CN107281474A

CN107281474A - Strengthen the New Immunity Strategy and immune composition of anti tumor immune response

Info

Publication number: CN107281474A
Application number: CN201610222062.6A
Authority: CN
Inventors: 冷启彬; 郭昵宁; 陈宪洋; 胡广
Original assignee: Institut Pasteur of Shanghai of CAS
Current assignee: Institut Pasteur of Shanghai of CAS
Priority date: 2016-04-11
Filing date: 2016-04-11
Publication date: 2017-10-24
Also published as: WO2017177910A1

Abstract

The invention provides the New Immunity Strategy and immune composition of enhancing anti tumor immune response.Specifically, the inventive method includes step：(a) the first tumour antigen and the second tumour antigen are provided, wherein described enhancing tumor immunity is tumor immune response enhanced, for the first tumour antigen；First tumour antigen is low immunogenicity；Second tumour antigen is and first tumor antigens are homologous and the polypeptide intersected or its immunogenic fragments；(b) by the immune object for being applied to described mammal of the second tumour antigen, the immune response for second tumour antigen is excited；(c) the first described tumour antigen is immunized and is applied to the described mammalian object excited through initial immunity, so as to produce the enhanced tumor immune response for being directed to the first tumour antigen.Immune composition and purposes for the inventive method is also provided.

Description

Strengthen the New Immunity Strategy and immune composition of anti tumor immune response

Technical field

The invention belongs to field of immunology；More particularly, to the New Immunity Strategy of enhancing anti tumor immune response And immune composition.

Background technology

Tumour is that current serious threatens human health and causes the principal disease of human death.Effective cancer is researched and developed to control It is imperative to treat medicine.

Therapeutic tumor vaccine can produce immune response, specificity to tumour antigen by human activin immune system Remove solid tumor tissue and form immunological memory and lifelong protection is provided patient.With traditional Cancer Treatment Regimens (surgery excision, radiotherapy and chemotherapy) and other immunization therapy modes are compared, and therapeutic tumor vaccine is made For a kind for the treatment of method of active, it is more easy to use with patient, and strong side effect will not be caused.

Although the research of therapeutic tumor vaccine has been achieved for certain progress, but totally apparently, obtain synthesis and face Bed experimental result data is still unsatisfactory.Low clinical efficacy is the subject matter faced during vaccine is researched and developed.2003 Before year, at least 200 kinds vaccines have carried out second phase or the clinical trial of three phases, and most of vaccine therein is facing It is proved to effectively to activate the specific for tumour antigen T cell in patient's body in bed experiment and produces immune response suppression Tumour growth processed, only 2.6% patient generates antineoplastic immune under vaccine effect

Tumour antigen has the problem of immunogenicity is poor as autoantigen always, have impact on therapeutic tumor vaccine Application and development clinically.

One major reason of influence therapeutic tumor vaccine validity is central tolerance mechanism to antineoplastic immune The inhibitory action of reaction.Central tolerance mechanism refers to the T cell of high-affinity during thymus development because contacting it The autoantigen of specific recognition and by Solid phase mechanism remove caused by immune tolerance.

Although developing multiple technologies in the prior art, such as using adjuvant, the tumour antigen using mutation, to Enhancing anti tumor immune response level is hoped, but the therapeutic effect in clinical trial is not obvious.

In summary, this area still lack satisfactorily, the tumor vaccine with efficient antitumous effect.Cause This, this area in the urgent need to exploitation it is new can effectively strengthen the method and composition of anti tumor immune response, so as to Obtain preferably antitumor prevention or therapeutic effect.

The content of the invention

It is an object of the invention to provide a kind of method and composition for strengthening anti tumor immune response and its application.

In the first aspect of the present invention, there is provided a kind of non-therapeutic or curative enhancing anti tumor immune response Method, including step：

(a) the first tumour antigen and the second tumour antigen are provided, wherein described enhancing tumor immunity is enhancing , tumor immune response for the first tumour antigen；

The first described tumour antigen is the natural polypeptides of the low immunogenicity from a mammal or it is immune Immunogenic fragment, and when first tumour antigen is immunized and is applied to described mammal, it is impossible to effectively production The raw anti-tumor immune response for being effectively directed to first tumour antigen；And

The second described tumour antigen is and first tumor antigens are homologous and the polypeptide intersected or it is immune Immunogenic fragment；

And first tumour antigen and the antigentic specificity CD8 from the mammal⁺T cell it is affine Power is Q1, second tumour antigen and the antigentic specificity CD8 from the mammal⁺T cell it is affine Power is Q2, and the ratio R 1 of the Q2/Q1 is 0.02-0.80；

(b) by the immune object for being applied to described mammal of the second described tumour antigen, excite for described The immune response of second tumour antigen, so as to obtain the mammalian object excited through initial immunity；

(c) applied in the immune of previous step afterwards after t days, wherein t is 3-60, by the first described tumour Antigen immune is applied to the described mammalian object excited through initial immunity, excites anti-for first tumour Former immune response, so that the mammalian object through immune stimulating again is obtained, wherein swashing in described be immunized again In the mammalian object of hair, the enhanced tumor immune response for being directed to the first tumour antigen is generated.

In another preference, described " the enhanced tumor immune response for being directed to the first tumour antigen " refers to and used First tumour antigen carries out initial immunity and applies and use first tumour antigen that the control applied is immunized again In the horizontal Yc of tumor immune response for the first tumour antigen compare, resisted with second tumour in step (c) Original carries out the pin that initial immunity applies and used first tumour antigen to be immunized again in the mammalian object applied Yc is significantly higher than to the horizontal Yv of tumor immune response of the first tumour antigen.

In another preference, described " Yv is significantly higher than the ratio R 2 >=1.5 that Yc " refers to Yv/Yc, preferably Ground >=2, more preferably >=5, more preferably >=10, most preferably >=20.

In another preference, described tumor immune response level is special for antigen in immune mammalian T cell Different in nature CD8⁺The quantity and/or ratio of T cell.

More preferably, described tumor immune response level includes：Mammal T is stimulated with the first antigen in vitro The secretion level of cell factor (such as interferon gamma) is detected after cell, or uses major histocompatibility complex (MHC) tetramer or polymer of-antigen epitope polypeptide are through flow cytometry analysis antigentic specificity CD8⁺T cell Quantity and ratio.

In another preference, described t is 4-45, and preferably t is 5-30, and more preferably t is 6-25, most Good ground t is 7-21.

In another preference, described Q2/Q1 ratio R 1 is 0.04-0.60, preferably 0.05-0.50, More preferably it is 0.06-0.40.

In another preference, the affinity Q1 of the first tumour antigen and T cell 9-100 nanomoles (uM) it Between, and/or the second tumour antigen and the affinity Q2 of T cell are between 0.01-10 nanomoles.

In another preference, described affinity is defined as follows：MHC- antigen epitope polypeptides and φt cell receptor Between the intensity that combines.

In another preference, described affinity is functional affinity or IC₅₀Inverse.

In another preference, described functional affinity refers to, the secretion of antigen epitope polypeptide stimulated in vitro T cell Cell factor (such as interferon gamma) reach the 50% of highest level required for peptide concentration.In another preference, Described " can not effectively produce effective antitumour immune response " refers to is applied to the lactation in described be immunized During animal, anti-tumor immune response rate≤25% for first tumour antigen is produced, preferably≤15%, more Goodly≤10%, more preferably≤5%, most preferably≤3%.

In another preference, the first described tumour antigen is that the wild type tumor from the mammal is related The epitope polypeptide (i.e. polypeptide fragment) of antigen (albumen).

In another preference, described " homologous and intersection " refers to described the second tumour antigen and described First tumour antigen has average 1-2 amino acid mutation on every 10 amino acid lengths, therefore can not only Cause the immune response for the second tumour antigen, and the intersection for first tumour antigen can be caused Immune response.

In another preference, the first described tumour antigen and the second tumour antigen are derived from consistent swollen Knurl related antigen.

In another preference, the first described tumour antigen and the second tumour antigen are derived from consistent swollen The same section of knurl related antigen.

In another preference, the first described tumour antigen and the second tumour antigen are that length is respectively n1 and n2 The epitope polypeptide of individual amino acid, wherein n1 and n2 are respectively 6-200, preferably 7-100, more preferably 8-50, Most preferably 8-25 or 8-12 positive integer.

In another preference, described mammal includes non-human mammal and people.

In another preference, described mammal includes Primate and rodent (such as mouse, rat).

In another preference, the first described tumour antigen is the tumor specific epitopes of central tolerance.

In another preference, the first described tumour antigen derives from the tumor associated antigen being selected from the group： NY-ESO-1, Her2, EGFR, CEA, GPC3, AFP, PAP, PSA, PSMA, PSCA or its combination.

In another preference, described " immune to apply " refers to by described tumour antigen or by the tumour antigen The BMDC (DC) of sensitization is directly or indirectly applied to the object, so as to trigger immune response.

In another preference, described " immune to apply " includes being administered in the form of vaccine combination.

In another preference, described vaccine combination contains：(i) (such as the first tumour resists the tumour antigen described in Former or the second tumour antigen) and/or by the BMDC of the tumour antigen sensitization；(ii) optional adjuvant；With (iii) acceptable carrier pharmaceutically or in immunology.

In another preference, described BMDC derives from same lactation with the first described tumour antigen Animal.

Strengthen the composition product of anti tumor immune response, the production there is provided a kind of in the second aspect of the present invention Product include：

(1) first chamber, described first chamber includes (i) the first tumour antigen and/or swollen by described first The BMDC of tumor antigen sensitization；(ii) optional adjuvant；(iii) is acceptable pharmaceutically or in immunology Carrier；

(ii) second chamber, described second chamber includes (i) the second tumour antigen and/or by described second The BMDC of tumour antigen sensitization；(ii) optional adjuvant；(iii) pharmaceutically or in immunology is subjected to Carrier；

Also, described first chamber and second chamber be it is independent,

Wherein, the first described tumour antigen be from a mammal low immunogenicity natural polypeptides or its Immunogenic fragments, and when first tumour antigen is immunized and is applied to described mammal, it is impossible to have Effect produces the anti-tumor immune response for being effectively directed to first tumour antigen；And

And first tumour antigen and the antigentic specificity CD8 from the mammal⁺T cell it is affine Power is Q1, second tumour antigen and the antigentic specificity CD8 from the mammal⁺T cell it is affine Power is Q2, and the ratio R 1 of the Q2/Q1 is 0.02-0.80,

And described enhancing tumor immunity is tumor immune response enhanced, for the first tumour antigen.

In another preference, described composition product is the knurl seedling or vaccine combination for treating or preventing tumour Thing.

In another preference, described vaccine combination also contains adjuvant.

In another preference, the vaccine combination is nucleic acid vaccine combination.

In another preference, described adjuvant includes aluminum oxide, saponin(e, muramyl dipeptide, mineral oil or plant Oil, the adjuvant based on vesica, non-ionic block copolymer or deae dextran, cell factor (including IL-1, IL-2, IFN-r, GM-CSF, IL-6, IL-12 and CpG).

In the third aspect of the present invention there is provided the purposes of the composition product described in second aspect of the present invention, they It is used for the medicine for preparing enhancing anti tumor immune response.

