WO2017177910A1

WO2017177910A1 - Novel immune strategy and immune composition for enhancing anti-tumour immune response

Info

Publication number: WO2017177910A1
Application number: PCT/CN2017/080156
Authority: WO
Inventors: 冷启彬; 郭昵宁; 陈宪洋; 胡广; 金兴魏
Original assignee: 中国科学院上海巴斯德研究所
Priority date: 2016-04-11
Filing date: 2017-04-11
Publication date: 2017-10-19
Also published as: CN107281474A

Abstract

Provided are a novel immune strategy and an immune composition for enhancing the anti-tumour immune response. In particular, the method of the present invention comprises the steps of: (a) providing a first tumour antigen and a second tumour antigen, wherein the enhanced tumour immune response is the enhanced tumour immune response against the first tumour antigen; the first tumour antigen has a low immunogenicity; and the second tumour antigen is a polypeptide that is homologous to and crossed with the first tumour immune antigen or an immunogenic fragment thereof; (b) immunising and administering the second tumour antigen to the mammalian subject, and stimulating an immune response against the second tumour antigen; and (c) immunising and administering the first tumour antigen to the mammalian subject that has undergone the primary immune challenge, and thereby producing an enhanced tumour immune response against the first tumour antigen. Also provided are an immune composition for use in the method of the present invention and the use thereof.

Description

Novel immunization strategies and immunological compositions that enhance anti-tumor immune responses

Technical field

The present invention is in the field of immunology; more specifically, it relates to novel immunization strategies and immunological compositions that enhance anti-tumor immune responses.

Background technique

Tumors are currently the main disease that poses a serious threat to human health and causes human death. It is imperative to develop effective cancer treatment drugs.

Therapeutic tumor vaccines can provide an immune response to tumor antigens by activating the body's immune system, specifically removing solid tumor tissue and forming immune memory to provide lifelong protection for patients. Compared with traditional cancer treatment methods (surgical resection, radiotherapy and chemotherapy) and other immunotherapy methods, therapeutic tumor vaccines are more likely to be used on patients without active side effects. .

Although research on therapeutic tumor vaccines has made some progress, overall, tumor vaccine vaccines have shown limited efficacy in clinical trials. Although many trials have shown that some patients with advanced cancer have good clinical outcomes, few trials are able to achieve partial or complete remission of 5% to 10%. Low clinical efficacy is a major problem in vaccine development. Before 2003, at least 200 vaccines were tested in Phase II or Phase III clinical trials, and most of the vaccines in clinical trials proved to be ineffective in activating tumor antigen-specific T cells in patients to produce immune response-suppressing tumors. Growth, only 2.6% of patients developed anti-tumor immunity under the action of vaccine

Tumor antigens as autoantigens have always had poor immunogenicity, which has affected the clinical application and development of therapeutic tumor vaccines.

An important reason for the effectiveness of therapeutic tumor vaccines is the inhibition of the central tolerance mechanism against tumor immune responses. The central tolerance mechanism refers to immune tolerance caused by high-affinity T cells being cleared by a negative selection mechanism during contact with their specifically recognized autoantigens during thymic development.

Although various techniques have been developed in the prior art, such as the use of adjuvants, the use of mutated tumor antigens, and the like, in order to enhance the level of anti-tumor immune response, the therapeutic effect in clinical trials is not significant.

In summary, there is a lack of satisfactory tumor vaccines with high anti-tumor effects in the art. Therefore, there is an urgent need in the art to develop new methods and compositions that are effective in enhancing anti-tumor immune responses in order to achieve better anti-tumor prophylaxis or therapeutic effects.

Summary of the invention

It is an object of the present invention to provide a method and composition for enhancing an anti-tumor immune response and uses thereof.

In a first aspect of the invention, there is provided a method of non-therapeutic or therapeutic enhancing an anti-tumor immune response comprising the steps of:

(a) providing a first tumor antigen and a second tumor antigen, wherein said enhanced tumor immune response is an enhanced tumor immune response against the first tumor antigen;

The first tumor antigen is a low immunogenic natural polypeptide derived from a mammal or an immunogenic fragment thereof, and is not efficiently produced when the first tumor antigen is immunologically administered to the mammal. An effective anti-tumor immune response against said first tumor antigen;

The second tumor antigen is a polypeptide homologous and intersecting with the first tumor immune antigen or an immunogenic fragment thereof;

And the affinity of the first tumor antigen to antigen-specific CD8 ⁺ T cells from the mammal is Q1, and the affinity of the second tumor antigen to antigen-specific CD8 ⁺ T cells from the mammal is Q2 And the ratio R1 of the Q2/Q1 is 0.02-0.80;

(b) immunizing said second tumor antigen to said mammalian subject, eliciting an immune response against said second tumor antigen, thereby obtaining a first immune challenged mammalian subject;

(c) immunizing said first tumor antigen to said primary immunized mammal after t days after immunization (or second tumor antigen administration) of the previous step, wherein t is 3-60 Subject, eliciting an immune response against the first tumor antigen to obtain a re-immunized mammalian subject, wherein in the re-immunized mammalian subject, an enhanced tumor immunity against the first tumor antigen is produced Answer.

In another preferred embodiment, the "enhanced tumor immune response against the first tumor antigen" refers to a control in which a primary immunization with the first tumor antigen is administered and re-immunized with the first tumor antigen. Comparing the tumor immune response level Yc of the first tumor antigen, the first tumor in the mammalian subject administered in the step (c) with the second tumor antigen for primary immunization and re-immunized with the first tumor antigen The tumor immune response level Yv of the antigen was significantly higher than Yc.

In another preferred embodiment, said "Yv is significantly higher than Yc" means that the ratio of Yv/Yc is R2 ≥ 1.5, preferably ≥ 2, more preferably ≥ 5, more preferably ≥ 10, most preferably ≥ 20 .

In another preferred embodiment, the tumor immune response level is the number and/or proportion of antigen-specific CD8 ⁺ T cells in the immunized mammalian T cells.

More preferably, the level of tumor immune response comprises: detecting the secretion level of a cytokine (such as gamma interferon) after stimulation of a mammalian T cell with the first antigen in vitro, or using a major histocompatibility complex (MHC) - The tetramer or multimer of the epitope polypeptide is analyzed by flow cytometry for the number and proportion of antigen-specific CD8 ⁺ T cells.

In another preferred embodiment, said t is 4-45, preferably t is 5-30, more preferably t is 6-25, and most preferably t is 7-21.

In another preferred embodiment, the ratio R1 of Q2/Q1 is from 0.04 to 0.60, preferably from 0.05 to 0.50, more preferably from 0.06 to 0.40.

In another preferred embodiment, the affinity of the first tumor antigen to the T cell is between 9 and 100 nanomolar (uM), and/or the affinity of the second tumor antigen to the T cell is between 0.01 and 10 nanomolar. .

In another preferred embodiment, the affinity is defined as follows: the strength of binding between the MHC-epitope polypeptide and the T cell receptor.

In another preferred embodiment, the functional affinity of the inverse of the affinity or IC _50.

In another preferred embodiment, the functional affinity refers to a polypeptide concentration required for the epitope polypeptide to stimulate T cells to secrete a cytokine (such as gamma interferon) to a maximum of 50%.

In another preferred embodiment, the "ineffective to produce an effective anti-tumor immune response" refers to an anti-tumor immune response rate against the first tumor antigen when the immune administration to the mammal is performed. ≤ 25%, preferably ≤ 15%, more preferably ≤ 10%, more preferably ≤ 5%, most preferably ≤ 3%.

In another preferred embodiment, the first tumor antigen is an epitope polypeptide (i.e., a polypeptide fragment) derived from a wild-type tumor-associated antigen (protein) of the mammal.

In another preferred embodiment, the "homologous and crossed" means that the second tumor antigen and the first tumor antigen have an average of 1-2 amino acid mutations per 10 amino acids in length, Thus, not only can an immune response against the second tumor antigen be caused, but also an immune response to the cross of the first tumor antigen can be caused.

In another preferred embodiment, the second tumor antigen is an antigen having high autoreactivity and high cross-reactivity.

In another preferred embodiment, the first tumor antigen and the second tumor antigen are derived or derived from the same tumor-associated antigen.

In another preferred embodiment, the first tumor antigen and the second tumor antigen are derived or derived from the same segment of the same tumor associated antigen.

In another preferred embodiment, the first tumor antigen and the second tumor antigen are epitope polypeptides of length n1 and n2 amino acids, respectively, wherein n1 and n2 are respectively 6-200, preferably 7-100, More preferably 8-50, optimally a positive integer of 8-25 or 8-12.

In another preferred embodiment, the mammal comprises a non-human mammal and a human.

In another preferred embodiment, the mammal comprises a primate and a rodent (e.g., mouse, rat).

In another preferred embodiment, the first tumor antigen is a centrally tolerated tumor-specific epitope.

In another preferred embodiment, the first tumor antigen is derived from a tumor-associated antigen selected from the group consisting of NY-ESO-1, Her2, EGFR, CEA, GPC3, AFP, PAP, PSA, PSMA, PSCA, or Its combination.

In another preferred embodiment, the "immunization administration" means that the tumor antigen or dendritic cells (DC) sensitized by the tumor antigen are directly or indirectly administered to the subject, thereby eliciting immunity. Respond to the reaction.

In another preferred embodiment, the "immune administration" includes administration in the form of a vaccine composition.

In another preferred embodiment, the vaccine composition comprises: (i) the tumor antigen (such as a first tumor antigen or a second tumor antigen) and/or dendritic cells sensitized by the tumor antigen (ii) an optional adjuvant; and (iii) a pharmaceutically or immunologically acceptable carrier.

In another preferred embodiment, the dendritic cells are derived from the same mammal as the first tumor antigen.

In a second aspect of the invention, there is provided a composition product for enhancing an anti-tumor immune response, the product comprising:

(1) a first composition comprising (i) a first tumor antigen and/or dendritic cells sensitized by said first tumor antigen; (ii) an optional adjuvant; And (iii) a pharmaceutically or immunologically acceptable carrier;

(ii) a second composition comprising (i) a second tumor antigen and/or dendritic cells sensitized by said second tumor antigen; (ii) an optional adjuvant; And (iii) a pharmaceutically or immunologically acceptable carrier;

And, the first composition and the second composition are independent,

Wherein the first tumor antigen is a low immunogenic natural polypeptide derived from a mammal or an immunization thereof An original fragment, and when the first tumor antigen is immunized to the mammal, is unable to effectively produce an effective anti-tumor immune response against the first tumor antigen;

And the affinity of the first tumor antigen to antigen-specific CD8 ⁺ T cells from the mammal is Q1, and the affinity of the second tumor antigen to antigen-specific CD8 ⁺ T cells from the mammal is Q2 And the ratio R1 of the Q2/Q1 is 0.02-0.80,

And the enhanced tumor immune response is an enhanced tumor immune response against the first tumor antigen.

In another preferred embodiment, the product further comprises: (iii) a third composition comprising an immune critical point inhibitor for enhancing an immune response.

In another preferred embodiment, the immune key inhibitor of the immune response comprises a CTLA-4 antibody, a PD-1 antibody, and a PD-L1/2 antibody.

In another preferred embodiment, the composition product is a tumor vaccine or vaccine composition for treating or preventing a tumor.

In another preferred embodiment, the vaccine composition further contains an adjuvant.

In another preferred embodiment, the vaccine composition is a nucleic acid vaccine composition.

In another preferred embodiment, the adjuvant comprises an adjuvant that is itself immunogenic and an adjuvant that is not immunogenic in itself. Preferably, the immunogenic adjuvant itself comprises B. pertussis, acid-fast bacilli (Mycobacterium tuberculosis), and Gram-negative bacilli, etc.; the non-immunogenic adjuvant itself comprises aluminum hydroxide Calcium phosphate, alum adjuvant, liposome, Freund's adjuvant, and various cytokines and chemokines.

In another preferred embodiment, the adjuvant comprises alumina, saponin, muramyl dipeptide, mineral oil or vegetable oil, vesicle-based adjuvant, nonionic block copolymer or DEAE dextran, cytokines (Including IL-1, IL-2, IFN-r, GM-CSF, IL-6, IL-12, IL-15, and CpG).

In another preferred embodiment, the adjuvant is selected from the group consisting of a cytokine, a chemokine, or a combination thereof.

In another preferred embodiment, the cytokine is IL-15.

In a third aspect of the invention, there is provided the use of a composition of the invention according to the second aspect of the invention for the preparation of a medicament for enhancing an anti-tumor immune response.

In another preferred embodiment, the medicament is for treating a tumor selected from the group consisting of liver cancer, lung cancer, stomach cancer, breast cancer, ovarian cancer, prostate cancer, skin cancer, melanoma, cervical cancer, brain cancer, thyroid cancer, and Cholangiocarcinoma, bladder cancer and pancreatic cancer.

In another preferred embodiment, the subject to be treated includes humans and non-human mammals.

