 7)：8)) stimulate, simultaneously will PMA (50ng/ml) and ionomycin (1ug/ml；Sigma-Aldrich) it is used as positive control.

A kind of empirical method for determining antigentic specificity response is used.The positive response of single HER2 peptides is defined as： (1) minimum threshold subtracted after unprovoked background in test hole is 20SFC/2x10⁵Cell, and (2) have with respect to background The growth of antigentic specificity SFC more than or equal to 2 times.The measurement of Th1 responses should for the anti-HER2 described in above-mentioned reference example Answer rate, the quantity and 6 kinds of peptide (SFC/10 of reaction peptide (group storehouse)⁶Cell) cumulative acknowledgements.Analyze and receive adjuvant HER2 pulses The ＜ pCR patients (n=4) of 1 type polarization BMDC (DC1) vaccine inoculation front and rear Th1 responses are being immunized.

As a result

Research includes 87 patients.After T/C treats (n=5), untreated HER2 before^posThe downward of-IBC patients Anti- HER2 Th1 responses can not improve comprehensively.Compared with adjuvant T/C, lower rectal cancer-T/C (61.5%) with it is higher Th1 groups storehouse (1.5 relative 0.8, p=0.048) is related.Although PCR patient (n=16) and ＜ PCR patients (n=24) population/ It is not different in Clinical symptoms, but pCR patient more likely there are ERneg tumours.Compared with ＜ PCR patients, pCR patient has height Anti- HER2 responsiveness (94% compares 33%, p=0.0002) much, group storehouse (3.3 compare 0.3, p=0.001) and accumulation should Answer (148.2 compare 22.4, p=0.0001).This inconsistency is by CD 4⁺T-bet⁺IFN-γ⁺Phenotype is reconciled, Immune failure to ＜ pCR patients, the horizontal T cell ineffectivity of host, or increased immunosuppressive quantity do not work. In 4 ＜ PCR patients, after HER2 pulse DC1 vaccine inoculations, Th1 groups storehouse (3.7 compare 0.5, p=0.014) and cumulative acknowledgements (192.3 compare 33.9, p=0.014) is significantly improved.

Conclusion

Anti- HER2 Th1 responses are a kind of new immunotherapies, and its pathology after being treated with lower rectal cancer T/C should Answer relevant.In ＜ pCR patients, received the patient with HER2 expression of lower rectal cancer, its Th1 response lowered can be with Recovered by HER2-Th1 immunologic interventions, and pCR or recurrence rate can be improved.

Therefore, the HER2 Th1 immunologic interventions targetted are added in lower rectal cancer T/C methods or are added to excessive risk During the adjuvant that is carried out in ＜ pCR subgroups is set it is verified that.In addition, according to the anti-of the downward confirmed in reference example The immune follow-up recurrences with the patient of adjuvant T/C treatments of HER2Th1 have relation, monitor the anti-HER2 of excessive risk ＜ pCR patients Real-time fluctuations immune Th1 can be as the supplement of existing radial imaging method inspection, and assists in and carry out in the treatment The crucial window of intervention.

To sum up shown, this can be used as HER2^pos- IBC patients are in anti-HER2 CD4⁺Th1 is immunized and lower rectal cancer T/C The description first of crucial contact between pCR afterwards.Although not can confirm that causality still, controlled in lower rectal cancer T/C The IFN-γ drastically observed in the ＜ pCR patients for the treatment of⁺Anti- HER2 Th1 losses increase is exempted from using what HER2 Th1 intervened Epidemic disease relief on the effect of improving high-risk patient as standard HER2 targeted therapies supplement possibility.

Test examples 2

The anti-HER2 CD4 lowered⁺1 type T aids in the recurrence rate of HER2 breast cancer patients with positive of the response with treating completely to have The new role of pass-immunoregulation

As used herein shown in the reference example of expected group, in HER2^posIn the tumorigenic whole continuous process of-BC, resist HER2 CD4⁺1 type (" Th1 ") T skeptophylaxises gradually lose, wherein HER2^pos- BC from healthy donors, to HER2^pos- DCIS and Finally extend to HER2^posThe BC patient of-infiltration, sees Datta, J., etc. Oncolmmunology 4：8 e1022301(2015) DOL 10.1080/2162402X.2015.1022301 (", wait .Oncolmmunology at Datta, J.).In addition, examination here Test the moderate resistance HER2 Th1 responses of example 1 and show new immune-related with pathology response, the immune response is in HER2^pos- Occur after BC lower rectal cancer trastuzumab/chemotherapy (" T+C ").But the anti-HER2 Th1 lowered are immunized and hand Related-unfavorable clinical pathology result of the residual disease of art.See Datta, J., et al., breast cancer research .17 (1)：71 (2015) (" Datta, etc. breast cancer research ").