In another preference, the medicine is used to treat the tumour being selected from the group：Liver cancer, lung cancer, stomach cancer, breast Gland cancer, oophoroma, prostate cancer, cutaneum carcinoma, melanoma, cervical carcinoma, the cancer of the brain, thyroid cancer and cholangiocarcinoma, Carcinoma of urinary bladder and cancer of pancreas.

Select and be directed to enhancing there is provided a kind of (nondiagnostic and non-therapeutic) in the fourth aspect of the present invention The method of the candidate polypeptide immunogene of the anti tumor immune response of the tumour antigen of low immunogenicity, including step：

(a) test group is provided, the test group includes the i polypeptide immunogens for treating selection, wherein described is each Polypeptide immunogen is homologous and intersection, and wherein i is >=1 positive integer,

Also, described " homologous and intersection " refers to a polypeptide immunogen (Staphylococal Protein A or native antigen) and institute State another polypeptide immunogen (B antigens or close antigen) in test group has averagely on every 10 amino acid lengths 1-2 amino acid mutation, therefore can not only cause for the immune response from antigen in mammal, and The immune response of the intersection for the close antigen can be caused；

Wherein, at least one is mutant polypeptides immunogene in described polypeptide immunogen；

(b) each polypeptide immunogen and the antigentic specificity CD8 each triggered are determined⁺The reaction parent of T cell And power, any positive integer in Qj, wherein j=1-i is designated as respectively；

(c) each Qj is ranked up, selected and sorted is located at middle polypeptide immunogen, as with enhancing For the candidate polypeptide immunogene of the anti tumor immune response of the tumour antigen of low immunogenicity；And/or

Each Qj and Qz is compared, the polypeptide immunogen that the ratio R for selecting the Qj/Qz is 0.02-0.80, As with enhancing for low immunogenicity tumour antigen anti tumor immune response candidate polypeptide immunogene, its In, Qz is first tumour antigen and the antigentic specificity CD8 from the mammal⁺T cell it is affine Power, and described the first tumour antigen be the low immunogenicity from mammal natural tumor antigens or its exempt from Epidemic disease immunogenic fragment, and when first tumour antigen is immunized and is applied to described mammal, it is impossible to effectively Produce the anti-tumor immune response for being effectively directed to first tumour antigen.

In another preference, in step (c), including：Each Qj is ranked up, by Qj most Big value is defined as Qmax, and selects the polypeptide immunogen that Qj/Qmax ratio R is 0.02-0.80, as with Candidate polypeptide immunogene of the enhancing for the anti tumor immune response of the tumour antigen of low immunogenicity.

In another preference, in step (c), including：Each Qj is ranked up, and selected and Q Arithmetic mean of instantaneous value Qaverage or median Qmean closest to (i.e. | Qaverage-Qj | or | Qmean-Qj | most It is small) s polypeptide immunogen, and s is ＜ i positive integer, and i is >=3 positive integers, and i-s >=2.

In another preference, s/i is 1/3 to 3/5.

In another preference, as i >=3, the polypeptide immunogen being selected do not include Qj it is minimum and Polypeptide immunogen maximum Qj.

In another preference, described Qj/Qz or Qj/Qmax ratio R are 0.04-0.60, preferably 0.05-0.50, is more preferably 0.06-0.40.

In another preference, described method also includes step：(d) candidate picked out for previous step Polypeptide immunogen, tests ability of its enhancing for the anti tumor immune response of the tumour antigen of low immunogenicity.

In another preference, in described step (d), including：The candidate polypeptide picked out with previous step is exempted from Epidemic focus HX, carries out initial immunity administration, so as to excite for the candidate polypeptide immunogene to the mammal HX immune response, so as to obtain the mammalian object excited through initial immunity；

And after described initial immunity applies operation after t days, wherein t is 3-60, by described first Tumour antigen is immune to be applied to the described mammalian object excited through initial immunity, excites swollen for described first The immune response of tumor antigen, and determine in the mammalian object through immune stimulating again for the first tumour antigen Tumor immune response level, so as to judge whether the candidate polypeptide immunogene HX there is enhancing to be directed to low immunogene The ability and/or enhanced degree of the anti tumor immune response of the tumour antigen of property.

In the fifth aspect of the present invention there is provided a kind of purposes of second tumour antigen, second tumour antigen is With the first tumor antigensIt is homologousAnd the polypeptide or its immunogenic fragments intersected, wherein the second described tumour resists Original be used to prepare a pharmaceutical composition, and described pharmaceutical composition (a) is used to strengthen mammal generation for described The anti tumor immune response of first tumor antigens；And/or (b) is anti-to itself for breaking through mammalian organism The immune tolerance of original simultaneously strengthens anti tumor immune response.

In another preference, described " homologous and intersection " refers to the first tumour antigen (Staphylococal Protein A) and second Tumour antigen (B antigens) has average 1-2 amino acid mutation in Matching band on every 10 amino acid lengths.

In another preference, second tumour antigen is the epitope polypeptide that length is n2 amino acid, wherein N2 is 6-200, preferably 7-100, more preferably 8-50, most preferably 8-25 or 8-12 positive integer.

Strengthen the treatment method of anti tumor immune response, including step there is provided a kind of in the sixth aspect of the present invention：

(a) lymphocyte from the separation from patient is provided, the lymphocyte is selected from the group：Tumors invading Lymphocyte (TIL), PBLC (PBMC) or its combination of profit；And the first tumour is provided Antigen and the second tumour antigen；

The first wherein described tumour antigen be from a mammal low immunogenicity natural polypeptides or its Immunogenic fragments, and when first tumour antigen is immunized and is applied to described mammal, it is impossible to have Effect produces the anti-tumor immune response for being effectively directed to first tumour antigen；And

The second described tumour antigen is and first tumor antigensIt is homologousAnd the polypeptide intersected or it is immune Immunogenic fragment；

(b) in the presence of the second tumour antigen, described TIL lymphocytes and/or PBMC lymphocytes are carried out Culture；Then in the presence of the first tumour antigen, cultivated again, so as to obtain the T cell through sensitization；

(c) optionally the lymphocyte of described sensitization is passed on, so as to obtain through passage, through sensitization T cell；

(d) T cell through sensitization for obtaining previous step, feeds back to patient.

In another preference, in step (b), described culture, which is additionally included in, is contained in BMDC, cell In the factor or the cultivating system of its combination, ex vivo T cell culture is carried out.

In another preference, after the neutralization of step (b), after the neutralization of step (c) or step (d) it It is preceding and among, methods described also includes：Detect the T cell through sensitization and the first tumour antigen and/or second The affinity of tumour antigen.

In another preference, first tumour antigen and the antigentic specificity CD8 from the mammal⁺ The affinity of T cell is Q1, second tumour antigen and the antigentic specificity CD8 from the mammal⁺T The affinity of cell is Q2, and the ratio R 1 of the Q2/Q1 is 0.02-0.80 (i.e. parents of the second tumour antigen It is less than the affinity of the first tumour antigen with power).

Strengthen the treatment method of anti tumor immune response, including step there is provided a kind of in the seventh aspect of the present invention：

(c) after the immune administration operation of previous step after t days, wherein t is 3-60, by described first Tumour antigen is immune to be applied to the described mammalian object excited through initial immunity, excites swollen for described first The immune response of tumor antigen, so that the mammalian object through immune stimulating again is obtained, wherein exempting from again described In the mammalian object that epidemic disease is excited, the enhanced tumor immune response for being directed to the first tumour antigen is generated.

It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and below (such as embodiment) It can be combined with each other between each technical characteristic of middle specific descriptions, so as to constitute new or preferred technical scheme. As space is limited, no longer tire out one by one herein and state.

Brief description of the drawings

Fig. 1 shows the structure of Ova native antigenic epitopes N4 and mutant antigen.

Fig. 2 shows the functional affinity of native antigen N4 and mutant antigen to the polyclonal T cell of endogenous. Wherein, (A) WT mouse tail vein injections infection Lm-N4 5000cfu/ are only latter 7th day, take spleen cell external use The native antigen and mutant antigen of gradient concentration stimulate (10^-12M-10^-6M), IFN-γ is detected⁺CD8⁺T cell ratio. According to its numerical value and corresponding irritaiting concentration fitted dose-effect curve (n >=4)；(B) every group of docs-effect is calculated bent The corresponding EC of line₅₀, inverted (1/EC₅₀) represent relative functional affinity (relative avidity)；(C)WT Mouse tail vein injection infection Lm-N4 5000cfu/ are only latter 7th day, take spleen cell external use native antigen or prominent Becoming the μ g/ml of antigen 2 stimulates culture, detects IFN-γ⁺CD8⁺T cell ratio.

Fig. 3 shows that central tolerance mechanism makes T cell storehouse be more likely to react medium affinity mutant antigen.Its In, the IFN of identification antigen in itself after (A) native antigen and mutant antigen infection_-γ⁺CD8⁺The statistics of T cell ratio Average value (n >=4).(B) native antigen and mutant antigen recognize N4 IFN-γ after infecting⁺CD8⁺T cell ratio Assembly average (n >=4).(C) WT mouse and Rip-mOva mouse are infected with Lm-N4 or mutant strain tail vein injection Afterwards, its IFN-γ reacted native antigen N4⁺CD8⁺T cell ratio and the IFN-γ that corresponding antigens are reacted⁺CD8⁺ Ratio (n >=4) between T cell ratio.

Fig. 4 shows the structure for the Rip-mOva murine melanoma models being resistant to based on Ova antigens.Wherein, (A) as shown, 2 × 10⁴, 10 × 10⁴, 50 × 10⁴The Mo5 difference subcutaneous vaccination WT mouse of cell number, every 3 days Measurement tumor size is simultaneously recorded.When tumour major diameter and the average value of minor axis length are more than 20mm, mouse is put to death, It is considered as dead mouse.(B)50×10⁴The Mo5 subcutaneous vaccination Rip-mOva mouse of cell number, measurement in every 3 days is swollen Knurl size is simultaneously recorded.When tumour major diameter and the average value of minor axis length are more than 20mm, mouse is put to death, is considered as small Mouse is dead.(C)50×10⁴After cell number inoculation, the survival curve of WT tumor-bearing mices and Rip-mOva tumor-bearing mices (n≥7)。(D)50×10⁴20 days after cell number inoculation, Rip-mOva tumor-bearing mices spleen and groin drainage are drenched Fawn on middle N4-tetramer⁺CD44⁺CD8⁺T cell ratio.(E)50×10⁴20 days after cell number inoculation, Rip-mOva tumor-bearing mices spleen and groin draining lymph node cells external use native antigen N4 or mutant antigen Y3 Stimulate, detect its CD8⁺The expression of cell factor IFN-γ and TNF-α in T cell.