In a fourth aspect of the invention, there is provided a method (non-diagnostic and non-therapeutic) for selecting a candidate polypeptide immunogen having an anti-tumor immune response against a tumor antigen of low immunogenicity, comprising the steps of:

(a) providing a test group comprising i polypeptide immunogens to be selected, wherein said polypeptide immunogens are homologous and intersecting, wherein i is a positive integer of ≥1,

Also, "homologous and crossed" refers to a polypeptide immunogen (A antigen or natural antigen) and the test Another polypeptide immunogen (B antigen or similar antigen) in the group has an average of 1-2 amino acid mutations per 10 amino acids in length, thus not only capable of eliciting an immune response against the antigen in mammals, but also a cross-over immune response of the adjacent antigen;

Wherein at least one of the polypeptide immunogens is a mutant polypeptide immunogen;

(b) determining the reaction affinities of the respective polypeptide immunogens and the antigen-specific CD8 ⁺ T cells elicited by each of the polypeptides, respectively, as Qj, wherein any positive integer of j = 1 - i;

(c) sorting each of the Qjs, selecting a polypeptide immunogen that is located in the middle, as a candidate polypeptide immunogen having an anti-tumor immune response that enhances the antigen against the low immunogenic tumor; and/or performing each Qj and Qz Comparing, the polypeptide immunogen having a ratio R of Qj/Qz of 0.02-0.80 is selected as a candidate polypeptide immunogen having an anti-tumor immune response against a low immunogenic tumor antigen, wherein Qz is the first tumor An affinity of an antigen to antigen-specific CD8 ⁺ T cells from said mammal, and said first tumor antigen is a low immunogenic natural tumor antigen derived from a mammal or an immunogenic fragment thereof, and When the first tumor antigen is immunized to the mammal, it is not effective to produce an effective anti-tumor immune response against the first tumor antigen.

In another preferred embodiment, in the step (c), the method comprises: sorting the respective Qjs, defining a maximum value in Qj as Qmax, and selecting a polypeptide immunogen having a ratio R of Qj/Qmax of 0.02-0.80. , as a candidate polypeptide immunogen with an anti-tumor immune response that enhances tumor antigens against low immunogenicity.

In another preferred embodiment, in step (c), the method comprises: sorting the respective Qjs and selecting the closest to the arithmetic mean Qaverage of Q or the median Qmean (ie, |Qaverage-Qj| or |Qmean -Qj|minimum) s polypeptide immunogens, and s is a positive integer <i, i is a positive integer ≥3, and is ≥2.

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

In another preferred embodiment, when i > 3, the selected polypeptide immunogen does not include the polypeptide immunogen with the smallest Qj and the largest Qj.

In another preferred embodiment, the ratio R of Qj/Qz or Qj/Qmax is from 0.04 to 0.60, preferably from 0.05 to 0.50, more preferably from 0.06 to 0.40.

In another preferred embodiment, the method further comprises the step of: (d) testing the candidate polypeptide immunogen selected in the previous step for its ability to enhance the anti-tumor immune response against a low immunogenic tumor antigen.

In another preferred embodiment, the step (d) comprises: firstly immunizing the mammal with the candidate polypeptide immunogen HX selected in the previous step, thereby stimulating the immunogen against the candidate polypeptide. The immune response of HX to obtain a mammalian subject that has been primed by primary immunization;

And after t days after the initial immunization administration operation, wherein t is 3-60, the first tumor antigen is immunized to the first immunized mammalian subject, and the first tumor is challenged The immune response of the antigen, and determining the level of tumor immune response to the first tumor antigen in the re-immunized mammalian subject, thereby determining whether the candidate polypeptide immunogen HX has an anti-tumor that enhances tumor antigens against low immunogenicity The ability and/or degree of enhancement of the immune response.

In a fifth aspect of the invention, there is provided a use of a second tumor antigen which is a polypeptide homologous and intersecting with a first tumor immune antigen or an immunogenic fragment thereof, wherein said A second tumor antigen is used to prepare a pharmaceutical composition (a) for enhancing an anti-tumor immune response against a first tumor immune antigen in a mammal; and/or (b) for breaking through breastfeeding The animal's immune tolerance to autoantigens enhances the anti-tumor immune response.

In another preferred embodiment, the phrase "homologous and crossed" means that the first tumor antigen (A antigen) and the second tumor antigen (B antigen) are in the matching region and have an average of 1 per 10 amino acids in length. - 2 amino acid mutations.

In another preferred embodiment, the second tumor antigen is an epitope polypeptide of n2 amino acids in length, wherein n2 is 6-200, preferably 7-100, more preferably 8-50, optimally 8- A positive integer of 25 or 8-12.

In a sixth aspect of the invention, a method of enhancing an anti-tumor immune response is provided, comprising the steps of:

(a) providing an isolated lymphocyte from a patient selected from the group consisting of tumor infiltrating lymphocytes (TIL), peripheral blood lymphocytes (PBMC), or a combination thereof; and providing a first tumor An antigen and a second tumor antigen;

Wherein the first tumor antigen is a low immunogenic natural polypeptide derived from a mammal or an immunogenic fragment thereof, and is ineffective when the first tumor antigen is immunologically administered to the mammal Producing an effective anti-tumor immune response against the first tumor antigen;

(b) culturing said TIL lymphocytes and/or PBMC lymphocytes in the presence of a second tumor antigen; and then culturing again in the presence of the first tumor antigen to obtain sensitized T cells;

(c) optionally passage of said sensitized lymphocytes to obtain passaged, sensitized T cells;

(d) The sensitized T cells obtained in the previous step are returned to the patient.

In another preferred embodiment, in the step (b), the culturing further comprises performing in vitro T cell culture in a culture system containing dendritic cells, cytokines or a combination thereof.

In another preferred embodiment, the method further comprises: detecting the sensitized T during and after step (b), after and after step (c), or before and during step (d) The affinity of the cells for the first tumor antigen and/or the second tumor antigen.

In another preferred embodiment, the affinity of the first tumor antigen to antigen-specific CD8 ⁺ T cells from the mammal is Q1, and the second tumor antigen is antigen-specific CD8 ⁺ from the mammal. The affinity of the T cells is Q2, and the ratio R1 of the Q2/Q1 is 0.02-0.80 (ie, the affinity of the second tumor antigen is lower than the affinity of the first tumor antigen).

In a seventh aspect of the invention, there is provided a method of enhancing an anti-tumor immune response comprising the steps of:

The first tumor antigen is a low immunogenic natural polypeptide derived from a mammal or an immunogenic fragment thereof, and is not efficiently produced when the first tumor antigen is immunologically administered to the mammal. Effective targeting An anti-tumor immune response of said first tumor antigen;

(c) after t days after the immunization administration operation of the previous step, wherein t is 3-60, immunizing said first tumor antigen to said primary immunized mammalian subject, eliciting said An immune response to a tumor antigen to thereby obtain a re-immunized mammalian subject, wherein in the re-immunized mammalian subject, an enhanced tumor immune response against the first tumor antigen is produced.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

DRAWINGS

Figure 1 shows the amino acid sequence of the Ova natural epitope N4 and the mutant antigen.

Figure 2 shows the functional affinity of native antigen N4 and mutant antigen for endogenous polyclonal T cells. Among them, (A) WT mice were injected with Lm-N4 5000cfu/day on the 7th day after the tail vein injection, and the spleen cells were stimulated with a gradient of natural antigen and mutant antigen (10 ^-12 M-10 ^-6 M) in vitro. IFN-γ ⁺ CD8 ⁺ T cell ratio. The dose-response curve (n ≥ 4) is fitted according to its value and the corresponding stimulus concentration; (B) the EC ₅₀ corresponding to each dose-effect curve is calculated, and the reciprocal (1/EC ₅₀ ) represents relative functional affinity (relative (C) WT mice were injected intravenously with Lm-N45000 cfu/day, and spleen cells were cultured in vitro with natural antigen or mutant antigen 2 μg/ml to detect the proportion of IFN-γ ⁺ CD8 ⁺ T cells.

Figure 3 shows that the central tolerance mechanism makes the T cell pool more prone to respond to moderate affinity mutant antigens. Among them, (A) the statistical mean value (n ≥ 4) of the ratio of IFN-γ ⁺ CD8 ⁺ T cells recognizing the antigen itself after infection of the natural antigen and the mutant antigen. (B) Statistical mean (n ≥ 4) of the ratio of IFN-γ ⁺ CD8 ⁺ T cells recognizing N4 after infection of the natural antigen and the mutant antigen. (C) IFN-γ ⁺ CD8 ⁺ T cell ratio of WT mouse and Rip-mOva mouse infected with natural antigen N4 after infection with Lm-N4 or mutant tail vein, and IFN-γ reacting with the corresponding antigen ⁺ Ratio of CD8 ⁺ T cell ratio (n ≥ 4).

Figure 4 shows the construction of a Rip-mOva mouse melanoma model based on Ova antigen tolerance. Here, (A) As shown, 2×10 ⁴ , 10×10 ⁴ , and 50×10 ⁴ cell numbers of Mo5 were subcutaneously inoculated into WT mice, and tumor size was measured every 3 days and recorded. When the average of the long diameter and short diameter length of the tumor exceeded 20 mm, the mice were sacrificed and the mice were considered dead. (B) 50×10 ⁴ cell number of Mo5 was subcutaneously inoculated into Rip-mOva mice, and tumor size was measured every 3 days and recorded. When the average of the long diameter and short diameter length of the tumor exceeded 20 mm, the mice were sacrificed and the mice were considered dead. (C) Survival curves of WT tumor-bearing mice and Rip-mOva tumor-bearing mice after inoculation with 50 × 10 ⁴ cells (n ≥ 7). (D) 50×10 ⁴ cell number The ratio of N4-tetramer ⁺ CD44 ⁺ CD8 ⁺ T cells in the spleen and inguinal draining lymph nodes of Rip-mOva tumor-bearing mice 20 days after inoculation. (E) 50×10 ⁴ cell number 20 days after inoculation, spleen and inguinal draining lymph node cells of Rip-mOva tumor-bearing mice were stimulated with natural antigen N4 or mutant antigen Y3 in vitro to detect cytokine IFN-γ in CD8 ⁺ T cells. And expression of TNF-α.

Figure 5: The central tolerance mechanism renders low-affinity autoreactive T cells unable to induce potent anti-tumor immune responses. Among them, (A) WT mice were infected with 5×10 ⁵ /Mo5 melanoma cells subcutaneously 7 days after infection of Lm-N4 or Lm-Y3 in the tail vein. On the 6th day after Mo5 inoculation, the tumor size was measured every 2 days, and the tumor volume was calculated. (B) Rip-mOva mice were subcutaneously inoculated with 5 x 10 ⁵ /Mo5 melanoma cells 7 days after infection of Lm-N4 or Lm-Y3 in the tail vein. On the 6th day after Mo5 inoculation, the tumor size was measured every 2 days, and the tumor volume was calculated. (C) WT mice and Rip-mOva mice were infected with Lm-N4 or Lm-Y3 in the tail vein 7 days later, and spleen cells were stimulated in vitro with a gradient of N4 (10 ^-12 M-10 ^-6 M) to detect IFN. -γ ⁺ CD8 ⁺ T cell ratio. A dose-response curve was fitted based on its value and the corresponding stimulus concentration. WT Sp. T cells: Lm-N4 infected WT mice; WT CR. T cells: Lm-Y3 infected WT mice; RO CR. T cells, Lm-Y3 infected Rip-mOva mice. (D) calculating for each dose - effect curve corresponding EC _50, taking the inverse (1 / EC ₅₀₎ representing the relative functional affinity.

Figure 6 shows the diversity of Vβ use of autoantigen-reactive T cell pools under central tolerance. Among them, (A) WT mice and Rip-mOva mice were injected intravenously with Lm-Y3 on the 7th day, N4-tetramer antibody and Y3-tetramer antibody were double-stained to detect the proportion of CD44 ⁺ CD8 ⁺ T cells recognizing different tetramers. . (B) Seven days after infection of Lm-N4 or Lm-Y3 in the tail vein of WT mice and Rip-mOva mice, spleen cells were cultured in vitro with N4, and the ratio of different Vβ was measured for IFN-γ ⁺ CD8 ⁺ T cells. WT Sp. T cells: Lm-N4 infected WT mice; WT CR. T cells: Lm-Y3 infected WT mice; RO CR. T cells, Lm-Y3 infected Rip-mOva mice. (C) Seven days after infection of Lm-Y3 in the tail vein of Rip-mOva mice, spleen cells were cultured in vitro with Y3, and the ratio of different Vβ ratios of IFN-γ ⁺ CD8 ⁺ T cells was examined. (D) Seven days after infection of Lm-Y3 in the tail vein of Rip-mOva mice, spleen cells were cultured in vitro with Y3, and the ratio of IFN-γ ⁺ CD8 ⁺ T cells using different Vβ was measured (n ≥ 8). (E) Comparison of different Vβ ratios between WT Sp. T cells and RO CR. T cells. (F) Comparison of WT CR. T cells and RO CR. T cells using different Vβ ratios.