In view of these are observed, it has been assumed that the immune HER2 that may also with treating completely of anti-HER2 Th1^posThe office of-BC patients Portion and/or distal end recur it is related.In exploration group, in order to identify the immune correlation with palindromia, checked recurrence with Disease-free HER2^posThe difference that the anti-HER2 Th1 of circulation between-BC patients are immunized.

Method

Research and design

In the approval by University of Pennsylvania's institutional review board, 95 HER2 are being obtained^posAfter-BC suffers from agreement It is enrolled into non-prejudice mode.Qualified patient has the infiltrative breast carcinoma (" IBC ") confirmed in institutional framework, HERI/ Neu is overexpressed (IHC 3+or 2+/FISH- are positive) and does not receive immunosuppressive drug.Untreated (that is, is being registered before When do not have determine treatment) stage I-III HER2^posThe anti-HER2 CD4 of-IBC patients (n=22)⁺Th1 responses were with receiving T + C treats (n=73；That is, lower rectal cancer or adjuvant T+C adds certainty to perform the operation) stage I-IV HER2^pos- IBC patients' Th1 responses are contrasted.In the patient of T+C treatments, analyze and be classified according to recurrence state.

HER2^pos- the recurrence confirmed is defined as any part (in breast/recurrent on chest wall, axillary failure) or distal end mammary gland Event (lung, bone, brain metastes etc.), or both have.In extra chemotherapy, HER2- targeted therapies or test procedure (example Such as, HER2 dendritic cells pulseds vaccine inoculation) start to recruit the patient of all recurrences before.The patient of only non-recurrence is minimum Disease-free follow-up 24 months, they, which are only, is adapted to analysis.Figure 14 is the flow chart to the study population of these test examples.

The test arrangement and dosage of trastuzumab and chemotherapeutic treatment protocols

The patient of T+C- treatments receives a kind of following scheme, before surgery or Post operation (1) AC/TH：Adriamycin/ Endoxan (every 2 Thursday circulation), subsequent Taxol and the trastuzumab (weekly, 12 weeks) received simultaneously；(2)TCH： Docetaxel/carboplatin and the trastuzumab (6 circulations in every three weeks) received simultaneously, or (3) TC-H：Docetaxel/endoxan with it is same When the trastuzumab (every three weeks 4 circulation) that receives.All patients individually to receive additional trastuzumab (every three weeks) with Complete annual treatment.

Immune response detects

Above with reference to described by example, for MHC II class peptides (42-56, the 98- in exogenic six kinds of HER2 sources 114,328-345,776-790,927-941,1166-1180) the unexpanded PMBC of external pulse, and with The mode of the secretion of ELISPOT detection IFN-γs checks the anti-HER2 CD4 of peripheral blood therein⁺Response；Also Datta, J. are seen, Deng Oncolmmunology；Datta, J., etc. breast cancer research；And Koski, etc..Briefly, participated in from each research The whole blood that person gathers 25-30mL is put into 5 test tube of hepari collecting pipes (BD Bioscience).(the ＜ within the short time after bloodletting 4-6 hours), according to manufacturing specification, use density-gradient centrifugation method separating periphery blood monocytic cell (that is, Ficoll-Paque Method；Fisher science and technology), it is with 10x10⁶Cell/mL concentration is in -80 DEG C of Cord bloods in 10%DMSO human serum. During the Cord blood of 4 weeks, all PMBCs of using up.Once it is 60%-80% to melt survival rate.Pvdf membrane Plate stays overnight coating with anti-IFN-γ coated antibody.The PMBC of Cord blood is defrosted the DMEM+5% of preheating In human serum, 1x10 is prepared in described culture medium⁶PMBC/mL.After culture plate is cleaned and closed, peripheral blood Monocyte is placed in culture plate (2x10 in triplicate⁵Cells/ is per hole), and with HER2 peptides (4 μ g；Genscript, Piscataway, NJ) or only culture medium (not having stimulated control) or positive control (anti-human CD33/CD28 antibody [0.5 μg/mL；BD antibody]) in 37 DEG C of hatching 24-48h.

In evaluable patient, according to the description of reference example, Candida albicans is stimulated to the memory of 1: 100 dilution The Th1 responses that [Allermed laboratories] and tetanus toxoid [Santa Cruz biotechnology] are made are detected.HER2- is special Property IL-4 and IL-10 production is determined, they are II types etc., ELISPOT described in breast cancer research by Datta, J. T aids in (Th2) and the T- cells (T of scalability_reg) function substitute.

Anti- HE2 Th1 reactivity is determined using the empirical method described in reference example.Subtract no stimulated After background, the positive/reactive response to single HER2 peptides is defined as in test hole >=20 spot formation cells [SFC]/2x10⁵PMBC.Determine the measurement of three anti-HER2 Th1 responses：(a) response rate is (at least to 6 kinds of peptides In a kind of Proportion of patients responded), (b) group storehouse (average number of reactive peptide) and (c) whole 6 kinds of peptides in accumulation Response (SFC/10⁶Cell).