Fig. 5：Central tolerance mechanism prevents the autoreactive T cell of low-affinity from inducing effective antitumour Immune response.Wherein, 7 days, subcutaneous vaccination 5 × 10 after (A) WT mouse tail veins infection Lm-N4 or Lm-Y3⁵/ Mo5 melanoma cells.Start within the 6th day after Mo5 inoculations, tumor size was measured every 2 days, calculate tumour Volume.(B) 7 days, subcutaneous vaccination 5 × 10 after Rip-mOva mouse tail veins infection Lm-N4 or Lm-Y3⁵/ Mo5 Melanoma cells.Start within the 6th day after Mo5 inoculations, tumor size was measured every 2 days, calculate gross tumor volume. (C) WT mouse and Rip-mOva mouse tail veins are infected after Lm-N4 or Lm-Y3 7 days, take spleen cell external use The N4 of gradient concentration stimulates (10^-12M-10^-6M), IFN-γ is detected⁺CD8⁺T cell ratio.According to its numerical value with it is corresponding Irritaiting concentration fitted dose-effect curve.WT Sp.T cells：Lm-N4 infects WT mouse；WT CR.T Cell：Lm-Y3 infects WT mouse；RO CR.T cells, Lm-Y3 infection Rip-mOva mouse.(D) calculate every The corresponding EC of group dose-effect curve₅₀, inverted (1/EC₅₀) represent relative functional affinity.

Fig. 6 shows that the autoantigen reaction-ive T cell storehouse V β under central tolerance use diversity.Wherein, (A) WT mouse and Rip-mOva mouse tail vein injections infection Lm-Y3 after the 7th day, N4-tetramer antibody and The double dyeing of Y3-tetramer antibody, the different tetramer of detection identification CD44⁺CD8⁺T cell ratio.(B)WT Mouse and Rip-mOva mouse tail veins are infected after Lm-N4 or Lm-Y3 7 days, take spleen cell external use N4 to pierce Swash culture, detect IFN-γ⁺CD8⁺T cell uses different V β ratio.WT Sp.T cells：Lm-N4 infects WT mouse；WT CR.T cells：Lm-Y3 infects WT mouse；RO CR.T cells, Lm-Y3 infection Rip-mOva Mouse.(C) 7 days after Rip-mOva mouse tail veins infection Lm-Y3, spleen cell external use Y3 is taken to stimulate culture, Detect IFN-γ⁺CD8⁺T cell uses different V β ratio schematic diagram.(D) Rip-mOva mouse tail veins infect 7 days after Lm-Y3, take spleen cell external use Y3 to stimulate culture, detect IFN-γ⁺CD8⁺T cell uses different V β Ratio (n >=8).(E) WT Sp.T cells and RO CR.T cells use the comparison of different V beta ratios.(F)WT CR.T cells and RO CR.T cells use the comparison of different V beta ratios.

Fig. 7, which shows that the repetition of isoantigen vaccine is immune, can not induce effective antitumour immune response.Wherein, (A) start within the 7th day after WT mouse inoculations melanoma cells Mo5, noted with the Lm bacterial strains abdominal cavity of expression correspondence antigen Capable treatment is injected, dosage is 10⁴Cfu/ is only.Treatment is repeated 3 times altogether, every minor tick one week.Mo5 inoculations 6 the 6th It starts, and measured tumor sizes every 2 days and calculates volume.(B) Rip-Ova mouse inoculations melanoma cells Start within the 7th day after Mo5, treated with the Lm bacterial strains intraperitoneal injection of expression correspondence antigen.Treatment repeats 3 altogether It is secondary, every minor tick one week.(C) start within the 7th day after WT mouse inoculations melanoma cells Mo5 to use Lm-N4 or Lm-Y3 Treatment, treatment is repeated 3 times altogether, every minor tick one week.Mouse is put to death when 30th day, spleen, abdomen stock is taken Ditch draining lymph node, Mesenteric lymph node cell, external use native antigen N4 are stimulated, and detect IFN-γ⁺CD8⁺T Cell proportion.(D) start within the 7th day after Rip-Ova mouse inoculations melanoma cells Mo5, activated with CpG DC load changing antigen Y3, foot-pad immunization is treated, and dosage is 10⁶Cell/only.Treatment is repeated 3 times altogether, Every minor tick one week.(E) start within the 7th day after Rip-Ova mouse inoculations melanoma cells Mo5, activated with CpG DC load changing antigen Y3, foot-pad immunization carry out first time treatment, dosage is 10⁶Cell/only；After one week, Second is carried out with Lm-Y3 intraperitoneal injections to treat, dosage is 10⁴Cfu/ is only.

Fig. 8 shows the dual alloimmunization strategy enhancing feature of autoreactive T cell of optimization and affine Power.Wherein, (A) Rip-Ova mouse are carried out immune for the first time with Lm-Y3 or DC-Y3；According to experiment description one week It is immunized again with expression mutant antigen Y3 of the same race afterwards.Take mouse spleen external use N4 to stimulate after 4 days, detect IFN-γ⁺ CD8⁺T cell, TNF-α⁺CD8⁺T cell and IFN-γ⁺TNF-α⁺CD8⁺The ratio (n >=4) of T cell. (B) described according to experiment, Rip-Ova mouse use dual alloimmunization strategy, be spaced one week, be immunized in two times. Take spleen external use N4 to stimulate after 2nd time immune 4 days, detect IFN-γ⁺CD8⁺T cell, TNF-α⁺CD8⁺ T cell and IFN-γ⁺TNF-α⁺CD8⁺The ratio of T cell.(C) Rip-Ova mouse describe to use respectively as shown Lm-Y3 is immune once, and Lm-Y3 is immune twice, and DC-Y3-Lm-Y3 is immunized, after DC-Y3-Lm-N4 is immune, spleen Cells in vitro is stimulated with gradient concentration N4, the IFN-γ of detection correspondence irritaiting concentration⁺CD8⁺T cell ratio, intends Close dose-effect curve.(D) the corresponding EC of every group of dose-effect curve in Fig. 6 C is calculated₅₀, inverted (1/EC₅₀) Represent relative functional affinity.(E) Rip-Ova mouse are with after DC- vaccine initial vaccinations, with the Lm- for changing antigen Vaccine immunity, detects CD8⁺The ratio of T cell expression cell factor IFN-γ and TNF-α.(F) Rip-Ova mouse After being immunized with DC-Y3-Lm-N4, spleen cell external use gradient concentration N4 is stimulated, and detects IFN-γ⁺CD8⁺T Each V β use ratio in cell.(G) Rip-Ova mouse are thin with the immune RO CR.T once produced afterwards of Lm-Y3 The V beta ratios (Fig. 4 B) and the IFN-γ with DC-Y3-Lm-N4 identification N4 after immune being inclined to use in born of the same parents⁺CD8⁺T Comparison in cell.

Fig. 9 shows that the autoreactive T cell of dual alloimmunization strategy activation has antitumor action.Wherein, (A) upper -14th day of Rip-Ova mouse with Lm-Y3 5000cfu/ only or DC-Y3 10⁶Cell/only exempt from advance, after 7 With Lm-Y3 5000cfu/ or Lm-N4 5000cfu/ mouse, Immune-enhancing effect reacts again.It is inoculated with the 0th day Melanoma cells Mo5 5 × 10⁵Cell/only.The 6th day, tumour was measured every 2 days since after Mo5 inoculations Size simultaneously calculates volume.(n=3 or 4).(B) after each immune group inoculation Mo5 of Rip-Ova mouse, at each time point The mean tumour volume size of every group of mouse.(C) Rip-Ova mouse DC-Y3-Lm-N4 immune groups, Lm-Y3-Lm-Y3 Survival rate after non-immune group mouse inoculation Mo5.

Figure 10 shows that dual alloimmunization can suppress tumour growth as therapeutic vaccine on tumor-bearing mice.Its In, melanoma Mo5 5 × 10 is inoculated with (A) Rip-Ova mouse⁵/ only, given for the first time according to description within the 7th day Immunization therapy, second of immunization therapy in the 14th day.DC vaccines foot-pad immunization is treated, dosage 1 × 10⁶/ only；Lm epidemic diseases Seedling intraperitoneal injection is immune, and dosage 10000cfu/ is only.(B) according to Figure 10 step As, dual alloimmunization treats lotus knurl Rip-Ova mouse.Mo5, which is inoculated with the 6th day, to be started, and was measured tumor sizes every 2 days and is calculated volume, determines small Mouse blood sugar concentration.(C) lotus knurl Rip-Ova mouse not treatment group, DC-Y3-Lm-Y3 treatment groups, DC-Y3-Lm-N4 Mean tumour volume size (n >=5) of the treatment group in every group of mouse of each time point.(D) lotus knurl Rip-Ova mouse are not controlled Treatment group, DC-Y3-Lm-Y3 groups, the mouse survival curve of DC-Y3-Lm-N4 groups.

It is immune anti-that Figure 11 shows that the dual alloimmunization strategy based on adjuvant can improve antineoplastic specificity T cell Should, and conspicuousness suppress tumour growth.

Embodiment

The present inventor, by largely screening and testing, develops a kind of enhancing first by in-depth study extensively The method of anti tumor immune response and the composition for this method.The inventive method passes through dual alloimmunization plan Slightly, the T cell of identification natural tumor antigens can be effectively activated under central tolerance environment, and effectively improves its parent With power and multifunctionality, so as to reach suppression tumour growth, extend survival period and other effects.Complete on this basis The present invention.

Specifically, specifically, Rip-mOva of the present inventor by the use of expression pattern antigen Ova as autoantigen Mouse constructs Ova (N4) specific T-cells central tolerance model, and with the restructuring of N4 and its 5 kinds of mutant antigens Liszt's bacterial strain have studied under central tolerance machining function as the pattern vaccine infection transgenic mice, body T Identification and reaction of the cell bank to natural (itself) antigen and mutant antigen.

The inventors discovered that, central tolerance not only completely inhibits reaction of the immune system to autoantigen N4, also portion Divide the reaction inhibited to mutant antigen.And the mutant antigen Y3 of medium affinity can induce most intersections to recognize Native antigen N4 T cell, i.e. autoreactive T cell.The immune response mechanism illustrates, because of tumour antigen For autoantigen, under central tolerance environment, effective antitumour immune response directly can not be induced with tumour antigen. Because the selective autoreactive T cell for removing high-affinity, Direct Recognition during thymus development The T cell storehouse very little of tumour antigen, and major part recognizes the T cell storehouse of mutant antigen by central tolerance mechanism Influence is limited, wherein still there is enough autoreactive T cells to tumour antigen with low-affinity, this Group's T cell relys more on some specific V β expression, it may be possible to because low using the TCR affinity of the V β, So being easier to flee from Solid phase.But this also have impact on the feature of autoreactive T cell, it is not had There is effective antitumour effect.Therefore, the T cell storehouse of intersection identification autoantigen is being induced using mutant antigen On the basis of, the affinity and feature of this group of autoreactive T cells of Selective long-range DEPT, to anti tumor immune response For it is particularly important.

The present inventor has found that the feature for strengthening autoreactive T cell is different by two kinds with affinity in an experiment Mechanism regulating.The change of immune carrier can influence the affinity of autoreactive T cell, and the change of antigen The functional cell factor expression of autoreactive T cell can be promoted.Carrier is used as using DC after CpG activation maturations Be immunized for the first time, using Lm as carrier, progress is immune for the second time, can effectively strengthen autoreactivity T The affinity of cell.And Lm or reverse DC and Lm are either all used twice as carrier using DC twice Using order then without same effect.