Figure 7 shows that repeated immunization with an alloantigen vaccine does not induce an effective anti-tumor immune response. Among them, (A) WT mice were treated with intraperitoneal injection of Lm strain expressing the corresponding antigen starting from the 7th day after inoculation of melanoma cells Mo5 at a dose of 10 ⁴ cfu/head. The treatment was repeated 3 times, one interval at a time. On day 6 of Mo5 inoculation, tumor size was measured every 2 days and the volume was calculated. (B) Rip-Ova mice were treated with intraperitoneal injection of Lm strain expressing the corresponding antigen starting on day 7 after inoculation of melanoma cells Mo5. The treatment was repeated 3 times, one interval at a time. (C) WT mice were treated with Lm-N4 or Lm-Y3 on the 7th day after inoculation of melanoma cells Mo5, and the treatment was repeated 3 times, one interval at a time. On the 30th day, the mice were sacrificed, and the spleen, inguinal draining lymph nodes, and mesenteric lymph node cells were taken and stimulated with natural antigen N4 in vitro to detect the proportion of IFN-γ ⁺ CD8 ⁺ T cells. (D) Rip-Ova mice were started on day 7 after inoculation of melanoma cells Mo5, and were treated with CpG-activated DC-loaded mutant antigen Y3, which was treated with footpad immunization at a dose of 10 ⁶ cells/cell. The treatment was repeated 3 times, one interval at a time. (E) Rip-Ova mice were started on day 7 after inoculation of melanoma cells Mo5, and CpG-activated DC-loaded mutant antigen Y3 was used for the first treatment with a footpad immunization at a dose of 10 ⁶ cells/room; one week later, Lm-Y3 was intraperitoneally injected for a second treatment at a dose of 10 ⁴ cfu/head.

Figure 8 shows that the optimized dual heterologous immunization strategy enhances the functionality and affinity of autoreactive T cells. Among them, (A) Rip-Ova mice were immunized with Lm-Y3 or DC-Y3 for the first time; one week after the experiment, the same mutant antigen Y3 was used for re-immunization. After 4 days, the mouse spleens were stimulated with N4 in vitro, and the ratio of IFN-γ ⁺ CD8 ⁺ T cells, TNF-α ⁺ CD8 ⁺ T cells and IFN-γ ⁺ TNF-α ⁺ CD8 ⁺ T cells was detected (n ≥ 4). (B) According to the experimental description, Rip-Ova mice were subjected to a dual heterologous immunization strategy at intervals of one week and in two immunizations. After 4 days of the second immunization, the spleen was stimulated with N4 in vitro, and the ratio of IFN-γ ⁺ CD8 ⁺ T cells, TNF-α ⁺ CD8 ⁺ T cells, and IFN-γ ⁺ TNF-α ⁺ CD8 ⁺ T cells was measured. (C) Rip-Ova mice were immunized once with Lm-Y3, twice with Lm-Y3, DC-Y3-Lm-Y3, DC-Y3-Lm-N4, and spleen cells in vitro. The concentration of IFN-γ ⁺ CD8 ⁺ T cells corresponding to the stimulation concentration was detected by gradient concentration N4 stimulation, and the dose-response curve was fitted. Effect curve corresponding EC _50, taking the inverse (1 / EC ₅₀₎ representing the relative functional affinity - each dose (D) is calculated in FIG. 6C. (E) Rip-Ova mice were immunized with a DC-vaccine, and immunized with an Lm-vaccine that altered the antigen to detect the ratio of CD8 ⁺ T cells expressing cytokines IFN-γ and TNF-α. (F) Rip-Ova mice were immunized with DC-Y3-Lm-N4, and spleen cells were stimulated in vitro with a gradient concentration of N4 to determine the ratio of use of each Vβ in IFN-γ ⁺ CD8 ⁺ T cells. (G) Proportion of Vβ prevalent in RO CR.T cells produced by immunization with Rm-Y3 in Rip-Ova mice (Fig. 4B) and IFN-γ ^{+ in} N4 after immunization with DC-Y3-Lm-N4 Comparison in CD8 ⁺ T cells.

Figure 9 shows that autoreactive T cells activated by dual heterologous immunization strategies have anti-tumor effects. Among them, (A) Rip-Ova mice were pre-exempted with Lm-Y3 5000 cfu/only or DC-Y3 10 ⁶ cells/day, and Lm-Y35000 cfu/only or Lm-N4 5000 cfu/mouse after 7 days. Re-immunization enhances the response. On day 0, melanoma cells were inoculated with Mo55×10 ⁵ cells/cell. Tumor size was measured every 2 days and volume was calculated starting from day 6 after Mo5 inoculation. (n=3 or 4). (B) The average tumor volume of each group of mice at each time point after inoculation of Mo5 in each immunization group of Rip-Ova mice. (C) Survival rate of Rip-Ova mouse DC-Y3-Lm-N4 immunized group, Lm-Y3-Lm-Y3 and non-immunized mice after inoculation with Mo5.

Figure 10 shows that dual heterologous immune energy acts as a therapeutic vaccine to inhibit tumor growth in tumor-bearing mice. Among them, (A) Rip-Ova mice were inoculated with melanoma Mo5 5×10 ⁵ /piece, the first immunotherapy was given on the 7th day, and the second immunotherapy was performed on the 14th day. DC vaccine footpad immunotherapy, the dose of 1 × 10 ⁶ / only; Lm vaccine was injected intraperitoneally, the dose was 10000 cfu / only. (B) Dual heterologous immunotherapy of tumor-bearing Rip-Ova mice according to the procedure of Figure 10A. On the 6th day after Mo5 inoculation, the tumor size was measured every 2 days and the volume was calculated, and the blood glucose concentration of the mouse was measured. (C) Mean tumor volume of mice in the Rip-Ova mice without treatment group, DC-Y3-Lm-Y3 treatment group and DC-Y3-Lm-N4 treatment group at each time point (n≥5) ). (D) Survival curves of mice in the untreated group of Rim-Ova mice, DC-Y3-Lm-Y3 group, and DC-Y3-Lm-N4 group.

Figure 11 shows the ability of the mouse melanoma natural antigen Trp2SD and the mutant antigen to induce a specific CD8 T cell immune response. Among them, (A) the amino acid sequence of the mouse melanoma natural antigen Trp2SD and the mutant antigen. (B) Statistical mean (n ≥ 8) of the ratio of IFN-γ ⁺ CD8 ⁺ T cells recognizing the antigen itself after immunization with the natural antigen and the mutant antigen. (C) Statistical mean (n ≥ 8) of the proportion of IFN-γ ⁺ CD8 ⁺ T cells recognizing Trp2SD after immunization with natural antigen and mutant antigen.

Figure 12 shows that the dual heterologous immunization strategy based on the melanoma antigen Trp2SD can increase the anti-tumor specific T cell immune response and significantly inhibit tumor growth. C57/B6 mice were immunized with DC-Trp2SK and DC-TrpSD for the first time; 4 after LPS+CpG+SK/SD for the second immunization. Seven days after the first immunization, the mouse spleens were stimulated with Trp2SD in vitro, and (A and D) were used to detect IFN-γ ⁺ CD8 ⁺ T cells, (B and E) TNF-α ⁺ CD8 ⁺ T cells and (C and F) IFN. -γ ⁺ TNF-α ⁺ CD8 ⁺ T cell ratio (n ≥ 4); (G) C57/B6 mice were inoculated with melanoma Mo5 5 × 10 ⁵ / only, on the 7th day with DC-Trp2SK, DC-TrpSD The first treatment was performed, the second treatment was performed with LPS+CpG+SK/SD on the 11th day, the third treatment was performed with LPS+CpG+SK/SD on the 18th day, and the second treatment was started on the 6th day of Mo5 vaccination. The tumor size was measured and the volume was calculated (n ≥ 10).

Figure 13 Figure 11 shows the ability of the mouse melanoma natural antigen gp100 and the mutant antigen to induce a specific CD8 T cell immune response. Among them, (A) the amino acid sequence of the mouse melanoma natural antigen gp100 and the mutant antigen. (B) Statistical mean (n ≥ 4) of the ratio of IFN-γ ⁺ CD8 ⁺ T cells recognizing the antigen itself after immunization with the natural antigen and the mutant antigen. (C) Statistical mean (n ≥ 4) of the ratio of IFN-γ ⁺ CD8 ⁺ T cells recognizing gp100 after immunization with natural antigen and mutant antigen.

Figure 14 shows that the dual heterologous immunization strategy based on the melanoma antigen gp100 can increase the anti-tumor specific T cell immune response and significantly inhibit tumor growth. C57/B6 mice were immunized with DC-gp100, DC-MK gp100 for the first time; 4 after LPS+CpG+gp100/MK gp100 for the second immunization. Seven days after the first immunization, the mouse spleens were stimulated with gp100 in vitro to detect (A) IFN-γ ⁺ CD8 ⁺ T cells, (B) TNF-α ⁺ CD8 ⁺ T cells, and (C) IFN-γ ⁺ TNF-α. ⁺ CD8 ⁺ T cell ratio (n ≥ 4); (D) C57/B6 mice were vaccinated with melanoma Mo5 5 × 10 ⁵ / only, and the first treatment with DC-gp100 or DC-MK gp100 on day 7 On the 11th day, LPS+CpG+gp100/MKgp100 was used for the second treatment. On the 18th day, LPS+CpG+gp100/MKgp100 was used for the third treatment. On the 6th day after Mo5 inoculation, the tumor size was measured every 2 days. Calculate the volume, (E) and record the survival rate of tumor-bearing mice.

Figure 15 shows the promotion of T cell responses by different cytokines. C57/B6 mice were immunized with DC-MK gp100 or DC-Trp2I2 for the first time, and then treated with DC+gp100+PBS/CCL3/IL12/IL15 or DC+Trp2SD+PBS/CCL3/IL12/IL15 for 4 days. Secondary immunization, 7 days after the first immunization, the mouse spleen was stimulated with gp100 in vitro, and (A and D) IFN-γ ⁺ CD8 ⁺ T cells, (B and E) TNF-α ⁺ CD8 ⁺ T cells and (C, respectively) were detected. And F) the ratio of IFN-γ ⁺ TNF-α ⁺ CD8 ⁺ T cells (n ≥ 4).

Figure 16 shows the effect on T cell immune response after deletion of regulatory T cells with CD25 antibody. C57/B6 mice were intraperitoneally injected with 250 μg CD25 antibody and their isotype control on the 4th day before immunization, and were immunized with DC-gp100 or DC-MK gp100 respectively. The spleen of the mice was stimulated with gp100 in vitro on the 7th day after immunization. (A) Ratio of IFN-γ ⁺ CD8 ⁺ T cells, (B) TNF-α ⁺ CD8 ⁺ T cells, and (C) IFN-γ ⁺ TNF-α ⁺ CD8 ⁺ T cells (n ≥ 4).

detailed description

The inventors have extensively and intensively studied, and through a large number of screenings and experiments, for the first time, a method for enhancing an antitumor immune response and a composition for the method have been developed. The method of the invention can effectively activate T cells recognizing natural tumor antigens in a central tolerance environment by a dual heterologous immunization strategy, and effectively improve the affinity and versatility thereof, thereby achieving the effects of inhibiting tumor growth and prolonging survival. The present invention has been completed on this basis.

Specifically, the present inventors constructed an Ova (N4)-specific T cell central tolerance model using Rip-mOva mice expressing the pattern antigen Ova as an autoantigen, and using the recombinant Listeria strain of N4 and its five mutant antigens as The model vaccine infects the transgenic mice and studies the recognition and response of the body's T cell pool to natural (self) antigens and mutant antigens under the mechanism of central tolerance.

The present inventors have found that central tolerance not only completely inhibits the immune system's response to the autoantigen N4, but also partially inhibits the response to the mutant antigen. The moderately affine mutant antigen Y3 can induce the most cross-recognition of the natural antigen N4 T cells, ie autoreactive T cells. The mechanism of the immune response indicates that the tumor antigen is self-antigen and is resistant to the central Under the environment, direct use of tumor antigens cannot induce an effective anti-tumor immune response. Because of the selective elimination of high-affinity autoreactive T cells during thymic development, the T cell pool that directly recognizes tumor antigens is small, and most T cell banks that recognize mutant antigens are limited by central tolerance mechanisms. There are still enough autoreactive T cells with low affinity for tumor antigens. This group of T cells is more dependent on the expression of certain Vβ, probably because the TCR affinity of the Vβ is low, so it is easier to escape the negative selection. However, this also affects the functionality of autoreactive T cells, making them less effective against tumors. Therefore, based on the use of mutant antigens to induce T cell pools that cross-recognize autoantigens, it is particularly important to selectively enhance the affinity and functionality of these autoreactive T cells against tumor immune responses.

The present inventors have found in experiments that enhancing the functionality and affinity of autoreactive T cells is regulated by two different mechanisms. Alterations in immune vectors can affect the affinity of autoreactive T cells, and antigenic changes can promote functional cytokine expression in autoreactive T cells. The first immunization with DC as a vector after activation of CpG and the second immunization with Lm as a vector can effectively enhance the affinity of autoreactive T cells. The use of Lm twice or twice, or the reverse order of DC and Lm as carriers does not have the same effect.

By studying the autoreactive T cell bank Vβ, the inventors have found that the central tolerance mechanism not only reduces the absolute number of autoreactive T cells, but also has a low affinity affinity T cell bank for recognizing autoantigens. Sex is also reduced. In the present inventors' system, the ratio of Vβ3, Vβ7, Vβ10b, and Vβ13 in low-affinity autoreactive T cells is higher than that of high-affinity T cells recognizing the same antigen, while other (undetected Vβ) is used. The proportion was significantly reduced, presumably because of the specific clearance of high-affinity T cell clones during thymic negative selection, leading to a decrease in the preference for certain low-affinity Vβ and overall cell bank diversity. After the optimized dual heterologous immunization strategy DC-Y3-Lm-N4 immunization, the proportion of Vβ3 in the T cell pool that recognizes autoantigen is significantly reduced, while the proportion of Vβ4 is significantly increased, ie, the autoreactivity T is effectively increased. The functional affinity of the cells. Interestingly, the use of Vβ4 in high-affinity T cell pools is not high. The present inventors speculate that the affinity and functionality of autoreactive T cells are increased after optimized dual heterologous immunization, mainly by specifically not amplifying certain low affinity and functional T cell clones, and adding other T The opportunity for cell clones to be selected.