Flow cytometry

The suspension PMBC in FACS buffer solution (PBS+1%FCS+0.01% azide).Such as Datta, J., etc., described in breast cancer research, mouse Anti-Human class CD3, CD4, CD25, T-bet are conjugated using PE/FITC/Cy5-, GATA-3, IFN-γ, or determine relative PBMC immunophenotypes with compare (flow cytometer) of subclass matching.Said according to manufacture Bright book fixes/intracellular dyeing of the permeabilization instrument (antibody) to anti-FoxP3 with FoxP3.Use BD LSR-II hemacytometers Implement analysis, and data analysis is carried out using CellQuest Pro softwares.

Th1 contributes compared to the feature of Th2 hypotypes

DMEM+5% human serums are with 1.2x10 in 24 orifice plates⁶PMBC is resuspended in cell/mL, with HE2-II classes Peptide mixer (24 μ g/mL) enters horizontal pulse.Do not have stimulated and anti-CD3/CD28 antibody-pulse periphery from each donor Blood monocyte each acts as negative and positive control.Hatch 6 hours at 37 DEG C, protein transport is suppressed into Brefeldin- A(Sigma Aldrich；10 μ g/ml) it is added to each sample, it is incubated overnight.After washing, contaminated at room temperature with anti-human CD4 Color 30 minutes.According to operation instructions, then wash cell twice ,/permeabilization kit (Billegend) is fixed using Foxp3 Be fixed with it is penetrating, and with anti-T-bet, anti-GATA-3 and anti-IFN-γ (Biolegend) dye 30 minutes.Hatched Afterwards, cell is washed, and is analyzed in BD LSR-II hemacytometers.

Statistical analysis

Descriptive statistic summarizes distribution and the immune response variable of patient characteristic.Do not recur and recur HER2^pos- Contrast between IBC groups is by hereafter being implemented：(1) 2- groups/unit-variable analysis shows-unpaired Student ' s t- tests are (continuous Parameter), Wilcoxon rank-sum tests (discontinuous parameter), and Chi-square Test (absolute)；(b) 2 groups of tests of ＞-use because Single variance analysis of fruit relation statistical analysis mode.P ＜ 0.1 variable unit-variable analysis shows enter forward, and multivariable logic is returned Return model related (binary outcome Yes/No) with the independence for determining to recurring (for entering p ＜ 0.05).

The science of law is sent out by Kaplan-Meier and detects single argument disease-free survival (" DFS ") evaluation, should by anti-HE2 Th1 Answer rate and other covariants are classified.The non-observation (subsequently minimum 24 months) for returning patient is in last known follow-up progress Examine.In order to analyze the moment risk of all variables and different subsequent controls, implement Cox proportional hazards models.To vacation If Cox models assessed, including reciprocation and the harm ratio with the time.P ＜ 0.05 are statistically considered as It is significant.All tests are two-sided.Analysis (IBM Corporation Armonk, NY) is completed with SPSS v22.

As a result

Patient characteristic

The demography and tumour correlated characteristic of whole groups (n=95) is described in detail in table 3 below.Treated in T+C Patient (n=73), median ages are 49 years old (scope, 24-85), and majority is white people's (87.5%).Most patients have Estrogen/PgR-the positive (ER/PR^pos) tumour (58.9%), and show Locally Advanced/lymph node positive disease and (face Bed stage I：9.6%, II：47.9%, III：42.5%).In 37 (50.7%) patients, implement lower rectal cancer T+C.Letter The most frequent execution (53.4%) of single or improvement radical cure mastectomy, and adriamycin/endoxan/taxol/He Sai Spit of fland is generally utilized (57.5%) in T+C schemes.

Table 3：The demography and tumour correlated characteristic of study population.The age of study population, race, AJCC pathology Stage, hormone receptor status, and the time from completion trastuzumab (when applicable).

* all groups of clinical stage is shown

Abbreviation：HER2^pos：HEGF

It is overexpressed；IBC：Infiltrative breast carcinoma；T+C：Trastuzumab and chemotherapy, AJCC：American cancer federation； ER：ERs；PR：PgR

Table 4 below shows local recurrence, and distal end is recurred, or both 25 (34.2%) patients for having；These patients In most of (n=21；84%) the Th1 responses of patient are measured in relapse diagnosis.

Abbreviation：T+C：Trastuzumab and chemotherapy；ER：ERs；PR：Flavones acceptor；Neoadj is newly aided in Treatment；

Adj：Adjuvant；MRM：Modified radical mastectomy；Simple：Simple mastectomy；BCS：Breast conservation surgery； ALND：Lymph node excision；Trast：Trastuzumab；AI：Aromatase inhibitor；DC1：1 type polarization BMDC epidemic disease Seedling.