By the research to autoreactive T cell storehouse V β, the inventors discovered that, central tolerance mechanism not only subtracts Lack the absolute quantity of autoreactive T cell, it is thin in the T of the identification autoantigen of the low-affinity remained Born of the same parents storehouse, its diversity is also reduced.In the system of the present inventor, in the autoreactive T cell of low-affinity V β 3, V β 7, V β 10b, and V β 13 T cell of the use ratio higher than the high-affinity for recognizing same antigen, And using the ratio of other (the V β not detected) to be then decreased obviously, this is probably because the mistake selected in thymic negative Meeting specificity removes high-affinity T cell clone in journey, and then result in the use to some low-affinity V β The multifarious reduction of preference and generally cell bank.And exempt from the dual alloimmunization strategy DC-Y3-Lm-N4 of optimization After epidemic disease, recognize that only V β 3 ratio is substantially reduced in the T cell storehouse of autoantigen, and V β 4 use ratio Substantially increase, that is, effectively increase the functional affinity of autoreactive T cell.It is interesting that affine in height V β 4 use ratio is not but high in power T cell storehouse.The present inventor speculates the dual alloimmunization by optimization The affinity of autoreactive T cell and feature increase, mainly do not expand some low parents by specific afterwards Cloned with the T cell of force and work energy, and add other T cells and clone selected chance.

The present inventor is inoculated with expression Ova melanoma cells Mo5 on Rip-mOva mouse, simulates people itself A situation arises for tumour, and then the dual alloimmunization strategy DC-Y3-Lm-N4 by the use of optimization is used as vaccine therapy lotus knurl Mouse, finds to compare control group, the immunization strategy after optimization can suppress tumour growth, effectively extends depositing for mouse Current.Illustrate that the efficiency of antitumor reaction is mainly also to rely on autoreactive T cell feature and affinity Improve.

Term

As used herein, term " tumour antigen of the present invention " refers to the second tumour antigen, i.e., exempt from first tumour Epidemic disease antigenIt is homologousAnd the polypeptide or its immunogenic fragments intersected.For example, as Trp2SD peptides (SEQ ID NO.:7) During for the first tumour antigen, a kind of second tumour antigen is Trp2SK (SEQ ID NO.:8).

As used herein, term " epitope (peptide) " refers to other albumen of plan induced animal generation immune response One section of peptide.Generally, epitope refers to the peptide fragment that targeting is intended in immune response, preferably from mammal (such as people) One section of peptide of albumen.

Central tolerance model

In the present invention, for central tolerance model it is demonstrated experimentally that the inventive method can effectively activate identification naturally The T cell of hypoimmunity antigen (including tumour antigen or self tumor antigen), so that effectively enhancing is for low immune The immune response of property antigen.

In the present invention, a kind of representational central tolerance model is Ova transgenic mice (Rip-mOva), should Transgenic mice is the artificial constructed central tolerance model for native antigen N4 specific T-cells.The mouse Ova is expressed in medullary substance thymic epithelial cells, pancreatic beta cell and renal proximal tubules as autoantigen, because This during the developing immune system central tolerance mechanism N4 specificity Ts are thin in selective clearing Mice Body Born of the same parents.The experiment of the present inventor shows, the N4 itself of low-affinity is still suffered from the central tolerance model mice body Reaction-ive T cell.

The present inventor utilizes the Liszt's bacterial strain (Lm-N4) or expression mutation Ova antigens for expressing natural Ova antigens Liszt's bacterial strain (Lm-A2~Lm-V4) infects Rip-mOva and wild type (WT) mouse, as a result finds, maincenter is resistance to By under machining function, the T cell storehouse of mouse is not reacted autoantigen, that is, native antigen, and is easier by Mutant antigen etc. affinity is activated.

Ovalbumin native antigen and its mutant antigen that is homologous and intersecting

The native antigen and series mutation antigen of model antigen ovalbumin (Ova) are always applied to t cell responses Research.Ova 257-264 amino acid sequence SIINFEKL (N4) can be by I type MHC molecules K^bRecognize and submission To T cell, so as to cause strong antigentic specificity CD8⁺T cell responses.In native antigen N4 sequence basis On single amino acids mutation is carried out according to amino acid correspondence position, obtain a series of mutant antigens.As described in Figure 1, N4 and its representational mutant antigen include：

Native antigen N4:	SIINFEKL	(SEQ ID NO.:1)
			Mutant antigen A2:	SAINFEKL	(SEQ ID NO.:2)
Mutant antigen Y3:	SIYNFEKL	(SEQ ID NO.:2)
			Mutant antigen Q4:	SIIQFEKL	(SEQ ID NO.:4)
Mutant antigen T4:	SIITFEKL	(SEQ ID NO.:5)
			Mutant antigen V4:	SIIVFEKL	(SEQ ID NO.:6)

This group of mutant antigen and native antigen are to murine antigen presenting cell MHC molecule K^bWith similar affinity, And to CD8⁺The functional affinity of φt cell receptor is then different.N4 is used to simulate first (tumour) antigen, and Y3 It is preferred homologous and intersection second (tumour) antigen.

Composition and application process

Present invention also offers a kind of composition (or composition product), it contains：(1) first chamber, it is described First chamber include (i) the first tumour antigen and/or the BMDC by the first tumour antigen sensitization； (ii) optional adjuvant；(iii) acceptable carrier pharmaceutically or in immunology；

(i i) second chamber, described second chamber includes (i) the second tumour antigen and/or by described second The BMDC of tumour antigen sensitization；(ii) optional adjuvant；(iii) pharmaceutically or in immunology is subjected to Carrier；

Also, described first chamber and second chamber is independent.

In the present invention, term " containing " represents that various composition can be applied to or be present in the composition of the present invention together In.Therefore, term " mainly by ... constitute " and " consist of " are included in term " containing ".

The composition of the present invention includes pharmaceutical composition and vaccine combination.

The composition of the present invention can be monovalent (only containing a kind of recombinant protein or polynucleotides) or many (the containing a variety of recombinant proteins or polynucleotides) of valency.

The pharmaceutical composition or vaccine combination of the present invention can be prepared into various regular dosage forms, including (but not It is limited to)：Injection, granula, tablet, pill, suppository, capsule, suspension, spray etc..

(1) pharmaceutical composition

First tumour antigen of the pharmaceutical composition of the present invention comprising (or containing) therapeutically effective amount and/or by described the The BMDC of one tumour antigen sensitization；And second tumour antigen and/or by the second tumour antigen sensitization BMDC.

Term " therapeutically effective amount " used herein refers to therapeutic agent treatment, alleviated or prevention target disease or situation Amount, or show the amount of detectable treatment or prevention effect.The effect can be detected for example, by antigen levels. Therapeutic effect also includes the reduction of physical symptoms.Accurate effective dose for a certain object depends on the body of the object The combination of therapeutic agent and/or therapeutic agent that type and health status, the nature and extent of illness and selection are given. Therefore, it is useless to preassign accurate effective dose.However, for certain given situation, can be with often Rule are tested to determine the effective dose.

For the purposes of the present invention, effective dosage is to give individual each about 0.001 mg/kg to 1000 Mg/kg, is preferably about the second tumour antigen described in 0.01 mg/kg to 100 mg/kg body weight And first tumour antigen.

Pharmaceutical composition can also contain pharmaceutically acceptable carrier.Term " pharmaceutically acceptable carrier " refers to use The carrier being administered in therapeutic agent (recombinant protein of the invention).The term refers to some such medicament carriers：They The antibody for producing and being harmful to the individual for receiving said composition itself is not induced, and does not have undue toxicity after administration.Close Suitable carrier can be big, be metabolized slow macromolecular, such as protein, polysaccharide, PLA (polylactic Acid), polyglycolic acid etc..These carriers are well known to those of ordinary skill in the art.In Remington's It can be found in Pharmaceutical Sciences (Mack Pub.Co., N.J.1991) on can pharmaceutically connect The carrier received or excipient are discussed fully.

The upper acceptable carrier of combination of traditional Chinese medicine may include liquid, such as water, salt solution, glycerine and ethanol.In addition, Complementary material, such as wetting agent or emulsifying agent, pH buffer substance are there is likely to be in these carriers.Generally, Composition can be made to injectable agent, such as liquid solution or suspension；It may also be fabricated which and be adapted to supplying solution before the injection Or the solid form of suspension, liquid excipient.Liposome is also included within the definition of pharmaceutically acceptable carrier.

(ii) vaccine combination

The vaccine (composition) of the present invention can be preventative (i.e. prevention disease) or curative (i.e. after illness Treat disease).

These vaccines include immunising antigen (i.e. the second tumour antigen and the first tumour antigen of the invention), and lead to Often combined with " pharmaceutically acceptable carrier ", these carriers are produced to receiving said composition including itself not inducing The harmful antibody of individual any carrier.Suitable carrier is typically big, is metabolized slow macromolecular, such as egg White matter, polysaccharide, PLA, polyglycolic acid, amino acid polymer, amino acid copolymer, lipid aggregates are (such as Oil droplet or liposome) etc..These carriers are well known to those of ordinary skill in the art.In addition, these carriers can Play immunostimulant (" adjuvant ").In addition, antigen can also with bacterial toxoid (such as diphtheria, lockjaw, The toxoid of the pathogen such as cholera, helicobacter pylori) coupling.

The preferred adjuvant of enhancing immune composition effect includes but is not limited to：(1) aluminium salt (alum), such as aluminium hydroxide, Aluminum phosphate, aluminum sulfate etc.；(2) oil-in-water emulsion formula, for example, (a) MF59 (referring to WO 90/14837), (b) SAF, and (c) Ribi^TMAdjuvant system (RAS) (Ribi Immunochem, Hamilton, MT), (3) saponin Adjuvant；(4) Freund Freund's complete adjuvants (CFA) and Freund Freund's incomplete adjuvants (IFA)；(5) cell factor, it is such as white Interleukin (such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12), interferon (such as interferon), Macrophage colony stimulatory factor (M-CFS), TNF (TNF) etc.；(6) bacterial ADPribosylating poison The detoxification variant of plain (such as E.coli LT LT)；And (7) strengthen group as immunostimulant The other materials of compound effect.

Vaccine combination including immunogenic composition is (for example, it may include antigen, pharmaceutically acceptable Carrier and adjuvant), usually contain diluent, such as water, salt solution, glycerine, ethanol etc..In addition, complementary thing Matter, such as wetting agent or emulsifying agent, pH buffer substance may be present in this kind of carrier.

More specifically, the vaccine including immunogenic composition, the immunogenicity comprising immunological effective amount is more Peptide, and above-mentioned other required components." immunological effective amount " refers to be given with single dose or a continuous agent part The amount of body is effective to treating or preventing.The consumption can according to treat individual health status and physiological status, Treat individual classification (such as people), the ability of individual immunity system synthesis antibody, required degree of protection, vaccine Preparation, treating physician to the assessments of medical conditions and other correlative factors depending on.It is expected that the consumption will be in phase To that in wider scope, can be determined by normal experiment.

Generally, injectable agent, such as liquid solution or suspension can be made in vaccine combination or immunogenic composition； It may also be fabricated which and be adapted to supplying solution or suspension, the solid form of liquid excipient before the injection.Said preparation is also emulsifiable Or be encapsulated in liposome, to strengthen adjuvant effect.