The present inventors inoculated Ova-expressing melanoma cells Mo5 on Rip-mOva mice to mimic the occurrence of human autologous tumors, and then used the optimized dual heterologous immunization strategy DC-Y3-Lm-N4 as a vaccine to treat tumor-bearing mice. It was found that the optimized immunization strategy can inhibit tumor growth and prolong the survival of mice compared with the control group. It is indicated that the efficiency of anti-tumor response is mainly dependent on the improvement of autoreactive T cell function and affinity.

the term

As used herein, the term "tumor antigen of the invention" refers to a second tumor antigen, ie, a polypeptide that is homologous and intersects with the first tumor immune antigen, or an immunogenic fragment thereof. For example, when the Trp2SD peptide (SEQ ID NO.: 7) is the first tumor antigen, one second tumor antigen is Trp2SK (SEQ ID NO.: 8).

As used herein, the term "epitope (peptide)" refers to a peptide of another protein that is intended to induce an immune response in an animal. Generally, an epitope refers to a peptide to be targeted by an immune response, preferably a peptide derived from a mammalian (e.g., human) protein.

Central tolerance model

In the present invention, experiments against the central tolerance model demonstrate that the method of the present invention can effectively activate T cells recognizing natural low-immunity antigens (including tumor antigens or auto-tumor antigens), thereby effectively enhancing immunity against low-immunity antigens. reaction.

In the present invention, a representative central tolerance model is Ova's transgenic mouse (Rip-mOva), which is an artificially constructed central tolerance model for native antigen N4-specific T cells. The mouse expresses Ova as a self-antigen in medullary thymic epithelial cells, pancreatic beta cells, and renal proximal tubules, so the central tolerance mechanism during the development of the immune system has selectively cleared N4-specific T cells in mice. Experiments by the inventors have shown that low affinity A4 autoreactive T cells are still present in the central tolerance model mice.

The present inventors infected Rip-mOva and wild type (WT) mice with a Listeria strain (Lm-N4) expressing a natural Ova antigen or a Listeria strain (Lm-A2 to Lm-V4) expressing a mutant Ova antigen, and found that Under the action of the central tolerance mechanism, the mouse T cell pool does not respond to the autoantigen, that is, the natural antigen, and is more easily activated by the medium affinity mutant antigen.

Ovalbumin natural antigen and its homologous and crossed mutant antigen

The natural antigen and a series of mutant antigens of the model antigen ovalbumin (Ova) have been used in the study of T cell responses. The amino acid sequence SIINFEKL (N4) at position 257-264 of Ova can be recognized by the MHC class I molecule K ^b and presented to T cells, resulting in a strong antigen-specific CD8 ⁺ T cell response. Based on the natural antigen N4 sequence, a single amino acid mutation is performed according to the corresponding position of the amino acid, and a series of mutant antigens are obtained. As shown in Figure 1, N4 and its representative mutant antigens include:

天然抗原N4:Natural antigen N4:	SIINFEKLSIINFEKL	(SEQ ID NO.:1)(SEQ ID NO.: 1)
突变抗原A2:Mutant antigen A2:	SAINFEKLSAINFEKL	(SEQ ID NO.:2)(SEQ ID NO.: 2)
突变抗原Y3:Mutant antigen Y3:	SIYNFEKLSIYNFEKL	(SEQ ID NO.:2)(SEQ ID NO.: 2)
突变抗原Q4:Mutant antigen Q4:	SIIQFEKLSIIQFEKL	(SEQ ID NO.:4)(SEQ ID NO.: 4)
突变抗原T4:Mutant antigen T4:	SIITFEKLSIITFEKL	(SEQ ID NO.:5)(SEQ ID NO.: 5)
突变抗原V4:Mutant antigen V4:	SIIVFEKLSIIVFEKL	(SEQ ID NO.:6)(SEQ ID NO.: 6)

This set of mutations native antigen and an antigen of mouse antigen-presenting cells having MHC K ^b molecules similar affinity, whereas functional affinity of CD8 ⁺ T-cell receptor is different. N4 is used to mimic the first (tumor) antigen, while Y3 is the preferred homologous and crossed second (tumor) antigen.

Composition and method of administration

The invention also provides a composition (or composition product) comprising: (1) a first composition comprising (i) a first tumor antigen and/or by said first a tumor antigen-sensitized dendritic cell; (ii) an optional adjuvant; and (iii) a pharmaceutically or immunologically acceptable carrier;

Also, the first composition and the second composition are independent.

In the present invention, the term "containing" means that the various ingredients may be applied together or in the composition of the present invention. Therefore, the terms "consisting essentially of" and "consisting of" are encompassed by the term "contains."

Compositions of the invention include pharmaceutical compositions and vaccine compositions.

The compositions of the invention (e.g., the first, second, and third compositions) may be monovalent (containing only one recombinant protein or polynucleotide) or multivalent (containing a plurality of recombinant proteins or polynucleosides) acid).

The pharmaceutical compositions or vaccine compositions of the present invention can be prepared in a variety of conventional dosage forms including, but not limited to, injections, granules, tablets, pills, suppositories, capsules, suspensions, sprays and the like.

(1) Pharmaceutical composition

The pharmaceutical composition of the present invention comprises (or comprises) a therapeutically effective amount of a first tumor antigen and/or dendritic cells sensitized by said first tumor antigen; and a second tumor antigen and/or by said second Tumor antigen-sensitized dendritic cells.

The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent that treats, alleviates or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect. This effect can be detected by, for example, antigen level. Therapeutic effects also include a reduction in physiological symptoms. The precise effective amount for a subject will depend on the size and health of the subject, the nature and extent of the condition, and the combination of therapeutic and/or therapeutic agents selected for administration. Therefore, it is useless to specify an accurate effective amount in advance. However, for a given situation, routine experimentation can be used to determine the effective amount.

For the purposes of the present invention, an effective dose is from about 0.001 mg/kg to 1000 mg/kg, preferably from about 0.01 mg/kg to 100 mg/kg body weight of the second tumor antigen and the first tumor, respectively, to the individual. antigen.

The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, such as a recombinant protein of the invention. The term refers to pharmaceutical carriers which do not themselves induce the production of antibodies harmful to the individual receiving the composition and which are not excessively toxic after administration. Suitable carriers can be large, slow-metabolizing macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, and the like. These vectors are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable carriers or excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N. J. 1991).

Pharmaceutically acceptable carriers in the compositions can include liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers. In general, the compositions may be formulated as injectables, such as liquid solutions or suspensions; they may also be in the form of solids suitable for solution or suspension, liquid excipient prior to injection. Liposomes are also included in the definition of pharmaceutically acceptable carriers.

(ii) vaccine composition

The vaccine (composition) of the present invention may be prophylactic (i.e., prevent disease) or therapeutic (i.e., treat disease after illness).

These vaccines comprise an immunological antigen (i.e., a second tumor antigen of the invention and a first tumor antigen) and are typically combined with a "pharmaceutically acceptable carrier" which includes itself not induced to be deleterious to the individual receiving the composition. Any carrier of the antibody. Suitable carriers are usually large, slow-metabolizing macromolecules such as proteins, polysaccharides, and polyemulsions. Acid, polyglycolic acid, amino acid polymer, amino acid copolymer, lipid agglutination (such as oil droplets or liposomes) and the like. These vectors are well known to those of ordinary skill in the art. Additionally, these carriers can function as immunostimulating agents ("adjuvants"). In addition, the antigen can also be coupled to bacterial toxoids such as toxoids of pathogens such as diphtheria, tetanus, cholera, and Helicobacter pylori.

Preferred adjuvants for enhancing the effectiveness of the immunological composition include, but are not limited to: (1) aluminum salts (alum) such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, and the like; (2) oil-in-water emulsion formulations, for example, (a) MF59 (see WO 90/14837), (b) SAF , and (c) Ribi ^TM adjuvant system (RAS) (Ribi Immunochem, Hamilton , MT), (3) saponin adjuvants; (4) Freund's complete adjuvant (CFA) and Freund incomplete adjuvant (IFA); (5) cytokines such as interleukins (eg IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12) , IL-15, etc., interferon (such as gamma interferon), macrophage colony-stimulating factor (M-CFS), tumor necrosis factor (TNF), etc.; (6) bacterial ADP-ribosylating toxin (such as Escherichia coli a detoxified variant of the heat labile toxin LT); and (7) other substances that act as immunostimulating agents to enhance the effect of the composition.

In the present invention, a preferred class of adjuvants are cytokines and chemokines or a combination thereof. Representative cytokines include, but are not limited to, IL-1, IL-2, IL-6, IL-7, IL-8, IL-11, IL-12, IL-14, IL-15, IL -18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL -30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, and the like, and combinations thereof. Representative chemokines include (but are not limited to): CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, and the like, and combinations thereof. In the present invention, a combination of a cytokine and a chemokine can be used as an adjuvant.

Vaccine compositions, including immunogenic compositions (e.g., can include antigens, pharmaceutically acceptable carriers, and adjuvants), typically contain diluents such as water, saline, glycerol, ethanol, and the like. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may be present in such carriers.

More specifically, vaccines, including immunogenic compositions, comprise an immunologically effective amount of an immunogenic polypeptide, as well as other desired components as described above. "Immunologically effective amount" means that the amount administered to a subject in a single dose or in a continuous dose is effective for treatment or prevention. The amount may be based on the health and physiological condition of the individual being treated, the type of individual being treated (eg, human), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the assessment of the medical condition by the treating physician, And other relevant factors. This amount is expected to be in a relatively wide range and can be determined by routine experimentation.

In general, the vaccine composition or immunogenic composition can be formulated as an injectable, such as a liquid solution or suspension; it can also be formulated in a solid form suitable for solution or suspension, liquid excipient prior to injection. The formulation may also be emulsified or encapsulated in liposomes to enhance the adjuvant effect.

Furthermore, the vaccine composition of the invention may be a monovalent or multivalent vaccine.

(iii) route of administration and dosage

Once formulated into a composition of the invention, it can be administered directly to the subject. The subject to be treated can be a mammal, especially a human.

When used as a vaccine, the recombinant protein of the present invention can be directly administered to an individual by a known method. Usually used These vaccines are administered by the same route of administration and/or mimicking the path of pathogen infection of conventional vaccines.

Routes for administering a pharmaceutical composition or vaccine composition of the invention include, but are not limited to, intramuscular, subcutaneous, intradermal, intrapulmonary, intravenous, nasal, oral or other parenteral routes of administration. If desired, the route of administration can be combined or adjusted depending on the condition of the disease. The vaccine composition can be administered in a single dose or in multiple doses and can include administration of a booster dose to elicit and/or maintain immunity.

The recombinant protein vaccine should be administered in an "effective amount", i.e., the amount of recombinant protein sufficient to elicit an immune response in the chosen route of administration, which is effective to promote protection of the host against the associated disease.

Representative diseases include (but are not limited to): tumors, etc., such as liver cancer, lung cancer, stomach cancer, breast cancer, ovarian cancer, prostate cancer, skin cancer, melanoma, cervical cancer, brain cancer, thyroid cancer and cholangiocarcinoma, bladder Cancer and pancreatic cancer.

The amount of recombinant protein selected for each vaccine formulation is based on the amount that will elicit an immunoprotective response without significant side effects. Typically, after infection of the host cell, the vaccine of each dose is sufficient to contain from about 1 μg to 1000 mg, preferably from 1 μg to 100 mg, more preferably from 10 μg to 50 mg of protein or polypeptide (including the second tumor antigen or the first tumor antigen). Standard assays including antibody titers and other reactions in the subject can be used to determine the optimal amount of a particular vaccine. The level of immunity provided by the vaccine can be monitored to determine if an increased dose is needed. After assessing antibody titers in serum, booster immunization may be required. Administration of an adjuvant and/or an immunostimulatory agent increases the immune response to the protein of the invention.

A preferred method is to administer the immunogenic composition by injection from the parenteral (subcutaneous or intramuscular) route.

Furthermore, the vaccine of the invention may be administered in combination with other immunomodulatory agents or with other therapeutic agents.

The main advantages of the invention include:

(a) The immunization strategies and compositions of the present invention are effective in breaking the body's immune tolerance to autoantigens.

(b) The immunization strategies and compositions of the present invention are effective in stimulating the body's immune response against tumor antigens.

(c) The present invention contributes to the targeted solution to the problem of the general inefficiency of current therapeutic tumor vaccines, and plays an important role in accelerating the development of tumor vaccines.

(d) The present invention can be applied to tumor immunotherapy, vaccine development and the like.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. Experimental methods in which the specific conditions are not indicated in the following examples are generally carried out according to the conditions described in conventional conditions, for example, Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions. The conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight and parts by weight.

All antibodies were commercially available antibodies from eBioscience or BD Bioscience. For example, Anti-mouse Vβ2 TCR (B20.6, eBioscience); Anti-mouse Vβ3 TCR (KJ25, BD Bioscience).

All reagents are commercially available unless otherwise stated.

Experimental animals: 6 to 8 weeks of wild-type C57BL/6 mice were purchased from SLAC, Shanghai Experimental Animal Center, Chinese Academy of Sciences Rip-mOva mice were obtained from the Howard Hughes Medical Institute at the University of Washington.