Anti- HER2 CD4⁺Th1 responses are lowered in the HER2pos-IBC patient of recurrence

In Figure 15 A, at untreated before (n=22), the non-recurrence (n=48) of T+C treatments, and T+C treatment recurrences are suffered from Anti- HER2 Th1 response rates are carried out between person (n=25), organize the comparison of storehouse and cumulative acknowledgements.Obtained from untreated patient before Anti- HER2 Th1 responses as immune " baseline ", compared with the patient of no recurrence, in the HER2 recurred^pos- IBC suffers from Anti- HER2 Th1 response rate (untreateds before are observed in person respectively：36.4% compared to recurrence：8.0% compares non-recurrence： 83.3%, P0.0001) (top), organize storehouse (0.6 ± 0.2 compares 1.5 ± 0.2, PO.0001 compared to 0.1 ± 0.1) (centre), and Cumulative acknowledgements (32.8 ± 4.7 compare 80.2 ± 11.0, P ＜ 0.0001 compared to 14.8 ± 2.0) (bottom) are significantly lowered.

In Figure 15 B, CD28 anti-to anti-CD3/ stimulates (P=0.57), tetanus toxoid (P=0.41) and false silk ferment The IFN-γ of female (P=0.74)⁺Response respectively it is non-recurrence and recurrence group be not significantly different, imply that observe anti-HER2 Th1 differences are not due to caused by the horizontal T- cell tolerances of host in unsuitable immune or recurrence patient.

The downward of the anti-HER2 T- cell responses of patients with recurrent is attributed to Th1 rather than Th2 or T_regPhenotype

Figure 15 C show the PMBC that HER2- is stimulated, and T-bet is co-expressed to it using flow cytometer (Th1 transcription factors (see, Szabo, S.J., et al, science 295 (5553)：338-342 (2002)) or GATA-3 (Th2 transcriptions The factor is (see Zheng, W., etc. cell 89 (4)：587-596 (1997)) (top graph) and intracellular IFN-γ assessed.With Non- recurrence group is compared, the special CD4 of the HER2- significantly lowered⁺bet⁺IFN-γ⁺(0.25 ± 0.1% compares 0.02 ± 0.01%, p =0.039) phenotype rather than CD4⁺GATA-3⁺IFN-γ⁺Or CD4⁺GATA-3⁺IFN-γ^-Phenotype ratio in recurrence group In be observed (centre).HER2- stimulates IL-4⁺Response rate (P=1.00), organize storehouse (P=0.84) and cumulative acknowledgements (P= 0.46) measurement of-Th2 functions-it is non-recurrence and recurrence group (bottom) be not different (bottom).

In addition, Figure 15 D show circulation T_reg(that is, CD4⁺CD25⁺Foxp3⁺) (1.3 ± 0.3 compare 1.6 ± 0.4, P to phenotype =0.61) absolute ratio is not different in non-recurrence and recurrence group respectively；HER2- stimulates IL-10⁺Response rate (P= I.00), storehouse (P=0.74) and cumulative acknowledgements (P=0.66)-T is organized_regThe measurement of function-between two groups is similar.

Anti- HER2 Th1 response rates are independent related to disease-free survival

By the control of the interference factor from related demography/clinical pathologic characteristic check anti-HE2 Th1 responses and Independent correlates between recurrence.Once unit-variable analysis shows, the group of recurrence and non-recurrence is in age, menopausal state, race, body Performance figure, disease, clinical stage, in the hormone receptor stage, LVI be present, cell Nuclear grading or whether need mastectomy not have Have any different.However, (P=0.006) still has in the patient for receiving adjuvant T+C (P=0.21) and after lower rectal cancer In the patient of residual disease, recurrence is more frequent.When by these correlated variables of multiple variables rehabilitation control, as shown in table 5, The order of anti-HER2 Th1 response rates rather than T+C treatment (P=0.304) keeps independent related to recurrence.

Figure 16 is shown after in (IQR31) moon of subsequent median 44, and analysis group is carried out by anti-HER2 Th1 response rates Classification, compared with Th1 response patients, the patient of the non-responses of Th1 demonstrates disease-free survival rate (" DFS ") (intermediate value being significantly deteriorated 47 compare 113 months, P ＜ 0.0001)；These correlations obtain the Hazard ratio of the non-response groups of confirmation-Th1 by Cox models： 15.2,95%CI 3.5-66.7 (P ＜ 0.001).

Discuss

In this analysis, anti-HER2 CD4 are circulated⁺Th1 responses are proved to be a kind of new and are immunized, and it is being controlled completely Treat HER2^posIt is interrelated with breast cancer relapse in-IBC patients.The anti-HER2 t cell responses of the downward of patients with recurrent are not Caused by the unsuitable immune or horizontal T- cell tolerances of host, it is mainly by Th1 drivings, rather than by Th2 Or T_regPhenotype driving.When controlling related demography/clinical pathological factors, anti-HER2 Th1 response rates are with recurring independently It is analytically related in Risk Adjusted；Compared with Th1 response patients, the non-response patients of Th1 are proved to have the DFS being significantly deteriorated.See Come this be first prove palindromia and host it is horizontal it is immune between correlation with it is carcinogenic in the generation of known tumor of breast Driving specificity is related, i.e. HER2/neu.