In addition, the vaccine combination of the present invention can be unit price or polyvaccine.

(iii) method of administration and dosage

Once being made into the composition of the present invention, object can be directly given by it.Object to be treated can be that lactation is moved Thing, especially people.

When as vaccine, can with known method by the present invention recombinant protein be directly applied to individual.Generally adopt These vaccines are applied with conventional vaccine identical route of administration and/or simulation pathogenic infection path.

Giving the approach of pharmaceutical composition of the present invention or vaccine combination includes (but being not limited to)：Intramuscular, subcutaneous, Intracutaneous, intrapulmonary, intravenous, intranasal, by oral administration or other parenteral route of administration.It is possible if desired to combine Method of administration, or be adjusted according to disease event.Vaccine combination can be given with single dose or multiple dose, and can To trigger including giving booster and/or maintain immunity.

The amount of recombinant protein vaccine, i.e. recombinant protein should be given with " effective dose " in selected administration routes mesopodium To trigger immune response, it can effectively promote the disease for protecting host's resistance related.

Representational disease includes (but being not limited to)：Tumour etc., such as liver cancer, lung cancer, stomach cancer, breast cancer, Oophoroma, prostate cancer, cutaneum carcinoma, melanoma, cervical carcinoma, the cancer of the brain, thyroid cancer and cholangiocarcinoma, bladder Cancer and cancer of pancreas.

The amount of selected recombinant protein in each vaccine dose part, be by can trigger protective immune response and without obvious Side effect amount depending on.Generally, after host cells infected, each dose of vaccine is enough containing about 1 μ g-1000mg, Preferably 1 μ g-100mg, more preferably 10 μ g-50mg protein or polypeptide (including the second tumour antigen or first Tumour antigen).It can be determined with the standard research techniques including the IgG titers in the object of observation and other reactions The optimum amount of specific vaccine.Reinforcing agent can be determined the need for by monitoring the immunity level of vaccine offer Amount.After the IgG titers in have evaluated serum, it may be necessary to from enhancing dose immunizations.Using adjuvant And/or immunostimulant can improve the immune response of the protein to the present invention.

Method for optimizing is to give immunogenic composition from parenteral (subcutaneously or intramuscularly) approach by injection.

In addition, the present invention vaccine can together be given with reference to other immunomodulators, or with other therapeutic agents one Rise and give.

Main advantages of the present invention include：

(a) immunization strategy and composition of the invention can effectively break through immune tolerance of the body to autoantigen.

(b) immunization strategy of the invention and composition can effectively immune response of the excitating organism for tumour antigen.

(c) the problem of present invention helps pointedly to solve Current therapeutic tumor vaccine general inefficiencies, to accelerating The research and development of tumor vaccine have important impetus.

(d) in terms of present invention can apply to immunotherapy of tumors, vaccine development.

With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate this Invention rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally According to normal condition, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) described in condition, or according to the condition proposed by manufacturer. Unless otherwise indicated, otherwise percentage and number are percentage by weight and parts by weight.

All antibody are commercial antibody, purchased from eBioscience or BD Bioscience companies.For example, Anti-mouse Vβ2TCR(B20.6,eBioscience)；Anti-mouse Vβ3TCR(KJ25,BD Bioscience)。

All reagents are unless otherwise indicated, otherwise commercially available.

Experimental animal：6 to 8 all wild type C57BL/6 mouse are purchased from Chinese Academy of Sciences's Shanghai Experimental Animal Center SLAC companies；Rip-mOva mouse are obtained from Howard Hughes Institute for Medical Research of University of Washington.

Universal method

(a) culture and infection of Listeria

The 1.-80 DEG C of Listeria line frozen applies TSB culture medium solid plates, and 37 DEG C of incubators stay overnight rear picking Monoclonal bacterium colony, 30 DEG C, 200rpm/min, shaking table culture was stayed overnight after 14 hours, according to 1：20 ratios switching bacterium In the fresh TSB fluid nutrient mediums of the μ l to 3ml of liquid 150,37 DEG C, 200rpm/min, shaking table culture 2.5~3 is small When after arrive exponential phase, survey OD600 values, now OD600 values are between 0.6~1.

2. taking 1ml bacterium solutions, 12000rpm/min centrifugation 10min, repetition is cleaned with 1ml phosphate buffer solutions Twice of centrifugation, bacterial sediment, OD600 and bacterial concentration are resuspended according to OD600 values phosphate buffer solution is measured Between corresponding relation be when OD600 values are 1, bacterial concentration is 10⁹cfu/ml。

3. according to 1：10 ratios take 100 μ l bacterium solutions to be diluted to 1ml phosphate buffer solutions successively, until bacterium solution Concentration dilution is 10⁵Cfu/ml, continues according to 1：4 dilution proportion bacterial concentrations are 2.5 × 10⁴Cfu/ml is used for small The tail vein injection of mouse.

(b) culture of bone marrow derived BMDC, activation and immune

1. the 1st day：The complete femur and shin bone for taking out 6 weeks to 8 weeks C57/BL6 female mice lower limb of age, The musculature of attachment is removed, is placed in precooling phosphate buffer solution, cell work platform is put in transfer.Use sterile PBS Rinse bone 3 times.Sterile scissors cut off bone two ends, 1ml irrigation with syringe ossis, until bone bleaches.Weight Phosphate buffer solution is suspended from, nylon membrane is filtered into single cell suspension.Supernatant is removed in 300g, 10min, 4 DEG C, centrifugation Afterwards, 1ml erythrocyte cracked liquids are added, adding 10ml phosphate buffer solutions after cracking 5 minutes on ice terminates. 300g, 10min, 4 DEG C of centrifugations go after supernatant, cell count to be resuspended in K medium 24 orifice plates of paving, cell concentration For 1 × 10⁶Cell/ml, 1ml/ holes.Move into cell culture incubator, 37 DEG C, 5%CO₂Under the conditions of cultivate.

2. the 3rd day：After 24 orifice plate small angle inclinations 10 minutes, liquid-transfering gun is drawn and removes the μ l of supernatant 500 in hole. The μ l of fresh culture 500 are supplemented, growth factor GM-CSF and IL-4,37 DEG C, 5%CO is added₂Culture.

3. the 5th day：All culture mediums and cell in 24 orifice plates are drawn, 4 DEG C, culture is gone in 300g, 10min centrifugations Base supernatant, supplement fresh culture 1ml/ holes, adds growth factor GM-CSF and IL-4, moves into cell culture incubator.

4. the 7th day：Suspension cells all in 24 orifice plates are collected in piping and druming, and 300g removes supernatant after 10min centrifugations, Fresh culture is resuspended in, cell concentration is 2 × 10⁶Cell/ml, adds CpG 800ng/ml, moves into cell Incubator, 37 DEG C stimulate activation 16 hours.

5.CpG removes supernatant after stimulating dendritic cells in mouse 300g, the 10min centrifugation after staying overnight, and is resuspended in fresh training Base is supported, cell concentration is 1 × 10⁶Cell/ml, adds the μ g/ml loads of test polypeptide 2,37 DEG C, 5%CO₂Training Support 2.5 hours.

6. remove supernatant, 10ml phosphate after 300g, 10min, 4 DEG C of centrifugations of dendritic cells in mouse after load polypeptide After cushioning liquid is cleaned 3 times, serum-free phosphate buffer solution is resuspended in, cell concentration is 20 × 10⁶Cell/ml, For mouse immune.

(c) culture of melanoma cells and the foundation of melanoma tumor model

K-1735 Mo5 cells (being obtained from the Chinese Academy of Sciences) recovery culture (first generation) from liquid nitrogen cryopreservation, It is thin for the Mo5 that 1mg/ml antibiotic G418 screens specific expressed Ova antigens that second generation culture is initially added into concentration Born of the same parents.Screening passage 3-4 times, forth generation or the 5th generation cell are collected when being in exponential phase of growth, are resuspended in phosphate In cushioning liquid or plasma-free DMEM medium.

Mouse is anaesthetized with 2.5% Avertin, and back unhairing hits exactly subcutaneous vaccination Mo5 cells, 5 × 10 in low back⁵ Cell/mouse.Since after inoculation the 6th day, every the 2 days length with vernier caliper measurement tumour major diameter and minor axis Degree, gross tumor volume is according to formula【Major diameter × (minor axis)²×π/6】Calculate⁴².When the average length of major diameter and minor axis After 2 centimetres, palliated the agonizing sufferings for the disconnected neck of mouse is put to death according to Animal Experimental Ethical criterion.

(d) immunoprophylaxis and immunization therapy of mouse tumor

The prevention of mouse tumor：Mouse exempts from expression antigen Listeria and/or dendritic cells epidemic disease according to experimental program in advance After seedling the 7th day, subcutaneous vaccination K-1735 Mo5 cells 5 × 10⁵Cell/mouse, observes and records swollen Knurl growing state.

The treatment of mouse tumor：After mouse hypodermic inoculation K-1735 Mo5, according to the therapeutic scheme of design, Listeria and dendritic cell vaccine treatment are given, observes and records tumour growth situation.

(e) the dendritic cells culture of derived from bone marrow

1. the 1st day：The complete femur and shin bone for taking out 6 weeks to 8 weeks C57/BL6 female mice lower limb of age, The musculature of attachment is removed, is placed in precooling phosphate buffer solution, cell work platform is put in transfer.Use sterile PBS Rinse bone 3 times.Sterile scissors cut off bone two ends, 1ml irrigation with syringe ossis, until bone bleaches.Weight Phosphate buffer solution is suspended from, nylon membrane is filtered into single cell suspension.Supernatant is removed in 300g, 10min, 4 DEG C, centrifugation Afterwards, 1ml erythrocyte cracked liquids are added, adding 10ml phosphate buffer solutions after cracking 5 minutes on ice terminates. 300g, 10min, 4 DEG C of centrifugations go after supernatant, cell count to be resuspended in K medium 24 orifice plates of paving, addition growth Factor GM-CSF and IL-4 50ng/ml, cell concentration is 1 × 10⁶Cell/ml, 1ml/ holes.Move into cell training Support case, 37 DEG C, 5%CO₂Under the conditions of cultivate.

5.CpG removes supernatant after stimulating dendritic cells in mouse 300g, the 10min centrifugation after staying overnight, and is resuspended in fresh training Base is supported, cell concentration is 1 × 10⁶Cell/ml, adds test polypeptide (such as Trp2SD or Trp2SK) 2 μ g/ml Load, 37 DEG C, 5%CO₂Culture 2.5 hours.

(f) statistical analysis

Except the place of specified otherwise, independent experiment is at least repeated 2 times, every group of total number of samples amount n >=5.Original number According to being checked rear with Prism 5.0 (GraphPad, the U.S.) software progress statistical analysis.Experimental data is with averagely Value ± standard error is represented.When P values less than 0.05 (confidence interval is 50%) are considered to have significant difference.

Embodiment 1

The identification of native antigen and mutant antigen to the polyclonal T cell functional affinity of endogenous

The present inventor takes spleen with the Listeria Lm-N4 tail vein injection infecting mouses of expression native antigen after 7 days Dirty cells in vitro is with the native antigen N4 of concentration or mutant antigen A2, Y3, Q4, T4, V4 etc. are pierced respectively in gradient Swash culture, detect IFN-γ⁺CD8⁺The ratio of T cell, does dose-response relationship curve (Fig. 2A).