General method

(a) Cultivation and infection of Listeria

1.-80 ° C frozen Listeria strained TSB medium solid plate, 37 ° C incubator overnight, pick the monoclonal colonies, 30 ° C, 200 rpm / min, shaker overnight for 14 hours, according to 1: 20 ratio of bacterial solution 150μl to 3ml fresh TSB liquid medium, 37 ° C, 200 rpm / min, shaken for 2.5 to 3 hours, to logarithmic growth phase, OD600 value, OD600 value of 0.6 to 1 .

2. Take 1ml of bacterial solution, centrifuge at 12000rpm/min for 10min, repeat the centrifugation with 1ml phosphate buffer solution twice, resuspend the cell pellet with phosphate buffer solution according to the measured OD600 value, and the corresponding relationship between OD600 and bacterial concentration. When the OD600 value is 1, the bacterial liquid concentration is 10 ⁹ cfu/ml.

3. Dilute 100 μl of the bacterial solution to 1 ml of phosphate buffer solution in a ratio of 1:10 until the concentration of the bacterial solution is diluted to 10 ⁵ cfu/ml, and continue to dilute the concentration of the bacterial solution to a ratio of 2.5×10 ⁴ cfu/ml according to the ratio of 1:4. For tail vein injection in mice.

(b) Culture, activation and immunization of mouse bone marrow-derived dendritic cells

1. Day 1: Completely remove the femur and tibia of the lower limbs of C57/BL6 female mice from 6 weeks to 8 weeks, remove the attached muscle tissue, place in the pre-cooled phosphate buffer solution, and transfer the cell table. The bones were rinsed 3 times with sterile PBS. Scissor scissors cut the ends of the bone and rinse the marrow cavity with a 1 ml syringe until the bone turns white. Resuspend in phosphate buffer solution and filter the nylon membrane into a single cell suspension. After 300 g, 10 min, 4 ° C, after centrifugation, add 1 ml of red blood cell lysate, lyse on ice for 5 minutes and then terminate by adding 10 ml of phosphate buffer solution. After 300 g, 10 min, the supernatant was centrifuged at 4 ° C, and the cells were counted and resuspended in a K medium-coated 24-well plate at a cell concentration of 1 × 10 ⁶ cells/ml, 1 ml/well. Transfer to a cell culture incubator and incubate at 37 ° C under 5% CO ₂ .

2. Day 3: After the 24-well plate was tilted at a small angle for 10 minutes, the pipette sucked 500 μl of the supernatant from the removal hole. 500 μl of fresh medium was added, and growth factors GM-CSF and IL-4 were added, and cultured at 37 ° C, 5% CO ₂ .

3. Day 5: Pipette all the medium and cells in the 24-well plate, centrifuge at 4°C, 300g, centrifuge for 10min, add 1ml/well of fresh medium, add growth factors GM-CSF and IL-4, and transfer Cell culture incubator.

4. Day 7: Blow all the suspended cells in the 24-well plate, 300g, centrifuge for 10min, remove the supernatant, resuspend in fresh medium, the cell concentration is 2×10 ⁶ cells/ml, add CpG 800ng/ml, Transfer to a cell culture incubator and stimulate activation at 37 ° C for 16 hours.

5. CpG stimulated the mouse dendritic cells 300g after overnight, centrifuged at 10min, removed the supernatant, resuspended in fresh medium, the cell concentration was 1×10 ⁶ cells/ml, and added the test peptide 2μg/ml load, 37 ° C, Incubate for 5% CO _{2 for} 2.5 hours.

6. 300g of mouse dendritic cells after loading the peptide, 10min, centrifuged at 4°C, remove the supernatant, wash 3 times with 10ml phosphate buffer solution, resuspend in serum-free phosphate buffer solution, the cell concentration is 20×10 ⁶ Cells/ml for immunization in mice.

(c) Culture of melanoma cells and establishment of melanoma model

The melanoma cell line Mo5 cells (obtained from the Chinese Academy of Sciences) were cultured from liquid nitrogen cryopreservation (first generation), and the second generation culture was started by adding a concentration of 1 mg/ml antibiotic G418 to screen for Mo5 cells that specifically expressed Ova antigen. Screening was passaged 3-4 times, and the fourth or fifth generation cells were collected in the exponential growth phase and resuspended in phosphate buffer solution or serum-free DMEM medium.

The mice were anesthetized with 2.5% Avertin, the back was depilated, and Mo5 cells were inoculated subcutaneously in the mid-lumbar region, 5 x 10 ⁵ cells/mouse. From the 6th day after inoculation, the length of the long diameter and the short diameter of the tumor were measured with a vernier caliper every 2 days. The tumor volume was calculated according to the formula [long diameter × (short diameter) ² × π / 6] ⁴² . When the average length of the long and short diameters exceeds 2 cm, the neck is sacrificed to relieve pain according to the ethical guidelines of animal experiments.

(d) Immunization and immunotherapy of mouse tumors

Prevention of mouse tumors: On the 7th day after the mouse was pre-expressed to express the Listeria and/or dendritic cell vaccine according to the experimental protocol, the melanoma cell line Mo5 cells 5×10 ⁵ cells/mouse were subcutaneously inoculated, observed and recorded. Tumor growth.

Treatment of mouse tumors: After subcutaneous inoculation of the melanoma cell line Mo5, the mice were treated with Listeria and dendritic cell vaccine according to the designed treatment protocol, and the tumor growth was observed and recorded.

(e) Bone marrow-derived dendritic cell culture

1. Day 1: Completely remove the femur and tibia of the lower limbs of C57/BL6 female mice from 6 weeks to 8 weeks, remove the attached muscle tissue, place in the pre-cooled phosphate buffer solution, and transfer the cell table. The bones were rinsed 3 times with sterile PBS. Scissor scissors cut the ends of the bone and rinse the marrow cavity with a 1 ml syringe until the bone turns white. Resuspend in phosphate buffer solution and filter the nylon membrane into a single cell suspension. After 300 g, 10 min, 4 ° C, after centrifugation, add 1 ml of red blood cell lysate, lyse on ice for 5 minutes and then terminate by adding 10 ml of phosphate buffer solution. After centrifugation at 300g, 10min, 4°C, the cells were counted and resuspended in K medium-coated 24-well plate. Growth factors GM-CSF and IL-4 50ng/ml were added, and the cell concentration was 1×10 ⁶ cells/ml, 1ml. /hole. Transfer to a cell culture incubator and incubate at 37 ° C under 5% CO ₂ .

5. CpG stimulated the mouse dendritic cells 300g after overnight, centrifuged at 10min, removed the supernatant, resuspend in fresh medium, the cell concentration was 1×10 ⁶ cells/ml, and added test peptide (such as Trp2SD or Trp2SK) 2μg/ The ml load was incubated at 37 ° C, 5% CO _{2 for} 2.5 hours.

(f) flow dyeing

1. Take the mouse spleen and place it in a pre-cooled phosphate buffer solution and grind it into a single cell suspension. After filtering with nylon membrane at 4 ° C, 320 g, centrifuge for 10 min, remove the supernatant, add 1 ml of red blood cell lysate, and lyse on ice for 5 minutes. The mixture was stopped by adding 10 ml of phosphate buffer solution, centrifuged at 4 ° C, 300 g, 10 min, and resuspended in 1 ml of 1640 medium.

2. Spleen cell suspension was plated into 96-well plates at 4 x 10 ⁶ cells/well. The polypeptide to be tested is added for stimulation (e.g., Trp2SD) at a polypeptide concentration of 2 g/ml. After incubation at 37 ° C, 5% CO _{2 for} 30 minutes, BFA was added at a concentration of 2.5 μg/ml. Continue to stimulate the culture for 5 hours.

3. The cells in the 24-well plate were transferred to a flow centrifuge tube, washed with a pre-cooled 1 ml flow staining solution, and centrifuged at 4 ° C, 320 g, 5 min to remove the supernatant.

4. The cells were placed on ice, and 1 μl of Fcblocker Anti-CD16/CD32 and 50 μl of flow staining solution were added to each tube, gently shaken and mixed, and incubated on ice for 10 min.

5. Flow-through antibody PE-cy7-CD8, PerCP-cy5.5-TCRβ, PE-CD44 and 50 μl flow staining solution were added to each tube, and the cells were shaken and mixed, protected from light, and incubated on ice for 30 min.

6. Each tube was washed twice with 1 ml of flow staining solution, and centrifuged at 4 ° C, 320 g for 10 min to remove the supernatant.

7. Add 250 μl of the cell fixative in each tube, mix well with the cells, protect from light, and incubate on ice for 50 min.

8. Each tube was added to the cell dialysis solution in the 1 ml kit, washed, shaken and mixed, and centrifuged at 4 ° C, 320 g, 10 min to remove the supernatant. Repeat the cleaning twice.

9. The cells were placed on ice, and 1 μl of Fcblocker Anti-CD16/CD32 and 50 μl of cell rupture solution were added to each tube, shaken and mixed, and incubated on ice for 10 min.

10. Flow tube antibody FITC-IFNγ, APC-TNFα and 50 μl cell disruption solution were added to each tube, and the cells were shaken and mixed, protected from light, and incubated on ice for 30 min.

11. Add 1 ml of cell membrane solution to each tube, mix well, shake at 4 ° C, 320 g, and centrifuge for 10 min to remove the supernatant. Finally, it was resuspended in 300 μl of flow staining solution and analyzed by flow cytometry.

(g) Statistical analysis

Except where specified, independent experiments are repeated at least 2 times, and the total number of samples in each group is n ≥ 5. The original data was checked and statistical analysis was performed using Prism 5.0 (GraphPad, USA) software. Experimental data is expressed as mean ± standard error. When the P value is less than 0.05 (the confidence interval is 95%), it is considered to be statistically different.

Example 1

Identification of Functional Affinity of Endogenous Polyclonal T Cells by Natural Antigen and Mutant Antigen

The present inventors infected mice with tail vein injection of Listeria Lm-N4 expressing natural antigen, and after 7 days, spleen cells were stimulated in vitro with a gradient of natural antigen N4 or mutant antigens A2, Y3, Q4, T4, V4, etc. Culture, detection of the proportion of IFN-γ ⁺ CD8 ⁺ T cells, do dose response curve (Figure 2A).

The result is shown in Figure 2. The inventors have found that the dose response curve for the native antigen N4 relative to the CD8 ⁺ T cell has a right shift in the dose response curve for other mutant antigens. The present inventors used the reciprocal of the half-maximum effect concentration (EC ₅₀ ) (1/EC ₅₀ ) to express the relative functional affinities of the native antigen and the modified antigen, and found that despite the function of the native antigen and the mutant antigen against the endogenous polyclonal T cell system. Sexual affinity decreased compared to OT-I T cells, but the order of affinity did not change. The native antigen N4 has the highest functional affinity for endogenous polyclonal Ova-specific T cells (EC ₅₀ = 27.58 pM), A2 is a highly functional affinity antigen (35.4 pM) next to N4, and Y3 and Q4 are moderately affinitive. Antigens (253.51 pM and 689.17 pM), T4 and V4 were low affinity antigens (3221 pM and 20930 pM) (Fig. 2B).

The difference in functional affinity may affect the cross-reactivity of the endogenous T cell bank that specifically recognizes N4 to the mutant antigen, and recognize the endogenous polyclonal T cell bank of N4 at a conventional antigen stimulation concentration of 2 μg/ml. The level of cross-reactivity to the mutant antigen is generally positively correlated with the functional affinity of the mutant antigen. The high affinity antigen is more cross-reactive with the N4-specific T cell pool than the low affinity antigen (Figure 2C).

Example 2

Central tolerance makes the mouse immune system more prone to respond to moderately affinitive mutant antigens

After identifying the functional affinities of the native antigen and the endogenous polyclonal T cell system specific for the Ova antigen, the inventors further studied the response of the mouse endogenous polyclonal T cell bank to the native antigen and the mutant antigen. And the impact of the central tolerance mechanism on this law.

WT mice and Rip-mOva mice were infected with Liszt strain expressing natural antigen N4 or mutant antigen, respectively. After 7 days, spleen cells were stimulated with antigens corresponding to the respective infected strains, and IFN-γ ⁺ CD8 ⁺ T cells were detected. proportion.

The result is shown in Figure 3. The T cell pool in WT mice can produce an immune response to both natural and mutant antigens; in Rip-mOva mice, the immune response to native antigen N4 and high affinity antigen A2 is almost due to the central tolerance mechanism. It was completely inhibited, and the immune responses to other medium-affinity and low-affinity mutant antigens were also reduced to varying degrees, while central tolerance had a lower effect on the level of immune responses induced by the three mutant antigens Y3, Q4, and V4.

This result indicates that in the presence of a central tolerance mechanism, the body's T cell bank cannot respond to autoantigens and high affinity mutant antigens, while the level of immune response to mutant antigens is inhibited, but some medium affinity and low affinity mutant antigens remain Activation can be induced to produce a certain number of antigen-specific T cell clones.

Since in the anti-tumor immune response, the group of T cells which recognize the natural antigen in the immune reaction induced by the mutant antigen plays a major role, in this embodiment, after further detecting the mutation of the mutant antigen, the T cell bank is resistant to the natural antigen. Cross reaction.