In nearest evidence interesting data suggest-in adjuvant (Perez, E.A., et al, J.Clin.Oncol.33 (7)：701-708 (2015) DOL 10.1200/JCO.2014.57.6298) (" Perez, is waited in both ") and lower rectal cancer (Gianni, L., etc. Cancer Res.72 (24Supplement)：S6-7 (2012)), from trastuzumab be benefited may by It is limited to immunity enrichment tumor group, wherein Th1 genes IFN-γ/TNF-α has leading role.See, Perez, etc..Altogether, seemingly The tumor levels Th1 gene expressions and the defective anti-HER2 Th1 of circulation lacked, which is immunized, may imply that HER2 targetings are controlled Failure in treatment.These observations may partly explain that evidence shows that Th1 cell factors IFN-γ/TNF-α exists with external evidence Trastuzumab-treatment HER2 is promoted to be overexpressed the special CD8 of HER2- of breast cancer⁺It is particularly important in the targeting of T cell mediation 's.See Datta, J., et al, Yale J.Biol.Med.87 (4)：491-518(2014)；Datta, J., etc., Front.Immunol.6：271(2015)；And Datta, J., etc. cancer immunity .Res.3 (5)：455-463(2015).For The HER2 treated completely^pos- BC patients monitor the fluctuation during anti-HER2 Th1 are immunized in real time, are lost accordingly, it is possible to disclose in clinical Lose the vulnerable crowd of risk.Such immunologic surveillance may be not only to supply current radiography, can also Crucial factor, such as the immunologic intervention that anti-HER2 Th1 are oriented to are determined for therapeutic intervention.For example, Datta, etc. breast cancer is ground Study carefully.

It is immune whether complete for the anti-HER2 Th1 of these patients because immune response is detected in palindromia Keep lowering after into instruction trastuzumab treatment or decline simultaneously with the development of recurrence, be still not clear.In future The Longitudinal Surveillance that HER2 targets anti-HER2 Th1 responses before or after scheme is completed in clinical test is desirable.Also its He must be mentioned limits value.Firstly, since the research and design property of this test examples, discovery here should be interpreted to assume With the extensive confirmation of needs.Second, this research can not handle that (for example, Lapatinib, pa is appropriate with selectable HER2 targeting agents Pearl monoclonal antibody etc.) the anti-HER2 Th1 immune responses that occur after treatment.Finally, to the anti-HER2 Th1 immunologic surveillance cases of these patients Example is additional, and this research can not be that numerical threshold is established in described monitoring.Those skilled in the art can pay attention to no matter entering one The foundation for examining whether that numerical value thresholding can be caused of step, i.e. medical practitioner can follow prison with blood testing described herein Survey, and be possible to the patient that treatment promotes treatment with anti-HER2-, such as the anti-HER2 of BMDC described here Vaccine is to recover anti-HER2 response rates.

Sum it up, the anti-HER2 CD4 lowered⁺Th1 responses are a kind of novel immunes, and it is completed with HER2- targeted therapies HER2 afterwards^posThe recurrence of-IBC patients has correlation.Data in this test examples highlight is treating completely HER2^posImmunologic surveillance in-IBC patients is supplying the monitoring of existing radiography and is determining there is clinical pathology failure wind Effect in the fragile crowd of danger.

Comprehensive summing up, in the immune dynamics shown here between recurrence of the anti-HER2 Th1 of trastuzumab treatment Posterior circle Correlation, and enlighten in the HER2 treated completely^posImmunologic surveillance and therapeutic choice have prospect in-BC patients.HER2- sun Property patient with breast cancer can be middle and carry out blood count afterwards with their immune state of monitoring before treatment so that Obtain physician and other medical practitioners measure the risk of recurrence, and reduced again using the immune treatments of anti-HER2 are improved The risk of hair, for example, DC vaccines or other cancer vaccines.

Test examples 3

Response of the anti-HER2 Th1 responses in the lower rectal cancer chemotherapy in HER2 breast cancer patients with positive Evaluation is superior to breast dedicated magnetic and shaken radiography

In HER2 positive breast cancers (" HER2⁺BC ") lower rectal cancer chemotherapy (" nCT ") from 40-67%.Yuan Y, Deng Am.J.Radiol.195 (1)：260-268 (2010) realizes complete pathology response (" pCR ").Successive treatment breast core Magnetic resonance imaging (PMRI) is presently considered to be high specific (90.7%) but the gold criterion of sensitiveness reduction (63.1%). Hylton, N.M., etc. radiology 263 (3)：663-72(2012 6).Anti- HER2 Th1 responses are in HER2⁺BC Datta, J., It is related to pathology response after nCT treatments in et patient.Breast cancer research 17：71.doi：10.1186/s13058-015- 0584-1(2015 Nay 23).It can help to change successive treatment with pCR identification patients.This guides new auxiliary to control for exploitation The sensitive instrument for the treatment of is vital.In this research will post processing MRI and anti-HER2 Th1 responses contrasted with HER2⁺Complete pathology response is assessed in BC patient.