As a result it is as shown in Figure 2.The inventors discovered that, relative to CD8⁺T cell is anti-to native antigen N4 dosage Curve is answered, is all moved to right for the dose-effect curve of other mutant antigens.The present inventor's half-maximal effect Concentration (EC₅₀) inverse (1/EC₅₀) the relative functional affinity of native antigen and modified antigen is represented, find to use up Pipe native antigen is compared to OT-I T with mutant antigen to the functional affinity of the polyclonal T cell system of endogenous Cell is reduced, but its affinity sequence does not change.Native antigen N4 Ovas polyclonal to endogenous Functional affinity highest (the EC of specific T-cells₅₀=27.58pM), A2 be only second to N4 high functionality it is affine Resist strenuously former (35.4pM), Y3 and Q4 are medium affinity antigen (253.51pM and 689.17pM), and T4 and V4 are Low-affinity antigen (3221pM and 20930pM) (Fig. 2 B).

The difference of functional affinity may influence specific recognition N4 endogenous T cells storehouse to mutant antigen Cross reactivity, under the conditions of the conventional μ g/ml of antigenic stimulus concentration 2, recognize the N4 polyclonal T of endogenous Functional affinity positive correlation of the cell bank to the cross reaction level of mutant antigen generally with mutant antigen.High parent It is higher than the antigen (Fig. 2 C) of low-affinity with the cross reactivity in N4 specific T-cells storehouse with the antigen of power.

Embodiment 2

Central tolerance makes mouse immune system be more likely to the mutant antigen reaction to medium affinity

Identify the function of native antigen and mutant antigen to the polyclonal T cell system of endogenous of Ova antigen-specifics Property affinity after, the polyclonal T cell storehouse of the further Study Mouse endogenous of the present inventor is anti-to native antigen and mutation Influence of the former reaction rule and central tolerance mechanism to the rule.

WT mouse are infected respectively with expression native antigen N4 or mutant antigen Liszt's bacterial strain and Rip-mOva is small Mouse, takes the corresponding antigen of each self-infection strain of spleen cell external use to stimulate respectively, detects IFN-γ after 7 days⁺CD8⁺T The ratio of cell.

As a result it is as shown in Figure 3.T cell storehouse in WT Mice Bodies can be produced to native antigen and mutant antigen exempts from Epidemic disease is reacted；And in Rip-mOva mouse, because the presence of central tolerance mechanism, to native antigen N4 and height parent Almost it is totally constrained with the immune response for resisting former A2 strenuously, to other medium affinity and low-affinity mutant antigen Immune response also have a different degrees of reduction, and central tolerance is exempted to tri- mutant antigens inductions of Y3, Q4, V4 The influence of epidemic disease reaction level is relatively low.

The result illustrates that in the presence of central tolerance mechanism the T cell storehouse of body is to autoantigen and high-affinity Mutant antigen can not be reacted, and the immune response level of mutant antigen is suppressed, but some medium affinity and The mutant antigen of low-affinity still can produce a number of T cells with antigenic specificity with induced activation and clone.

Due in anti tumor immune response, play a major role be mutant antigen induction immune response in recognize day That a group T cell of right antigen, thus in the present embodiment, after further detection mutant antigen is immune, T cell Cross reaction of the storehouse to native antigen.

It is immunized respectively after WT mouse and Rip-mOva mouse with the Liszt's bacterial strain for carrying mutant antigen, takes spleen thin Cell space external natural antigen N4 stimulates culture, detects IFN-γ⁺CD8⁺The ratio of T cell.

In WT mouse, the ratio for the T cell reacted native antigen is below what mutant antigen was reacted in itself The ratio of T cell, the cross reaction to native antigen of mutant antigen induction and the functional affinity of mutant antigen Generally correlation, the mutant antigen of high-affinity can induce higher proportion of native antigen reactivity T thin Born of the same parents, the mutant antigen of low-affinity tends to induce the native antigen reaction-ive T cell of minor proportion.And In Rip-mOva mouse, because native antigen is autoantigen, immune only produce of other mutant antigens seldom can be right Native antigen reaction T cell, only mutant antigen Y3 can induce significantly, native antigen cross reactivity T Cell (average proportions are 0.9%), that is, autoreactive T cell.But it is interesting that the mutation of high-affinity The native antigen cross reactivity T cell produced after antigen A 2 is immune accounts for the ratio of all antigen reactivity T cells Close to 100% in WT mouse, illustrate that the specific T cells of nearly all A2 can be reacted native antigen, this Individual ratio is 55% in Rip-mOva mouse, is reduced close to half.And Y3 it is immune after cross reacting rate from 29% in WT mouse is reduced to 19% in Rip-mOva mouse, reduced by only about 30% (Fig. 3).

These experimental results illustrate that, when native antigen is exogenous antigen, body T cell storehouse is to mutant antigen Immune response level is mainly related to its functional affinity；And when native antigen is autoantigen, central tolerance Mechanism removes most of autoreactive T cell and the T cell reacted high functionality affinity mutant antigen, So that T cell storehouse is more likely to the mutant antigen reaction to medium affinity.And be immunized with Lm-Y3 in Rip-mOva The obvious T cell reacted native antigen is detected in mouse, is illustrated in central tolerance mechanism to autoreactivity Property T cell removing it is not thorough, still suffered from T cell storehouse sufficient amount of clone can recognize response itself resist It is former.And under central tolerance mechanism, recognize the t cell responses level of autoantigen and the relation of cross reacting rate Not substantially, illustrate to determine that the factor of antitumor response availability is still what the low-affinity existed in vivo was cloned Absolute quantity, high cross reacting rate, which is not intended that, can activate more autoreactive T cells.

Embodiment 3

Melanoma tumor model is built on WT mouse and Rip-mOva mouse

Identify under central tolerance machining function, the induction that the mutant antigen Y3 of medium affinity can be best from Body reaction-ive T cell, therefore with potential antitumor action.For follow-up experiment, in the present embodiment, With the expression Ova of various dose melanoma cells Mo5 subcutaneous vaccination WT mouse, it is outer to set up tumour antigen The non-Tolerance Model of melanoma (Fig. 4 A) of source property antigen, it is determined that 5 × 10⁵Cell/only inoculum concentration when, can To ensure that every mouse forms the homogeneous melanoma tumors of growth.

With 5 × 10⁵Tumour antigen is in a cell/dose inoculation Rip-mOva mouse, simulation human tumour generating process The situation of autoantigen, melanoma Mo5 can on mouse normal growth, therefore set up right Ova antigens from The melanoma tumor model (Fig. 4 B) of body tolerance, its survival curve is slightly above WT mouse (Fig. 4 C).Further look at The T cell (Fig. 4 D and E) of specific for tumour antigen whether can be produced after Rip-mOva mouse inoculation tumours, used The specific tetramer detections of N4 find all not recognize itself in Rip-mOva mouse spleens and draining lymph node Antigen N4 body cell (Fig. 4 D), external use N4 is stimulated and is also nearly free from IFN-γ⁺CD8⁺T cell.It is interesting , spleen cell and draining lymph node cells are stimulated in vitro with Y3, generate a number of expression IFN-γ With the CD8 of TNF-α⁺T cell ratio (Fig. 4 E).This prompting, internal T cell storehouse is easier to mutant antigen Y3 reacts.

Embodiment 4

The autoreactive T cell of medium affinity mutant antigen induction can not effectively suppress tumour growth

In the presence of central tolerance mechanism, medium affinity mutant antigen Y3 can be produced at most in inducing T cell storehouse Autoantigen reaction-ive T cell, and, it was also found that small in the detection of Ova antigen central tolerance mouse-borne tumor models There is the T cell reacted mutant antigen Y3 in mouse body.However, further investigations have shown that Lm-Y3 infection Immune not have obvious inhibitory action to the tumour to the natural Ova antigens of expression, antitumor reaction efficiency is low. In the present embodiment, exempt from WT mouse and Rip-mOva mouse in advance respectively with Lm-N4 and Lm-Y3, be then inoculated with table Up to the K-1735 Mo5 of Ova antigens.

As a result it is as shown in Figure 5.Although Lm-Y3 can suppression black more preferable than Lm-N4 on Rip-mOva mouse Plain knurl grows (Fig. 5 B), but still significantly lower than the inhibition (Fig. 5 A) of two kinds of antigen on WT mouse.This is dark Show, during antitumor, what is played a major role is still the autoreactive T cell for native antigen N4, Rather than that group of cells only reacted mutant antigen Y3.

Then, the present inventor with native antigen Lm-N4 and mutant antigen Lm-Y3 infect respectively WT mouse and Rip-mOva mouse, draw N4- specific T-cells (WT Sp.T cells) and the N4- cross reactions of WT mouse Property T cell (WT CR.T cells), and Rip-mOva mouse N4- cross reactivities T cell (RO CR.T Cell.Because existing, ratio is too low to be calculated RO Sp.T cells) dose-effect curve (Fig. 5 C), and calculate relative Functional affinity.

As a result show, compared with WT Sp.T cells, the relative functional affinity of WT CR.T cells have dropped About 109 times, and the affinity of RO CR.T cells have dropped 490 times (Fig. 5 D).Illustrate not deposit in central tolerance In case, the T cell storehouse of the identification native antigen of mutant antigen induction only has relatively low affinity；And in In the presence of pivot tolerance mechanism, the T cell storehouse of the identification native antigen of mutant antigen activation is autoreactive T cell, Its affinity scope is further reduced.

It these results suggest that, the low-affinity autoreactive T cell that mutant antigen is activated under central tolerance environment is not Effective immune response can be carried out to tumour antigen, suppresses its growth.

Embodiment 5

The autoreactive T cell storehouse and high-affinity T cell storehouse deviation for recognizing same antigen use different V β

In the polyclonal immune system of endogenous, the functional affinity between antigen and T cell, actual is T cell The Average functionality affinity of the T cell clone of the same antigen of response is recognized in storehouse.Because the acceptor of single T cell V β genes can produce different V β chains and then be combined as different TCR and form huge by segment rearrangements The T cell storehouse constituted is cloned by different.

In the present embodiment, further the T cell storehouse of the identification autoantigen of research mutant antigen activation and identification are same Difference between the high-affinity T cell storehouse of one antigen.

WT mouse and Rip-mOva mouse are immunized respectively with mutant antigen Y3, with N4-tetramer antibody and Y3-tetramer antibody dyes the T cell storehouse for detecting that it is recognized respectively to Y3 and N4 altogether.

As a result it is as shown in Figure 6.Intersect identification native antigen N4 T cell storehouse and identification Y3 T in WT mouse Cell bank overlaps, N4-tet⁺Y3-tet⁺CD8⁺T cell ratio is 3.20%, the CD8 for still having 1.05%⁺T Cell only recognizes N4 and 15.1% CD8⁺T cell only recognizes Y3；And in Rip-mOva mouse, recognize N4 T cell storehouse in 84% both be from identification mutant antigen Y3 T cell storehouse (Fig. 6 A).