After immunizing WT mice and Rip-mOva mice with Liszt strain carrying mutant antigen, spleen cells were cultured in vitro with natural antigen N4, and the ratio of IFN-γ ⁺ CD8 ⁺ T cells was detected.

In WT mice, the proportion of T cells that respond to natural antigens is lower than that of T cells that respond to the mutant antigen itself. The cross-reactivity of the mutant antigen induced by the mutant antigen and the functional affinity of the mutant antigen are generally positively correlated. Relationship, high-affinity mutant antigens can induce a higher proportion of native antigen-reactive T cells, and low-affinity mutant antigens tend to induce a lower proportion of native antigen-reactive T cells. In Rip-mOva mice, since the natural antigen is a self-antigen, the immunization of other mutant antigens produces only T cells that rarely respond to natural antigens, and only the mutant antigen Y3 can induce distinct, natural antigen cross-reactive T cells. (The average ratio is 0.9%), which is autoreactive T cells. Interestingly, however, the ratio of natural antigen cross-reactive T cells produced by high-affinity mutant antigen A2 to all antigen-reactive T cells is close to 100% in WT mice, indicating that almost all A2-specific T cells are It is able to respond to natural antigens, which is 55% in Rip-mOva mice, which is reduced by nearly half. The cross-reaction rate after Y3 immunization decreased from 29% in WT mice to 19% in Rip-mOva mice, only about 30% lower (Fig. 3).

The results of these experiments indicate that when the natural antigen is an exogenous antigen, the level of immune response of the body T cell bank to the mutant antigen is mainly related to its functional affinity; when the natural antigen is autoantigen, the central tolerance mechanism clears most of the Autoreactive T cells and T cells that respond to high functional affinity mutant antigens make T cell pools more prone to respond to moderately affinitive mutant antigens. However, Tm-YO immunization detected a significant T cell response to natural antigen in the Rip-mOva mice, indicating that the clearance of autoreactive T cells in the central tolerance mechanism is not complete, and there is still enough in the T cell pool. A number of clones can recognize the response to the self antigen. Under the central tolerance mechanism, the relationship between T cell response level and cross-reaction rate of self-antigen is not obvious, indicating that the factor determining the effectiveness of anti-tumor response still lies in the absolute number of low-affinity clones present in the body, high. Cross-reaction rates do not indicate that more autoreactive T cells can be activated.

Example 3

Construction of a melanoma model in WT mice and Rip-mOva mice

It was identified that the medium-affinity mutant antigen Y3 can best induce autoreactive T cells under the action of the central tolerance mechanism, and thus has a potential anti-tumor effect. For subsequent experiments, in this example, WT mice were inoculated subcutaneously with different doses of Ova-expressing melanoma cells Mo5 to establish a melanoma non-tolerance model with tumor antigens as exogenous antigens (Fig. 4A). At an inoculation amount of 5 × 10 ⁵ cells/head, it was ensured that each mouse developed a melanoma tumor with uniform growth.

Rip-mOva mice were inoculated with 5×10 ⁵ cells/dose to simulate the tumor antigen as autoantigen during human tumorigenesis. Melanoma Mo5 can grow normally in mice, thus establishing Osa antigen self-tolerance. The melanoma model (Fig. 4B) had a slightly higher survival curve than WT mice (Fig. 4C). It was further observed whether Rip-mOva mice produced tumor antigen-specific T cells after inoculation of tumors (Fig. 4D and E). N4-specific tetramer detection revealed that Rip-mOva mice did not recognize themselves in the spleen and draining lymph nodes. Somatic cells of antigen N4 (Fig. 4D), IFN-γ ⁺ CD8 ⁺ T cells were hardly produced by stimulation with N4 in vitro. Interestingly, spleen cells and draining lymph node cells were stimulated in vitro with Y3, producing a certain number of CD8 ⁺ T cells expressing IFN-[gamma] and TNF-[alpha] (Fig. 4E). This suggests that the T cell pool in vivo is more susceptible to the mutant antigen Y3.

Example 4

Autoreactive T cells induced by moderate affinity mutant antigens are not effective in inhibiting tumor growth

In the presence of a central tolerance mechanism, the medium-affinity mutant antigen Y3 can induce the most autoantigen-reactive T cells in the T cell pool, but it is also found in the mouse model of the Ova antigen central tolerant mouse tumor-bearing model. T cells that respond to mutant antigen Y3. However, further studies have shown that Lm-Y3 infection immunization has no significant inhibitory effect on tumors expressing native Ova antigen, and the anti-tumor response is inefficient. In the present example, WT mice and Rip-mOva mice were pre-inactivated with Lm-N4 and Lm-Y3, respectively, and then inoculated with the Ova antigen-expressing melanoma cell line Mo5.

The result is shown in Figure 5. Although Lm-Y3 can inhibit melanoma growth better than Lm-N4 in Rip-mOva mice (Fig. 5B), it is still significantly lower than the inhibitory effect of both antigens on WT mice (Fig. 5A). This suggests that in the anti-tumor process, the main role is still autoreactive T cells against the natural antigen N4, rather than only the mutation resistance The group of cells that were originally reacted by Y3.

Next, the present inventors infected WT mice and Rip-mOva mice with the natural antigen Lm-N4 and the mutant antigen Lm-Y3, respectively, and mapped N4-specific T cells (WT Sp. T cells) and N4- in WT mice. Cross-reactive T cells (WT CR.T cells), and N4-cross-reactive T cells of Rip-mOva mice (RO CR.T cells. RO Sp.T cells cannot be calculated due to the low ratio) The dose response curve (Fig. 5C) and calculate the relative functional affinity.

The results showed that the relative functional affinity of WT CR. T cells decreased by about 109-fold compared to WT Sp. T cells, while the affinity of RO CR. T cells decreased by 490-fold (Fig. 5D). This indicates that in the absence of central tolerance, the T cell pool that recognizes the natural antigen induced by the mutant antigen has only a low affinity; while the central tolerance mechanism exists, the T cell library that recognizes the natural antigen activated by the mutant antigen is itself Reactive T cells have a further reduced range of affinity.

The above results indicate that low-affinity autoreactive T cells activated by mutant antigens in a central tolerance environment are unable to effectively immunoreact with tumor antigens and inhibit their growth.

Example 5

Autoreactive T cell bank and high affinity T cell bank that recognize the same antigen are biased to use different Vβ

In an endogenous polyclonal immune system, the functional affinity between an antigen and a T cell is actually the average functional affinity of a T cell clone that recognizes the same antigen in the T cell pool. Since the receptor Vβ gene of a single T cell is rearranged by a fragment, different Vβ chains can be produced and combined into different TCRs to form a huge T cell bank composed of different clones.

In this example, the difference between the mutant cell-activated T cell bank that recognizes the autoantigen and the high affinity T cell bank that recognizes the same antigen is further studied.

WT mice and Rip-mOva mice were separately immunized with the mutant antigen Y3, and the T cell pools recognized by Y3 and N4, respectively, were detected by co-staining with N4-tetramer antibody and Y3-tetramer antibody.

The result is shown in Figure 6. The T cell pool that cross-recognizes the natural antigen N4 in WT mice partially overlaps with the T cell pool that recognizes Y3. The ratio of N4-tet ⁺ Y3-tet ⁺ CD8 ⁺ T cells is 3.20%, and there are still 1.05% of CD8 ⁺ T cells. Only N4 and 15.1% of CD8 ⁺ T cells were recognized to recognize only Y3; whereas in Rip-mOva mice, 84% of the N cell-recognized T cell pool was derived from the T cell pool recognizing mutant antigen Y3 (Fig. 6A).

Further detection of Vβ expression in WT Sp. T cells, WT CR. T cells and RO CR. T cells revealed that although the functional affinity of WT CR. T cells was significantly lower than that of WT Sp. T cells, both Vβ use bias and T cell pool diversity were basically the same. On the contrary, although the affinity was only about 4 times different, the T cell pool diversity of RO CR.T cells was significantly different from the other two (Fig. 6B). The T cell pool that recognizes Y3 in Rip-mOva mice is largely unaffected by central tolerance (Fig. 6C). This indicates that after the central tolerance, the remaining T cell pool is mainly directed to the mutant antigen, and most of the activated autoreactive T cells are derived from the mutant antigen-specific T cell bank.

It has also been found that the diversity of the autoreactive T cell pool is reduced compared to the other two, and it is more likely to use certain specific Vβ. By comparing the Vβ usage rates, it was found that the use rates of Vβ3, Vβ7, Vβ10b and Vβ13 in RO CR.T cells were significantly higher than those in WT mice, and the proportion of undetected Vβ(Others) was significantly decreased (Fig. 6D and E). This result suggests that, due to central tolerance-selective clearance of high-affinity autoreactive T cell clones, the remaining T cells that recognize autoantigens are more dependent on the use of certain low affinity specific Vβs in the mutant antigen T cell pool.

Example 6

Repeated immunotherapy with the same mutant antigen under central tolerance cannot effectively inhibit tumor growth

Due to the inhibitory effect of central tolerance on autoantigen-reactive T cell pools, autoreactive T cells produced by immunization of mutant antigens have only a very low affinity. Since the mutant antigen-induced autoantigen T cell response is the basis of effective anti-tumor immunity, the present inventors attempted to use the repeated immunomutation antigen as a therapeutic tumor vaccine to see if it can enhance the anti-tumor immune response and inhibit tumor growth.

The present inventors inoculated Ova-expressing melanoma cells Mo5 on Rip-Ova mice to establish a central tolerance model of natural tumor antigen N4; and inoculated the same tumor on WT mice as a non-central tolerance of natural tumor antigens. model. On the 7th day after inoculation of the tumor, the present inventors treated each group of mice with a Liszt strain expressing Lm-N4 or a mutant antigen of a natural antigen, and the treatment was performed three times in total at intervals of one week.

The result is shown in Figure 7. In the non-central-tolerant WT mouse tumor-bearing therapeutic model, the inventors found that repeated treatment with natural antigen N4 and high affinity mutant antigen A2 can effectively inhibit tumor growth, and other medium-affinity and low-affinity mutant antigens have no therapeutic effect. (Fig. 7A).

By detecting Lm-N4 and Lm-A2-treated tumor-bearing WT mice, the inventors found that the spleen, mesenteric lymph nodes, and inguinal lymph nodes of the mice all produced a certain number of T cells that responded to the natural antigen N4. (Fig. 7C).

In the central tolerance model based on Rip-Ova mice, all antigen therapy, including the mutant antigen Y3, which induces autoreactive T cells in Rip-Ova mice, does not produce effective antibiotics. Tumor action inhibits tumor growth (Fig. 7B).

Considering that there may be a peripheral tolerance mechanism to Ova antigen in Rip-Ova mice, in which immature DC-presenting antigens in vivo may induce inhibition of autoreactive T cells, for which the present inventors constructed A new immunization strategy: CpG is used to stimulate mature bone marrow-derived dendritic cells loaded with Y3 (DC-Y3) to treat tumor-bearing Rip-Ova mice as a vaccine.

The inventors first stimulated bone marrow-derived DC cells with different concentrations of CpG to detect the expression of the activating molecules CD80 and CD86.

As a result, it was found that DC cells were most preferably activated under the stimulation of a concentration of 2 μg/ml. However, neither DC-Y3 repeat immunotherapy (Fig. 7D) nor DC-Y3 combined with Lm-Y3 dual immunotherapy strategy could effectively inhibit tumors (Fig. 7E).

These experimental results indicate that traditional alloantigen repeated immunotherapy is not sufficient to generate sufficient anti-tumor immune response based on mutant antigen Y3, and new immune strategies need to be designed to improve autoantigen-reactive T cell responses under central tolerance. It is possible to be effective in treating cancer.

Example 7

Dual heterologous immunity can effectively improve immune response under central tolerance

In this embodiment, the present inventors further utilized mature dendritic cells to load different antigens, construct a dendritic cell vaccine, and bind Listeria cells expressing different antigens to immunize Rip-Ova mice, and detect their activation recognition itself. The T cell immune response of the antigen.

The present inventors first tested a vector (Listeria monocytogenes or mature dendritic cells) that only presented antigens in two immunizations without changing the immune antigen, and self-antigen-specific T cells in a central tolerance environment. Whether the reaction has an effect. The present inventors immunized Rip-Ova mice with a dendritic cell vaccine (DC-Y3) or Lm-Y3 loaded with mutant antigen Y3, and then re-immunized each group with DC-Y3 or Lm-Y3 according to experimental design. Mice were stimulated with native antigen N4 in vitro 4 days later to detect the expression of cytokines IFN-γ and TNF-α in CD8 ⁺ T cells.

The result is shown in Figure 8. Without changing the immune antigen, the proportion of IFN-γ ⁺ CD8 ⁺ T cells produced by each group of mice after two immunizations did not increase in the proportion after immunization with Lm-Y3 only, but decreased. Approximately 50%, while the ratio of TNF-α ⁺ CD8 ⁺ T cells and IFN-γ ⁺ TNF-α ⁺ CD8 ⁺ T did not change significantly (Fig. 8A). However, the inventors found that the spleen cells of the immunized mice were dosed with a gradient concentration of N4 in vitro (Fig. 8C) and calculated to compare the relative functional affinities (Fig. 8D), and the inventors found that, and only the mutant antigen Y3 was used for immunization. Compared with autoreactive T cells produced after one time, the relative functional affinities of autoreactive T cells produced by two Lm-Y3 immunizations and DC-Y3-Lm-Y3 immunization were significantly increased, respectively, 2.6 times and 15 times. Interestingly, the latter's relative functional affinity even exceeds the relative affinity of WT CR.T cells.