Method

In Univ Pennsylvania USA, 30 patients look back IBC the and HER2/neu mistakes for thinking to confirm in institutional framework Expression.In any patient not evidence suggests far-end transfer and without reception immunosuppressive drug.With anti-HER2 TH1 points The patient of analysis is implemented with non-prejudice type.Collect original post processing MRI reports and by for pCR and immune response not The mammary gland radiologist of understanding is evaluated imaging.Measured RECIST standards, assessing the tumour based on imaging should Answer, thus it is similar to entity damage, improve with the non-substantial increase comprising assessment.In the mastectomy sample of pathologic finding excision With the lymph node (that is, ypTO/Tis ypNO) of sampling, complete pathology response is defined as lacking invasive cancer residual.Such as With reference to the anti-HER2 CD4 of circulation described by example, checked in not having stimulated PMBC⁺Th1 responses are used Carry out MHC II classes peptide (peptide 42-56, peptide 98-114, peptide 328-345, peptide 776-790, the peptide in exogenic six kinds of HER2 sources 927-941, and peptide 1166-1180) (serial ID NOS：1-6) pulse, surveyed by enzyme-linked disposable adsorption measurement (ELISPOT) The secretion of IFN-γ is determined to determine cumulative acknowledgements.Implemented ELISPOT with reference to as described in example.MRI and anti-HER2 Th1 responses with The diagnostic criteria of pathology response and calculating measurement is related.

The most frequently used therapeutic scheme is adriamycin/endoxan/taxol/Trastuzumab.The most frequently used operation intervention is breast Room resection.The characteristics of patient, is being illustrated below：

As a result

The regularity of distribution of the HER2 cumulative acknowledgements of subject with complete pathological reaction (" PathCR ") is 13 patients With PathCR (average value ± SD=167.0 ± 115.6SFC/10⁶Cell, minimum value=53.0SFC/10⁶, maximum= 418.0SFC/10⁶Cell), 17 patients do not have PathCR (average value ± SD=23.9 ± 15.2SFC/10⁶Cell, minimum value =0.4SFC/10⁶, maximum=53.3SFC/10⁶Cell).With 50SFC/10⁶Cell is cut point, and patient is by two points (" low " ＜ 50, " height " >=50).

The diagnostic characteristic of MRI and HER2 cumulative acknowledgements in HER2 patient is being illustrated below.PPV=positive predictive values； NPV=negative predictive values

1.MRI results compare pathology results (golden standard), N=30 patient

A.13 it is individual that there is PathCR

B.17 individual no PathCR

2.ER is negative, N=11 patient

A.8 it is individual that there is PathCR

B.3 individual no PathCR

3.ER is positive, N=19 patient

A.5 it is individual that there is PathCR

B.14 individual no PathCR

4. the HER2_ cumulative acknowledgements result of binary compares pathology results (golden standard), N=30 patient

A.13 it is individual that there is PathCR

B.17 individual no PathCR

C.16 it is individual to be classified with " The low response " ＜ 50

D.14 it is individual to be classified with " high response " >=50

5.ER is negative, N=11 patient

A.8 it is individual that there is PathCR

B.3 individual no PathCR

C.3 it is individual to be classified with " The low response " ＜ 50

D.8 it is individual to be classified with " high response " >=50

6.ER is positive, N=19 patient

A.5 it is individual that there is PathCR

B.14 individual no PathCR

C.13 it is individual to be classified with " The low response " ＜ 50

D.6 it is individual to be classified with " high response " >=50

7. assessing MRI (" new MRI ") result recently compares pathological examination (golden standard), N=30 patient

A.13 it is individual that there is PathCR

B.17 individual no PathCR

8.ER is negative, N=11 patient

A.8 it is individual that there is PathCR

B.3 individual no PathCR

9.ER is positive, N=19 patient

A.5 it is individual that there is PathCR

B.14 individual no PathCR

Following table summarizes the data of all patients：

As shown above, compared to anti-HER2 Th1 responses, original pMRI has much lower pCR diagnostic results. In the subgroup that blind 28 patients for commenting pMRI form, pCR diagnostic result is substantially inferior to anti-HER2 Th1 response (susceptibilitys 46.2% compares 100.0%, and specificity 64.7% compares 94.1%, and overall accuracy 56.7% is compared to 96.7%).When patient according to Similar result is able to observe that during the state classification of ER, refers to the data of above-mentioned numbering 2,3,4,7 and 9.