By further expressing feelings to V β in WT Sp.T cells, WT CR.T cells and RO CR.T cells The detection of condition is found, although the functional affinity of WT CR.T cells is substantially less than WT Sp.T cells, both V β use tendencies and T cell storehouse diversity it is basically identical, although on the contrary, affinity is only differed about 4 times, the T cell storehouse diversity of RO CR.T cells with it is another both compare significantly different (Fig. 6 B), and Rip-mOva Recognize that Y3 T cell storehouse is not influenceed (Fig. 6 C) substantially but by central tolerance in mouse.This explanation, central tolerance Afterwards, remaining T cell storehouse is mainly for mutant antigen, and the autoreactive T cell activated is largely from prominent Become the T cell storehouse of antigentic specificity.

It has also been found that the diversity in autoreactive T cell storehouse is more likely to using some specific compared to both another reductions V β.By the comparison to V β utilization rates, as a result find, compared to WT mouse, in RO CR.T cells To V β 3, V β 7, V β 10b and V β 13 utilization rate significantly rises, and V β (Others) ratio not detected shows Write and decline (Fig. 6 D and E).The result implies, due to central tolerance selectivity removing high-affinity itself is anti- Answering property T cell is cloned, and the T cell of the identification autoantigen remained, which is relied more on, uses mutant antigen T cell The specific V β of some low-affinities in storehouse.

Embodiment 6

The repetition immunization therapy of mutant antigen of the same race can not effectively suppress tumour growth under central tolerance

Because central tolerance acts on the inhibitory action to autoantigen reaction-ive T cell storehouse, produced after mutant antigen is immune Raw autoreactive T cell only has extremely low affinity.Because the autoantigen T cell of mutant antigen induction Reaction is the immune basis of effective antitumour, and the present inventor is attempted with the immune mutant antigen of repetition as therapeutic swollen Knurl vaccine, whether can strengthen anti tumor immune response, suppress tumour growth if seeing.

The present inventor is inoculated with expression Ova melanoma cells Mo5 on Rip-Ova mouse, sets up native tumor Antigen N4 central tolerance model；And same tumour is inoculated with WT mouse, it is used as the non-of natural tumor antigens Central tolerance model.Start within the 7th day after inoculated tumour, Lm-N4 or mutation of the present inventor with expression native antigen Liszt's bacterial strain treatment each group mouse of antigen, treatment is carried out three times altogether, each time interval one week.

As a result it is as shown in Figure 7.Non- central tolerance WT mouse-borne tumors treatment model in the inventors discovered that, day Right antigen N4 and high-affinity mutant antigen A2 repetitive therapy can effectively suppress tumour growth, other medium parents Mutant antigen with power and low-affinity is then without therapeutic effect (Fig. 7 A).

By being detected to the lotus knurl WT mouse after Lm-N4 and Lm-A2 treatments, the inventors discovered that mouse Spleen, lymphonodi mesenterici, groin draining lymph node, are all generated a number of anti-to native antigen N4 The T cell (Fig. 7 C) answered.

And in the central tolerance model built based on Rip-Ova mouse, including can be best in Rip-Ova mouse Including the mutant antigen Y3 for inducing autoreactive T cell, all antigen therapies can not all produce effective anti- Function of tumor, suppresses tumour growth (Fig. 7 B).

In view of perhaps there is the peripheral tolerance mechanism to Ova antigens in Rip-Ova mouse, wherein in vivo not Ripe DC present antigens perhaps can induce the inhibitory action to autoreactive T cell, therefore, the present inventor Construct new immunization strategy：Ripe derived from bone marrow dendritic cells load Y3 (DC-Y3) is stimulated using CpG, is made For vaccine therapy lotus knurl Rip-Ova mouse.

The present inventor stimulates the DC cells of derived from bone marrow culture with the CpG of various concentrations first, detects its activation point Sub- CD80 and CD86 expression.

As a result find, in the case where 2 μ g/ml concentration is stimulated, what DC cells can be best is activated.But, nothing By the Double immune therapeutic strategy of the repetition immunization therapy (Fig. 7 D) for being DC-Y3, or DC-Y3 combinations Lm-Y3, It all can not effectively suppress tumour (Fig. 7 E).

These test result indicates that, based on mutant antigen Y3, traditional isoantigen repeats immunization therapy and is not enough to Enough anti tumor immune responses are produced, and need to design new immunization strategy to improve oneself under the conditions of central tolerance Body antigen reactivity t cell responses, are only possible to effective to oncotherapy.

Embodiment 7

Dual alloimmunization can effectively improve the immune response under central tolerance

In the present embodiment, the present inventor further loads not synantigen using ripe dendritic cells, builds dendron Cell vaccine, and expression not synantigen Listeria combination Rip-Ova mouse are immunized, detect its activation Identification autoantigen t cell immune response.

The present inventor is detected in the case of immunizing antigen is immovable first, only changes present antigen in being immunized twice Carrier (dendritic cells of Listeria or maturation), it is anti-to the autoantigen-specificity T cell under maincenter tenable environment Whether should have an impact.The present inventor is divided with load changing antigen Y3 dendritic cell vaccine (DC-Y3) or Lm-Y3 Not Mian Yi Rip-Ova mouse, after 7 days, further according to experimental design with DC-Y3 or Lm-Y3 again be immunized each group it is small Mouse, is stimulated with native antigen N4 in vitro after 4 days, detects CD8⁺Cell factor IFN-γ and TNF-α in T cell Expression.

As a result it is as shown in Figure 8.In the case where not changing immunizing antigen, each group mouse produces after being immunized twice IFN-γ⁺CD8⁺T cell ratio does not increase quantitatively compared to the ratio after being only immunized once with Lm-Y3, Reduce about 50% on the contrary, and TNF-α⁺CD8⁺T cell and IFN-γ⁺TNF-α⁺CD8⁺T ratio does not have Substantially change (Fig. 8 A).But, by the way that the N4 of the spleen cell external use gradient concentration of immunized mice is stimulated Draw dose-effect curve (Fig. 8 C) and calculate relatively with respect to functional affinity (Fig. 8 D) afterwards the inventors discovered that, Only compared with the immune autoreactive T cells once produced afterwards of mutant antigen Y3, twice Lm-Y3 it is immune and The relative functional affinity of the autoreactive T cell produced after DC-Y3-Lm-Y3 combined immunes significantly increases Plus, respectively 2.6 times^With15 times.It is interesting that the relativity functional affinity of the latter, has been even more than WT The relativity affinity of CR.T cells.

As a result show, in the case of immunizing antigen is constant, the raising of autoreactive T cell affinity is depended on The influence of vaccine carrier, and the such combinations of DC-Lm can effectively improve the affinity of autoreactive T cell.

Further, the present inventor changes immunizing antigen on the basis of being immunized twice using different carriers combination, Whether the expression and functional affinity for detecting the functional cell factor of T cell are affected.According to experimental design With shown in Fig. 8 B, the present inventor is with two kinds of carriers DC and Lm, the difference with native antigen N4 and mutant antigen Y3 Rip-Ova mouse are immunized for 7 days in combination, interval twice, detection identification native antigen N4 after being immunized 4 days for the second time Autoreactivity CD8⁺The cytokine-expressing and functional affinity (Fig. 8 B, C and D) of T cell.The present invention People has found that combination is immunized as only DC-Y3-Lm-N4 can produce obvious IFN-γ⁺CD8⁺T cell, Although its ratio 0.68%, the immune IFN-γs once produced afterwards of still slightly below Lm-Y3⁺CD8⁺The ratio of T cell Example 0.93%；But, its TNF-α produced⁺CD8⁺T cell ratio is combined apparently higher than others are immune, and About 3 times of ratio after Lm-Y3 is immunized once is exceeded；And the IFN-γ that it is produced⁺TNF-α⁺CD8⁺T is thin The ratio of born of the same parents is about 4 times after Lm-Y3 is immunized once, has exceeded the minimum immune combination of wherein level The IFN-γ that Lm-N4-DC-Y3 is produced⁺TNF-α⁺CD8⁺13 times of T cell ratio (Fig. 8 B)；In addition, its own The relative functional affinity of reaction-ive T cell compared to the affinity after Lm-Y3 primary immune responses although significantly increase 10.5 times, but compared with the DC-Y3-Lm-Y3 functional affinities for combining immune rear autoreactive T cell, There is no obvious difference (Fig. 8 C and D).These results illustrate, on the basis of carrier is determined, autoreactivity Property T cell feature influenceed by immunizing antigen, the combination of first mutant antigen native antigen again can promote from Body reaction-ive T cell expresses cytokine profiles.

The dual alloimmunization strategy being combined using carrier with antigen, first reuses native antigen using mutant antigen Immune, this combinations of DC-Y3-Lm-N4 can strengthen the multifunctionality and feature of autoreactive T cell simultaneously Affinity.The present inventor further detects the V β service conditions of the autoreactive T cell of this group of activation, with true Whether determine the diversity in T cell storehouse has change.By to IFN-γ⁺CD8⁺The V β of T cell are contaminated with specific antibodies Found after color, each V beta ratios of the RO CR.T cells after being immunized once with Lm-Y3 are compared (Fig. 4 B), double Although others V β use ratios are only slightly increased in Rip-Ova mouse after weight alloimmunization, T cell storehouse is more Sample reduces (Fig. 8 F), and autoreactive T cell expression V β 3 ratio with the former but compared to being only remarkably decreased About 80%, its ratio is similar to the ratio of V β 3 in the WT Sp.T cells of high-affinity, in addition, expression The ratio of V β 4 autoreactive T cell is also significantly increased about 3.4 times (Fig. 8 F and G).The result is said Bright, second immune with native antigen, and the autoreactive T cell storehouse of mutant antigen immune activation is sieved Choosing.

To sum up, the inventors discovered that using specific dual alloimmunization strategy, can increase under central tolerance suppression The immune response of strong autoreactive T cell, multifunctionality can be promoted by first reusing native antigen using mutant antigen The generation of T cell, and the functional affinity of T cell can effectively be improved by changing immunizing antigen delivery vehicle.It is logical Cross and V β are done to autoreactive T cell storehouse found using analysis, the low-affinity produced after mutant antigen is immune from Body reaction-ive T cell may tend to use some specific V β, and it is anti-to optimize itself that produced after immunization strategy In answering property T cell storehouse, do not select what the T cell using these specific V β was cloned, it may be possible to autoreactivity The reason for T cell feature and affinity increase.

Embodiment 8

The dual alloimmunization of optimization can effectively activate tumor-specific immunity reaction under central tolerance mechanism, suppress Tumour growth

Based on mutant antigen Y3, inventors determined that under under central tolerance machining function, optimization it is dual different Autoantigen reaction-ive T cell in the most effective activation Rip-Ova Mice Bodies of the immune combination DC-Y3-Lm-N4 energy in source, And then there may be potential anti tumor immune response.

The present inventor further detection when tumour antigen is autoantigen, the induction of dual alloimmunization strategy itself Whether reaction-ive T cell has antitumor action, and whether can effectively suppress tumour growth.

Rip-Ova mouse are immunized respectively with DC-Y3 or Lm-Y3, are immunized again with Lm-Y3 or Lm-N4 after 7 days. Mo5 melanoma cells are inoculated with after immune 7 days for the second time, and observe tumour growth.