The results showed that the increase in the affinity of autoreactive T cells under the constant immunization antigen depends on the influence of the vaccine vector, and the combination of DC-Lm can effectively increase the affinity of autoreactive T cells.

Further, the present inventors changed the immune antigen based on the combination of two different combinations of vectors, and examined whether the expression of functional cytokines and functional affinities of T cells were affected. According to the experimental design and Figure 8B, the inventors used two vectors DC and Lm, different combinations of natural antigen N4 and mutant antigen Y3, immunized Rip-Ova mice twice every 7 days, and after 4 days of the second immunization. Cytokine expression and functional affinity of autoreactive CD8 ⁺ T cells recognizing native antigen N4 were detected (Fig. 8B, C and D). The present inventors have found that only immunological combinations such as DC-Y3-Lm-N4 are capable of producing distinct IFN-γ ⁺ CD8 ⁺ T cells, although the ratio is 0.68%, which is still slightly lower than that produced by Lm-Y3 immunization once. The ratio of γ ⁺ CD8 ⁺ T cells was 0.93%; however, the proportion of TNF-α ⁺ CD8 ⁺ T cells produced was significantly higher than that of other immune combinations, and it was about 3 times higher than that after Lm-Y3 immunization; The ratio of IFN-γ ⁺ TNF-α ⁺ CD8 ⁺ T cells produced was about 4 times higher than that after Lm-Y3 immunization, exceeding the IFN-γ ⁺ TNF produced by the lowest level of the immunological combination Lm-N4-DC-Y3. -α ⁺ CD8 ⁺ T cell ratio 13 times (Fig. 8B); in addition, the relative functional affinity of its autoreactive T cells, although significantly increased by 10.5 times compared to the affinity of Lm-Y3 after one immunization, and DC-Y3 There was no significant difference in the functional affinity of autoreactive T cells after the -Lm-Y3 combination immunization (Fig. 8C and D). These results indicate that, based on the identification of the vector, the functionality of autoreactive T cells is affected by the immune antigen, and the combination of the first mutant antigen and the natural antigen can promote the expression of various cytokines by autoreactive T cells.

The dual heterologous immunization strategy combining the vector and the antigen is firstly immunized with the mutant antigen and then with the natural antigen, and the combination of DC-Y3-Lm-N4 can simultaneously enhance the versatility and functional affinity of the autoreactive T cells. The inventors further tested the V[beta] use of this population of activated autoreactive T cells to determine if there was a change in the diversity of the T cell pool. By staining for Vβ of IFN-γ ⁺ CD8 ⁺ T cells with a specific antibody, it was found that compared with the Vβ ratio of RO CR.T cells after Lm-Y3 immunization once (Fig. 4B), after double heterologous immunization in Rip In the Ova mice, although the proportion of other Vβ used was only slightly increased, the diversity of the T cell pool was reduced (Fig. 8F), and the proportion of Vβ3 expressed by autoreactive T cells was significantly reduced by about 80% compared with the former alone. The ratio was similar to the ratio of Vβ3 in high-affinity WT Sp. T cells, and in addition, the proportion of autoreactive T cells expressing Vβ4 was also significantly increased by about 3.4-fold (Fig. 8F and G). This result indicated that the second time, immunized with the natural antigen, the autoreactive T cell pool immunoactivated by the mutant antigen was screened.

In conclusion, the present inventors have found that the use of a specific dual heterologous immunization strategy can enhance the immune response of autoreactive T cells under central tolerance inhibition, first using mutant antigens and then using natural antigens to promote the production of multifunctional T cells. However, changing the immune antigen-presenting vector is effective to increase the functional affinity of T cells. By analyzing the Vβ use of autoreactive T cell pools, it was found that low-affinity autoreactive T cells produced by immunization of mutant antigens may tend to use certain Vβ, and the autoreactive T cell bank generated after optimization of the immunization strategy. The lack of selection of T cell clones using these specific Vβ may be responsible for the increased functionality and affinity of autoreactive T cells.

Example 8

Optimized dual heterologous immune energy activates tumor-specific immune responses and inhibits tumor growth under central tolerance mechanisms

Based on the mutant antigen Y3, the inventors determined that the optimized dual heterologous immunological combination DC-Y3-Lm-N4 can most effectively activate autoantigen-reactive T cells in Rip-Ova mice under the action of the central tolerance mechanism. , in turn, may produce a potential anti-tumor immune response.

The present inventors further examined whether the autoreactive T cells induced by the dual heterologous immunization strategy have an anti-tumor effect when the tumor antigen is an autoantigen, and whether it can effectively inhibit tumor growth.

Rip-Ova mice were immunized with DC-Y3 or Lm-Y3, respectively, and re-immunized with Lm-Y3 or Lm-N4 7 days later. Mo5 melanoma cells were inoculated 7 days after the second immunization, and tumor growth was observed.

The result is shown in Figure 9. On Rip-Ova mice immunized with double heterologous combination DC-Y3-Lm-N4, the growth rate of tumors was significantly slower than that of the same combination Lm-Y3-Lm-Y3 and only the combination of DC-Y3-Lm- Tumor growth rate on Rip-Ova mice after Y3 immunization (Fig. 9A). By comparing the average tumor size, the inventors found that the combined immune-induced immune response of DC-Y3-Lm-N4 can effectively inhibit the growth of melanoma (Fig. 9B), compared to the unimmunized mice at the same time. The survival rate increased by 50% (Fig. 9C). The above results indicate that autoreactive T cells activated by the dual heterologous immunization strategy can specifically recognize and kill tumor cells, and have significant anti-tumor effects.

Example 9

Optimized dual heterologous immune energy as a therapeutic tumor vaccine inhibits tumor growth in tumor-bearing mice

The present inventors have demonstrated that dual heterologous immunocombination DC-Y3-Lm-N4 activated autoreactive T cells can kill tumor cells and inhibit tumor growth on Rip-Ova mice. In this example, it was further tested whether a dual heterologous immunotherapy strategy can be used as a therapeutic vaccine in a constructed central tolerant tumor-bearing mouse model, inhibiting tumors and prolonging the survival of mice.

Rip-Ova mice were inoculated subcutaneously with Ova-expressing melanoma cells Mo5, the first treatment was performed on day 7, and the second treatment was performed on day 14 (Fig. 10A).

The results showed that DC-Y3-Lm-N4 was significantly more effective than the untreated control group and other dual heterologous immunotherapy groups. More effective inhibition of tumor growth (Fig. 10B). Compared with the alloantigen immunotherapy group DC-Y3-Lm-Y3 (Fig. 5D), the double heterologous immunization combination was also more effective in inhibiting tumor growth (Fig. 10C) and increasing the survival rate of the mice (Fig. 10C). Figure 10D). The average survival of mice in the untreated group was 26.25 days, and the mean survival time in the DC-Y3-Lm-Y3 treatment group was 26.4 days, while the average survival time of DC-Y3-Lm-N4 treatment was 32 days. The other two groups prolonged the survival of the mice for nearly 1 week.

Example 10

Adjuvant-based dual heterologous immunization enhances anti-tumor specific T cell immune response and inhibits tumor growth

As described above, the present inventors have verified that autoreactive T cells induced by activation of the dual heterologous immunological combination DC-Y3-Lm-N4 can inhibit tumor growth on Rip-Ova mice. However, Ova is not a true melanoma antigen. In practice, melanoma cells do not induce Ova-specific T cell responses and do not kill tumor cells. In order to test whether the dual heterologous immunization strategy of the present invention is effective for a true tumor antigen, the T cell response to a true tumor antigen and its ability to inhibit tumors, in this example, the epitope of the melanoma antigen Trp2 The Trp2SD bit was studied as a research object. As shown in Fig. 11A and the following table, a series of mutant antigen polypeptides obtained by Trp2SD were synthesized, and their mutation sites and mutated amino acids were named I2, SE, SN, SK, SR, respectively.

天然抗原SD：Natural antigen SD:	SVYDFFVWLSVYDFFVWL	SEQ ID NO.:7SEQ ID NO.: 7
突变抗原I2：Mutant antigen I2:	SIYDFFVWLSIYDFFVWL	SEQ ID NO.:9SEQ ID NO.: 9
突变抗原SE：Mutant antigen SE:	SVYEFFVWLSVYEFFVWL	SEQ ID NO.:10SEQ ID NO.: 10
突变抗原SN：Mutant antigen SN:	SVYNFFVWLSVYNFFVWL	SEQ ID NO.:11SEQ ID NO.: 11
突变抗原SK：Mutation antigen SK:	SVYKFFVWLSVYKFFVWL	SEQ ID NO.:8SEQ ID NO.: 8
突变抗原SR：Mutation antigen SR:	SVYRFFVWLSVYRFFVWL	SEQ ID NO.:12SEQ ID NO.: 12

On the basis of this, the self-reactivity ability of these multi-mutation antigen polypeptides and the cross-reactivity ability against the native antigen SD were identified. I2, SE, and SN have better self-reactivity, but because SD can escape immune tolerance through various ways, its own response ability is better (0.6%), and its own reactivity has reached 0.6%. Although the ratio of autoreactive CD8 T cells of polypeptides such as I2, SE, and SN was higher than that of SD, the cross-reactivity was not significantly superior to that of SD (I2, SE slightly increased, Figs. 11B and 11C). None of these polypeptides significantly increased the cross-reactivity to native antigen SD (Figures 11B and 11C).

To validate the dual heterologous immunization strategy, an immunological combination of DC-SK-LPS + CpG + SD (SK-SD) was employed. This combination uses LPS+CpG as an adjuvant instead of Listeria + SD instead of SD-loaded Listeria, because of the lack of Listeria-loaded Listeria, and Listeria as an antigen carrier, its main effect is to induce body production. To verify the inflammatory environment, LPS+CpG can play a similar role and have the same effect, and LPS+CpG has a lower risk of causing infection to the body than Listeria.

As shown in Figure 12A, SK-SD immunization induced a stronger specific IFN-γ response than DC-SK-LPS + CpG + SK (SK-SK) immunization. The SK-SK immune-induced IFN-γ response was around 0.5%, while the SK-SD immune-induced IFN-γ response was as high as 5.6%. In addition, the SK-SD immune-induced TNF-α response and the versatile CD8 T cell response were also significantly higher than SK-SK (Figs. 12B and 12C). Replace the LPS with an adjuvant MPL that has been clinically approved. The CD8 T cell response induced by the SK-SD (DC-SK-MPL+CpG+SD) immunization group was also higher than that of the SD-SD (DC-SD-MPL+CpG+SD) control group.

Further use of clinically approved adjuvants? ? MPL replaces LPS in order to obtain better clinical application value. As shown in Fig. 12D, the immunological combination of DC-SK-MPL+CpG+SD is more sensitive than DC-SD-MPL+CpG+SD in inducing IFN-γ. The ability of positive CD8 T cells to respond slightly increased, but there was no significant difference, probably because the Trp2SD antigen itself has a stronger ability to induce T cell responses, while Trp2SK is not an ideal mutant antigen, and its induced cross-reactivity is better than Trp2SD-induced itself. The reaction is even weaker (Fig. 11C).

To further investigate the ability of the SK-SD immunological combination to inhibit tumor growth inhibition, melanoma cells were inoculated on wild-type mice, and the first treatment was performed on day 7 using DC-SK, and LPs were used on day 14. The second treatment was performed with +CpG+SK or LPS+CpG+SD, and tumor volume was measured every three days from the start of day 6 from the inoculation of Mo5.

The results showed that the tumor volume of the SK-SD treatment group was significantly smaller than that of the SK-SK treatment group and the non-treatment control group (Fig. 12EE). This suggests that dual heterologous immunization strategies can more effectively inhibit the growth of real tumor cells.

Example 11

Dual heterologous immunization enhances specific T cell immune responses of low-affinity tumor antigens and inhibits tumor growth

In order to study the effect of the dual heterologous immunization strategy on low-affinity tumor antigens, in this example, gp100 of the melanoma epitope was selected, and the mutant polypeptides MK gp100 (MK) and HM gp100 (HM) were synthesized. , A2, KV, P3 (Fig. 13A).

天然抗原gp100：Natural antigen gp100:	EGSRNQDWLEGSRNQDWL	SEQ ID NO.:13SEQ ID NO.: 13
突变抗原MK：Mutant antigen MK:	KGPRNQDWLKGPRNQDWL	SEQ ID NO.:14SEQ ID NO.: 14
突变抗原HM：Mutant antigen HM:	KVPRNQDWLKVPRNQDWL	SEQ ID NO.:15SEQ ID NO.: 15
突变抗原A2：Mutant antigen A2:	EASRNQDWLEASRNQDWL	SEQ ID NO.:16SEQ ID NO.: 16
突变抗原KV：Mutant antigen KV:	KVSRNQDWLKVSRNQDWL	SEQ ID NO.:17SEQ ID NO.: 17
突变抗原P3：Mutant antigen P3:	EGPRNQDWLEGPRNQDWL	SEQ ID NO.:18SEQ ID NO.: 18

As a result, it was identified that both MK and A2 antigens have a very good autoimmune response (Fig. 13B), in which MK has a significantly increased cross-reaction against the native antigen gp100, which is about 3.5-fold higher than that induced by gp100 itself (Fig. 13C), so MK antigen was selected for subsequent experiments.