Conclusion

Compared with the MRI after treatment, anti-HER2 Th1 immune responses show obvious accurate diagnostic metrics.Assessing In HER2+BC pCR, the presence of high anti-HER2 Th1 responses is better than the use of MRI after treatment.The general technology people of this area Member can utilize the HER2 for receiving NACT in assessment using method provided herein⁺During the response of BC patient, patient is monitored Immune response.

Each and each patent of the disclosure, patent application and the publication quoted herein pass through entire contents It is incorporated herein by reference.

This application provides the description of this invention for the purpose that illustrates and describe, but have no intention exhaustive or limit System.Many modifications and variations will become readily apparent to those skilled in the art.Embodiment is chosen and description is so as to best Principle and practical application is explained, and causes others of ordinary skill in the art's understanding there are the various embodiments of different amendments Open, these amendments are suitable to the use specifically considered.

Although embodiment illustrated of the present invention is described herein referring to the drawings, it is to be understood that of the invention and unlimited In these definite embodiments, those skilled in the art can be carried out each on the premise of without departing substantially from the scope or spirit of the invention The other changes of kind and change.The appended claims of the present invention are it is generally understood that to cover in all such embodiments and equal Change.

Claims (23)

1. diagnosis or treatment suffer from cancer or have the method for the mammalian subject for developing into risk of cancer, including：

CD4 from antigen presenting cell (" APCs ") or its precursor and in the sample from subject's blood⁺T- is thin Born of the same parents generation causes interferon gamma (" IFN-γ ") secretion circulation anticancer CD4⁺Th1 responses；The described anticancer CD4 with detection⁺ Th1 responses are to determine whether the response lowers.

2. according to the method for claim 1, wherein further comprising the step of described generation：

Unexpanded PMBC (" PBMCs ") is separated from the blood sample；With

With the PMBC described in composition pulse and APC- precursors monocyte therein, the composition includes base In the immunogenicity II class MHC binding peptides for making the haunted cancer types of subject, so as to activate the CD4 in the PBMC⁺ Th1 cells are with secretion of gamma-IFN；With

The detecting step includes the secreted IFN-γ of detection.

3. according to the method for claim 1, wherein further comprising the step of described generation：

The CD4 that will be purified in subject's sample⁺T cell with it is from subject's sample, with the APC of composition pulse not Ripe or ripe BMDC (" DCs ") co-incubation, so as to activate CD4⁺T cell is with secretion of gamma-IFN, the combination Thing is included based on the immunogenicity II class MHC binding peptides for making the haunted cancer types of subject；With

The detecting step includes the secreted IFN-γ of detection.

4. according to the method for claim 1, wherein the cancer is selected from by breast cancer, the cancer of the brain, carcinoma of urinary bladder, the cancer of the esophagus, lung Cancer, cancer of pancreas, liver cancer, prostate cancer, oophoroma, colorectal cancer and stomach cancer or its be combined formed group.

5. according to the method for claim 4, wherein the cancer is expression HER2.

6. according to the method for claim 2, wherein the cancer is HER2- positive breast cancers, the subject is the mankind Women, and the immunogenicity II classes MHC binding peptides are based on HER2 molecules.

7. according to the method for claim 3, wherein the cancer is HER2- positive breast cancers, the subject is the mankind Women, and the immunogenicity II classes MHC binding peptides are based on HER2 molecules.

8. according to the method for claim 6, wherein described composition further comprises HER2 II class MHC binding peptides, HER2 II class MHC binding peptides include：

Peptide 42-56：HLDMLRHLYQGCQVV(SEQ ID NO：1)；

Peptide 98-114：RLRIVRGTQLFEDNYAL(SEQ ID NO：2)；

Peptide 328-345：TQRCEKCSKPCARVCYGL(SEQ ID NO：3)；

Peptide 776-790：GVGSPYVSRLLGICL(SEQ ID NO：4)；

Peptide 927-941：PAREIPDLLEKGERL(SEQ ID NO：5)；With

Peptide 1166-1180：TLERPKTLSPGKNGV(SEQ ID NO：6).

9. according to the method for claim 7, wherein the composition further comprises HER2 II class MHC binding peptides, HER2 II class MHC binding peptides include：

Peptide 42-56：HLDMLRHLYQGCQVV(SEQ ID NO：1)；Peptide 98-114：RLRIVRGTQLFEDNYAL(SEQ ID NO：2)；With peptide 328-345：TQRCEKCSKPCARVCYGL(SEQ ID NO：3)；Peptide 776-790：GVGSPYVSRLLGICL (SEQ ID NO：4)；Peptide 927-941：PAREIPDLLEKGERL(SEQ ID NO：5)；With peptide 1166-1180： TLERPKTLSPGKNGV(SEQ ID NO：6).