As a result it is as shown in Figure 9.On Rip-Ova mouse after dual Heterologous combinations DC-Y3-Lm-N4 is immune, tumour The speed of growth change considerably slower than combination Lm-Y3-Lm-Y3 of the same race and only carrier combination DC-Y3-Lm-Y3 it is immune Tumor growth rate (Fig. 9 A) afterwards on Rip-Ova mouse.By comparing average tumor size, human hair of the present invention The immune response of existing DC-Y3-Lm-N4 combination immune induction can effectively suppress the growth (Fig. 9 B) of melanoma, Between at the same time in section, compared to the mouse not being immunized, its survival rate adds 50% (Fig. 9 C).Result above is said It is bright, the autoreactive T cell of dual alloimmunization strategy activation can specific recognition and killing tumor cell, With significant antitumor action.

Embodiment 9

The dual alloimmunization of optimization can suppress tumour growth as therapeutic tumor vaccine on tumor-bearing mice

The present inventor has been verified that the autoreactive T cell of dual alloimmunization combination DC-Y3-Lm-N4 activation Tumour cell can be killed, tumour growth is suppressed on Rip-Ova mouse.Therefore, in the present embodiment, one is entered Step detection is in the central tolerance bearing mouse model of structure, and can dual alloimmunization therapeutic strategy as therapeutic Vaccine, suppresses tumour and extends the survival period of mouse.

With expression Ova melanoma cells Mo5 subcutaneous vaccination Rip-Ova mouse, carry out within the 7th day controlling for the first time Treat, carry out within the 14th day second for the treatment of (Figure 10 A).

As a result show, compared to the control group do not treated, and other dual alloimmunization treatment groups, DC-Y3-Lm-N4 It substantially can more effectively suppress tumour growth (Figure 10 B).With the isoantigen immunization therapy group for changing carrier DC-Y3-Lm-Y3 compares (Fig. 5 D), and dual alloimmunization combination also can more effectively suppress tumour growth (figure 10C), and the survival rate (Figure 10 D) of mouse is improved.The mouse average survival time for the treatment of group is not 26.25 days, The average survival time of DC-Y3-Lm-Y3 treatment groups is 26.4 days, and the average survival time of DC-Y3-Lm-N4 treatments can Reach 32 days, compared to nearly 1 week of another two groups of extension mouse storaging currents.

Embodiment 10

Dual alloimmunization based on adjuvant improves antineoplastic specificity t cell immune response and suppresses tumour growth

(a) mouse immune

1. the 1st day, take 50 μ l (1 × 10⁶Cell) load Trp2SD or Trp2SK dendritic cells, sole 6 weeks to 8 weeks injecting immune age C57/BL6 female mices, each 25 μ l of left and right sole.

2. the 5th day, using Trp2SD or Trp2SK (50 μ g/ are only) immune mouse of intraperitoneal injection, with LPS (5 μ g/ are only) and CpG (10 μ g/) be used as adjuvant.

3. the 12nd day, repeat step 2.

4. the 19th day, mouse is put to death, Mouse spleen cells, streaming staining analysis are taken.

Wherein, Trp2SD:SVYDFFVWL(native)(SEQ ID NO.:7)；

Trp2SK:SVYKFFVWL(mutated)(SEQ ID NO.:8)

(b) streaming is dyed

1. taking mouse spleen to be placed in precooling phosphate buffer solution is ground to single cell suspension, 4 after nylon membrane filtration DEG C, 320g removes supernatant after 10min centrifugations, adds 1ml erythrocyte cracked liquids, is added after cracking 5 minutes on ice 10ml phosphate buffer solutions are terminated, 4 DEG C, 300g, and supernatant is removed in 10min centrifugations, is resuspended in 1ml 1640 and is cultivated In base.

2. spleen cell suspension is spread into 96 orifice plates, 4 × 10⁶Cell/well.Adding polypeptide stimulates (Trp2SD), Peptide concentration is 2 μ g/ml.37 DEG C, 5%CO₂Culture adds BFA after 30 minutes, concentration is 2.5 μ g/ml. Continue to stimulate culture 5 hours.

3. 24 orifice plate inner cells are transferred to flow centrifugal pipe, the 1ml streamings dyeing liquor for adding precooling is cleaned, 4 DEG C, Supernatant is removed in 320g, 5min centrifugation.

4. cell is placed on ice, often pipe adds 1 μ l Fcblocker Anti-CD16/CD32 and 50 μ l stream Formula dyeing liquor, soft concussion is mixed, and 10min is incubated on ice.

5. often pipe adds streaming antibody PE-cy7-CD8, PerCP-cy5.5-TCR β, PE-CD44 and 50 μ l streams Formula dyeing liquor, cell concussion is mixed, and lucifuge is incubated 30min on ice.

6. often pipe adds the cleaning of 1ml streamings dyeing liquor twice, 4 DEG C, 320g, supernatant is removed in 10min centrifugations.

7. the cell fixer that often pipe is added in 250 μ l kits, cell concussion is mixed, lucifuge is incubated on ice 50min。

8. the cell rupture of membranes liquid cleaning that often pipe is added in 1ml kits, concussion is mixed, 4 DEG C, 320g, 10min Supernatant is removed in centrifugation.Repeated washing 2 times.

9. cell is placed on ice, 1 μ l of often pipe addition Fcblocker Anti-CD16/CD32 and 50 μ l is thin Born of the same parents' rupture of membranes liquid, concussion is mixed, and lucifuge is incubated 10min on ice.

10. often pipe adds streaming antibody FITC-IFN γ, APC-TNF α and 50 μ l cell rupture of membranes liquid, cell Concussion is mixed, and lucifuge is incubated 30min on ice.

11. often pipe adds the cleaning of 1ml cell ruptures of membranes liquid, concussion is mixed, 4 DEG C, 320g, and 10min centrifugations are gone Clearly.300 μ l streaming dyeing liquor is finally resuspended in, is analyzed through flow cytometer loading.

Experimental result：

Initial immunity uses load changing peptide Trp2SK dendritic cells, is immunized uses Trp2SD and adjuvant again Mouse be designated as SK-SD mouse, initial immunity is immunized using load changing peptide Trp2SK dendritic cells, again The immune mouse using Trp2SK and adjuvant is designated as SK-SK mouse, and initial immunity uses load changing peptide Trp2SD Dendritic cells be immunized, be immunized be designated as SK-SD mouse using the mouse of Trp2SD and adjuvant again.

The cd8 t cell immune response of SD-SD mouse is relatively low, and the immune response of SK-SK mouse is relatively low, with SD-SD Mouse is significantly higher than SD-SD mouse and SK-SK mouse (Figure 11) without significant difference, the immune response of SK-SD mouse.

Result above is demonstrated again that, only immune equal using mutant peptide (Trp2SK) using native peptides (Trp2SD) or only The immune response of cd8 t cell can not be efficiently induced, first using mutant peptide amplification T cell storehouse, then using natural The immunization strategy of inducing peptide cross reaction is effective.

Discuss

Tumour antigen has the problem of immunogenicity is poor as autoantigen always, have impact on therapeutic tumor vaccine Application and development clinically.T cell epitope on tumour antigen is carried out amino acid modified to improve it and exempt from Epidemic focus, strengthens anti tumor immune response level, but the therapeutic effect in clinical trial is not obvious.Past Research show that modified antigen causes immune response to the cross reactivities of natural tumor antigens under central tolerance environment It is low be currently based on modified antigen therapeutic tumor vaccine effect it is poor the reason for one of, and to causing this problem point Handset system is still not very clear at present.

The present inventor utilizes the Listeria for expressing ovalbumin model antigen natural T-cell epitope or modification epitope Strain infection wild-type mice and the discovery of ovalbumin model antigen transgenosis Rip-mOva mouse, central tolerance mechanism The lower immune system of effect is not reacted native antigen, and the modified antigen of only medium affinity can induce machine after immune Body immune system produces the low-affinity T cell for intersecting identification native antigen, and T cell storehouse diversity significantly drops It is low.

By dual alloimmunization strategy, the T of identification natural tumor antigens can be activated under central tolerance environment Cell, effectively improves its affinity and multifunctionality, suppresses tumour growth, extends mouse storaging current.The present invention is The antigen optimization and screening of therapeutic tumor vaccine, and the design of immunization strategy provide theoretical foundation and instruct to anticipate Justice.

According to above-mentioned experimental result, the structure for summing up therapeutic tumor vaccine is preferably based on following several principles：

1. the selection of suitable mutant antigen, enables it as initial immunity antigen and activates quantity foot in T cell storehouse Enough autoantigen reaction-ive T cells；

2. initial immunity carrier and the again combination and change of immune carrier, it is affine to improve autoreactive T cell Power is main purpose and criterion；

3. the efficiency that native antigen is screened to T cell storehouse in being immunized again, makes the autoreactive T cell after screening More preferable expressive function cell factor.

All documents referred in the present invention are all incorporated as reference in this application, just as each document coverlet Solely it is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, this area Technical staff can make various changes or modifications to the present invention, and these equivalent form of values equally fall within right appended by the application Claim limited range.

Claims

1. the method for the enhancing anti tumor immune response of a kind of non-therapeutic, it is characterised in that including step：

2. the method as described in claim 1, it is characterised in that described " enhanced first tumour that is directed to resists Former tumor immune response ", which refers to, to be applied with carrying out initial immunity with first tumour antigen and uses first tumour The horizontal Yc of tumor immune response for the first tumour antigen that antigen is immunized in the control applied again is compared, and is walked Suddenly initial immunity is carried out in (c) with second tumour antigen to apply and applied with first tumour antigen is immune again The horizontal Yv of tumor immune response for the first tumour antigen in mammalian object is significantly higher than Yc.

3. the method as described in claim 1, it is characterised in that described " homologous and intersection " refers to institute The second tumour antigen and the first described tumour antigen stated have average 1-2 on every 10 amino acid lengths Amino acid mutation, therefore the immune response for the second tumour antigen can not only be caused, and can cause and be directed to The immune response of the intersection of first tumour antigen.

4. the method as described in claim 1, it is characterised in that the first described tumour antigen is central tolerance Tumor specific epitopes.

5. a kind of strengthen the composition product of anti tumor immune response, it is characterised in that the product includes：

Also, described first chamber and second chamber be it is independent,

6. composition product as claimed in claim 5, it is characterised in that described composition product is to be used for Treat or prevent the knurl seedling or vaccine combination of tumour.

7. the purposes of the composition product described in a kind of claim 5, it is characterised in that anti-for preparing enhancing The medicine of tumor immunity.

8. a kind of select many for the candidate of the anti tumor immune response of the tumour antigen of low immunogenicity with enhancing The method of peptide based immunogens, it is characterised in that including step：

9. method as claimed in claim 8, it is characterised in that in step (c), including：Will be described each Qj is ranked up, and the maximum in Qj is defined as into Qmax, and select Qj/Qmax ratio R for 0.02-0.80 Polypeptide immunogen, be used as with enhancing for low immunogenicity tumour antigen anti tumor immune response candidate Polypeptide immunogen.

10. method as claimed in claim 8, it is characterised in that described method also includes step：(d) it is right The candidate polypeptide immunogene picked out in previous step, tests its enhancing for the tumour antigen of low immunogenicity The ability of anti tumor immune response.