In addition, a dual heterologous immunization strategy was used to immunize wild-type mice with DC-MK-LPS+CPG+gp100 (MK-gp100), DC-gp100-LPS+CpG+gp100 (gp100-gp100) immunization and DC-MK- LPS + CPG + MK (MK-MK) immunization was used as a control. The response of IFN-γ-positive CD8 T cells induced by MK-gp100 immunization was significantly higher than that of DC-gp100-LPS+CpG+gp100(gp100-gp100) control group (Fig. 14A), which induced TNF-α positive CD8T. The response of the cells and the response of the multifunctional CD8 T cells were also higher than those of the control group (Figs. 14B and 14C). Compared with gp100-gp100 and MK-Mk control group DC-MK-LPS+CPG+MK(MK-MK), the immunological combination of MK-gp100 can inhibit the tumor growth and inhibit the survival rate of mice. Tumor volume and prolonged survival of mice (Figures 14D and 14E).

These results fully demonstrate that dual heterologous immunization can increase the specific T cell immune response of low affinity tumor antigens and inhibit tumor growth.

Example 12

Cytokine IL15 significant T cell immune response

Although the dual heterologous immunization strategy has been successful in inhibiting tumor growth in a mouse model, its vaccine component, Listeria, has a risk of infection in the body, thus limiting its use in humans. Although LPS+CpG can partially replace the action of Listeria, the inflammatory reaction caused by it may cause side effects to the human body. Therefore, it is necessary to find a suitable adjuvant to replace Listeria and LPS+CpG. Listeria infection can cause a variety of cytokines to react. In this example, CCL3, IL12, and IL15, which have enhanced effects on T cell responses, were selected to replace Listeria.

In the dual heterologous immunization strategy, the second immunization is necessary to use Listeria or LPS+CpG as an adjuvant, and its main function is to recruit inflammatory factors to the immunization site to assist in the recognition of TCR and low-affinity antigens. Reduce the threshold for T cell responses. In order to find out which cytokines have similar adjuvant effects, we immunized mice with DC-MK gp100 as the first immunization, and the second immunization with DC+ different cytokines + gp100, and detected gp100 specificity after 7 days of the first immunization. CD8 T cell response.

Experimental Results As shown in Figure 15, a combined dual heterologous immunization strategy using IL15 as an adjuvant significantly increased IFN-γ (Figure 15A), TNF-α (Figure 15B), and versatility (Figure 15C) CD8 T cells. Reaction. In another experiment with DC-Trp2I2 as a priming and DC + different cytokine + Trp2SD as a secondary immunoassay, we found that CCL3 also has a role in promoting CD8 T cell responses (Fig. 15D, 15E and 15F). The effect of which cytokine as an adjuvant in this immunization strategy, and the combined use of multiple cytokines as adjuvants remains to be further verified experimentally.

Example 13

Deletion of Treg can significantly increase the immune response of low-affinity T cells

Regulatory T cells (Tregs) play a key role in central immune tolerance, which inhibits the immune response of low-affinity T cells, a mechanism by which central tolerance avoids autoimmune responses. In the anti-tumor immune response, the inhibitory effect of Treg on low-affinity autoantigens adversely. Tregs were temporarily deleted using antibodies to CD25 to increase the anti-tumor immune response. On the fourth day before immunization, wild-type mice were administered CD25 antibody and the corresponding isotype control antibody, and then mice were immunized with DC-gp100 and DC-MK gp100, respectively, and the immune response against the native antigen gp100 was detected 7 days later.

The result is shown in Fig. 16. The gp100 immunized group did not induce a better specific CD8 T cell response regardless of Treg deletion, but the MK gp100 immunized group, after Treg deletion, specific IFN-γ (Fig. 16A), TNF-γ (Fig. 16B) And the multifunctional CD8T cell response was significantly improved. This suggests that deletion of Treg can significantly increase the immune response of T cells with low affinity natural antigens, provided that they are immunized with a mutant antigen that is better cross-reactive with the native antigen.

discuss

Tumor antigens as autoantigens have always had poor immunogenicity, affecting therapeutic tumor vaccines. Bed application and development. Amino acid modification of T cell epitopes on tumor antigens can improve their immunogenicity and enhance the level of anti-tumor immune response, but the therapeutic effect in clinical experiments is not obvious. Past studies have shown that the low cross-reactivity of modified antigens to the natural tumor antigens caused by modified antigens in the central tolerance environment is one of the reasons for the poor efficacy of therapeutic antigen vaccines based on modified antigens, and the molecular mechanism that causes this problem is currently Still not very clear.

The present inventors infected a wild type mouse and an ovalbumin model antigen transgenic Rip-mOva mouse with a Listeria strain expressing a natural T cell epitope or a modified epitope of an ovalbumin model antigen, and found that the immune system under the mechanism of central tolerance Non-reactive to natural antigens, only medium-affinity modified antigens can induce the body's immune system to produce low-affinity T cells that cross-recognize natural antigens, and the T cell pool diversity is significantly reduced.

Through the dual heterologous immunization strategy, T cells that recognize natural tumor antigens can be activated in a central tolerance environment, which effectively enhances their affinity and versatility, inhibits tumor growth, and prolongs mouse survival. In addition, the dual immunization strategy combined with key point blockade therapy can play a better anti-tumor effect. The invention provides theoretical basis and guiding significance for antigen optimization and screening of therapeutic tumor vaccines and design of immune strategies.

Based on the above experimental results, it is concluded that the construction of therapeutic tumor vaccines should be based on the following principles:

1 selection of a suitable mutant antigen to enable it to activate a sufficient number of autoantigen-reactive T cells in the T cell bank as a primary immunizing antigen;

2 The combination and alteration of the primary immunized vector and the re-immunized vector to improve the affinity of the self-reactive T cell is the main purpose and measurement standard;

3 The efficiency of screening the T cell library by the natural antigen in the re-immunization, so that the self-reactive T cells after screening can better express the functional cytokines.

More than 4 immune strategies to improve T cell response can play a better anti-tumor effect.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

A non-therapeutic method for enhancing an anti-tumor immune response, comprising the steps of:

(a) providing a first tumor antigen and a second tumor antigen, wherein said enhanced tumor immune response is an enhanced tumor immune response against the first tumor antigen;

The first tumor antigen is a low immunogenic natural polypeptide derived from a mammal or an immunogenic fragment thereof, and is not efficiently produced when the first tumor antigen is immunologically administered to the mammal. An effective anti-tumor immune response against said first tumor antigen;

The second tumor antigen is a polypeptide homologous and intersecting with the first tumor immune antigen or an immunogenic fragment thereof;

And the affinity of the first tumor antigen to antigen-specific CD8 + T cells from the mammal is Q1, and the affinity of the second tumor antigen to antigen-specific CD8 + T cells from the mammal is Q2 And the ratio R1 of the Q2/Q1 is 0.02-0.80;

(b) immunizing said second tumor antigen to said mammalian subject, eliciting an immune response against said second tumor antigen, thereby obtaining a first immune challenged mammalian subject;

(c) after t days after the immunization administration of the previous step, wherein t is 3-60, immunizing said first tumor antigen to said first immunized mammalian subject, eliciting said first An immune response to the tumor antigen, thereby obtaining a re-immunized mammalian subject, wherein in the re-immunized mammalian subject, an enhanced tumor immune response against the first tumor antigen is produced.
The method of claim 1 wherein said first tumor antigen is a centrally tolerated tumor-specific epitope.
The method of claim 1 wherein said first tumor antigen is a native polypeptide derived from a mammal or an immunogenic fragment thereof.
The method of claim 1 wherein said second tumor antigen is a polypeptide homologous and intersecting said first tumor immune antigen or an immunogenic fragment thereof.
The method of claim 1 wherein said "homologous and crossed" means said second tumor antigen and said first tumor antigen have an average of 1 per 10 amino acids in length - 2 amino acid mutations, thus not only capable of eliciting an immune response against a second tumor antigen, but also eliciting a cross-over immune response to the first tumor antigen.
The method according to claim 1, wherein said "enhanced tumor immune response against said first tumor antigen" means initial administration with said first tumor antigen and re-use said first tumor antigen Mammalian subject for primary immunization with the second tumor antigen and re-immunized with the first tumor antigen in step (c) compared to the tumor immune response level Yc for the first tumor antigen in the immunoadministered control The tumor immune response level Yv against the first tumor antigen was significantly higher than Yc.
The method of claim 1 wherein the pharmaceutical composition or vaccine composition of the invention is administered Routes include, but are not limited to, intramuscular, subcutaneous, intradermal, intrapulmonary, intravenous, nasal, oral or other parenteral routes of administration. If desired, the route of administration can be combined or adjusted depending on the condition of the disease.
The method of claim 1 wherein the vaccine composition is administered in a single dose or in multiple doses and can include administering a booster dose to elicit and/or maintain immunity.
The method according to claim 1, wherein said tumor comprises a tumor selected from the group consisting of liver cancer, lung cancer, gastric cancer, breast cancer, ovarian cancer, prostate cancer, skin cancer, melanoma, cervical cancer, brain Cancer, thyroid and cholangiocarcinoma, bladder cancer and pancreatic cancer.
A method for selecting a candidate polypeptide immunogen having an anti-tumor immune response against a tumor antigen of low immunogenicity, comprising the steps of:

(a) providing a test group comprising i polypeptide immunogens to be selected, wherein said polypeptide immunogens are homologous and intersecting, wherein i is a positive integer of ≥1,

Also, "homologous and crossed" refers to a polypeptide immunogen (A antigen or natural antigen) and another polypeptide immunogen (B antigen or similar antigen) in the test group at every 10 amino acids in length. Has an average of 1-2 amino acid mutations, and thus is capable of eliciting an immune response against the antigen not only in the mammal, but also causing a cross-over immune response against the adjacent antigen;

Wherein at least one of the polypeptide immunogens is a mutant polypeptide immunogen;

(b) determining the reaction affinities of the respective polypeptide immunogens and the antigen-specific CD8 + T cells elicited by each of the polypeptides, respectively, as Qj, wherein any positive integer of j = 1 - i;

(c) sorting each of the Qjs, selecting a polypeptide immunogen that is located in the middle, as a candidate polypeptide immunogen having an anti-tumor immune response that enhances against a low immunogenic tumor antigen; and/or

Comparing each Qj with Qz, selecting a polypeptide immunogen having a ratio R of Qj/Qz of 0.02-0.80 as a candidate polypeptide immunogen having an anti-tumor immune response against a low immunogenic tumor antigen, wherein Qz Is the affinity of the first tumor antigen to antigen-specific CD8 + T cells from the mammal, and the first tumor antigen is a low immunogenic natural tumor antigen derived from a mammal or an immunogen thereof A fragment, and when the first tumor antigen is immunized to the mammal, is not effective to produce an effective anti-tumor immune response against the first tumor antigen.
The method according to claim 10, wherein in the step (c), the method comprises: sorting the respective Qjs, defining a maximum value in Qj as Qmax, and selecting a ratio R of Qj/Qmax to be 0.02. A polypeptide immunogen of -0.80 as a candidate polypeptide immunogen with an anti-tumor immune response that enhances tumor antigens against low immunogenicity.
The method of claim 10, wherein said method further comprises the step of: (d) testing the candidate polypeptide immunogen selected in the previous step, and enhancing its resistance against tumor antigens of low immunogenicity The ability of a tumor to respond to an immune response.
A composition product for enhancing an anti-tumor immune response, characterized in that the product comprises:

(1) a first composition comprising (i) a first tumor antigen and/or dendritic cells sensitized by said first tumor antigen; (ii) an optional adjuvant; And (iii) a pharmaceutically or immunologically acceptable carrier;

(ii) a second composition comprising (i) a second tumor antigen and/or dendritic cells sensitized by said second tumor antigen; (ii) an optional adjuvant; And (iii) a pharmaceutically or immunologically acceptable carrier;

And, the first composition and the second composition are independent,

Wherein the first tumor antigen is a low immunogenic natural polypeptide derived from a mammal or an immunogenic fragment thereof, and is ineffective when the first tumor antigen is immunologically administered to the mammal Producing an effective anti-tumor immune response against the first tumor antigen;

The second tumor antigen is a polypeptide homologous and intersecting with the first tumor immune antigen or an immunogenic fragment thereof;

And the affinity of the first tumor antigen to antigen-specific CD8 + T cells from the mammal is Q1, and the affinity of the second tumor antigen to antigen-specific CD8 + T cells from the mammal is Q2 And the ratio R1 of the Q2/Q1 is 0.02-0.80,

And the enhanced tumor immune response is an enhanced tumor immune response against the first tumor antigen.
The composition product of claim 13 further comprising: (iii) a third composition comprising an immunological critical point inhibitor for enhancing an immune response.
The composition product according to claim 14, wherein said immunologically important immunosuppressive point inhibitor comprises a CTLA-4 antibody, a PD-1 antibody, and a PD-L1/2 antibody.
A composition product according to claim 13 wherein said composition product is a tumor vaccine or vaccine composition for treating or preventing a tumor.
The composition product of claim 13 wherein said adjuvant is selected from the group consisting of a cytokine, a chemokine, or a combination thereof.
The composition product of claim 17 wherein said cytokine is IL-15.
Use of a composition product according to claim 13 for the preparation of a medicament for enhancing an anti-tumor immune response.
The use according to claim 19, wherein the subject to be treated comprises a human and a non-human mammal.