10. according to the method for claim 1, wherein the secretion of the IFN-γ is by IFN-γ enzyme-linked immunospot assay (" ELISPOT ") measure.

11. recover HER2- specific Cs D4 in the HER2- breast cancer patients with positive for have demand⁺The method of Th1 immune responses, bag Include：

To the dendritic cell vaccine of patient therapeuticallv's effective dose, the dendritic cell vaccine includes using immunogenicity The autologous dendritic cell (" dendritic cell vaccine inoculation ") of HER2 II class MHC binding peptide pulses is to improve the patient The CD4 of anti-HER 2⁺Th1 responses；With

It is described before and after patient vaccination's dendritic cell vaccine to determine according to the method for claim 8 Anti- HER2 CD4⁺Th1 responses are to determine the incrementss in the response.

12. recover HER2- specific Cs D4 in the HER2- breast cancer patients with positive for have demand⁺The method of Th1 immune responses, bag Include：

To the dendritic cell vaccine of patient therapeuticallv's effective dose, the BMDC includes using immunogenicity HER2 The autologous dendritic cell (" dendritic cell vaccine inoculation ") of II class MHC binding peptide pulses is to improve the anti-of the patient HER 2 CD4⁺Th1 responses；With

According to the method for claim 9 come determine the patient receive dendritic cell vaccine inoculation before and after institute State anti-HER2 CD4⁺Th1 responses are to determine the incrementss in the response.

13. according to the method for claim 11, further comprise：

Connect by implementing the method described in claim 8 at one or more additional period intervals to determine dendritic cell vaccine The recovery state of the CD4+Th1 responses of the patient after kind, recovered with monitoring the response.

14. according to the method for claim 12, further comprise：

It is inoculated with to determine dendritic cell vaccine by the method for implementing claim 9 at one or more additional period intervals The CD4 of the patient afterwards⁺The recovery state of Th1 responses, recovered with monitoring the response.

15. mammary gland or the method for other cancers are screened for individual, including：

Detect the individual anti-HER2 CD4 according to the method for claim 1⁺Th1 responses, to determine and health Whether body phase ratio, the response lower.

16. screening has the individual method for developing into breast cancer or other risk of cancer, including：

Detect the individual anti-HER2 CD4 according to the method for claim 1⁺Th1 responses, to determine and health Whether body phase ratio, the response lower.

It is good 17. whether prediction HER- breast cancer patients with positive treatment such as chemotherapy and trastuzumab nonimmune to standard replys Good method, including：

Detect the anti-HER2 CD4 of the patient according to the method for claim 1⁺Th1 responses.

18. in positive-aggressive the breast cancer (" HER2 of HER2- treated with chemotherapy and trastuzumab^pos- IBC ") in patient The method for predicting new mammary gland event, including：

Determine the anti-HER2 CD4 of the patient according to the method for claim 1⁺Th1 responses, to determine the response Whether lower.

19. predict HER2- breast cancer patients with positive HER2- after new auxiliary trastuzumab and regimen chemotherapy (" T/C ") The method of the pathology response of positive breast cancer, including：

According to the method for claim 1 anti-HER2 CD4 are determined in the patient after T/C treatments⁺The degree of Th1 responsiveness, With determine the response whether be with the complete response of lower rectal cancer pathology (after surgery in pathology noresidue wellability mammary gland Cancer) related notable elevated anti-HER2 CD4⁺Th1 responses, or the relatively low of right and wrong pathology complete response correlation are answered Answer.

20. the method according to claim 11, wherein：In the case of the non-pathology complete response of the patient, institute State the anti-HER2 CD4 of patient⁺Dendritic cell vaccine inoculation in the Th1 response method according to claim 11 is able to Recover.

21. diagnosis or treatment suffer from cancer or have the method for the mammalian subject for developing into risk of cancer, including：

Blood is obtained from the subject；

In conjunction with inhibition, blood testing is carried out, anticancer CD4 is determined based on it⁺Suppression in Th1 responses；

From selected from normal by the cancers such as dendritic cell vaccine, trastuzumab targeting cancer therapy, chemotherapy, operation and radiation etc. The cancer drug of effective dose in the group that rule cancer therapy is formed is applied to the subject.

22. monitor HER2 after the targeting additional chemotherapy of breast cancer treatment is completed^posThe method of-IBC patients, to assess The risk of cancer return is stated, including：Determine the anti-HER2 CD4 of patient after the treatment according to the method for claim 1⁺The degree of Th1 response rates with determine the response whether be the notable downward related to the recurrence of the cancer anti-HER2 CD4⁺Response or whether the anti-HER2 CD4 of the related up-regulation of right and wrong recurrence⁺Th1 responses.

23. according to the method for claim 22, wherein the targeted therapy, which is included to the patient, imposes the bent appropriate list of medicine It is anti-.