CN110804594B

CN110804594B - Engineered antigen presenting cells and their use for activating CD3+ immune cells

Info

Publication number: CN110804594B
Application number: CN201911145779.5A
Authority: CN
Inventors: 蒋俊; 文高柳; 林鑫
Original assignee: Qichensheng Biotechnology Zhuhai Co ltd
Current assignee: Qichensheng Biotechnology Zhuhai Co ltd
Priority date: 2019-11-21
Filing date: 2019-11-21
Publication date: 2020-11-20
Anticipated expiration: 2039-11-21
Also published as: CN110804594A

Abstract

The invention discloses an engineered antigen presenting cell and application thereof in activating CD3+ immune cells. The engineered antigen presenting cell is obtained by introducing exogenous genes into a wild-type antigen presenting cell through an engineering means so as to over-express recombinant interleukin-12 and soluble CD 80. The invention promotes the activation of CD3+ immune cells through the combined synergistic effect of recombinant interleukin-12 and soluble CD 80.

Description

Engineered antigen presenting cells and their use for activating CD3+ immune cells

Technical Field

The invention relates to the fields of immunology and medicine, in particular to an engineered antigen presenting cell and application thereof in activating CD3+ immune cells.

Background

Interleukin-12 (IL-12) is produced by antigen presenting cells and B cells, is a pro-inflammatory cytokine in the form of a heterodimer, and is secreted extracellularly in this form. IL-12 can induce IFN-gamma production, in vivo immune response (especially in bacterial or parasitic infection) it is also IFN-gamma production required.

DC cells were the first cells to synthesize IL-12 and found to be dependent on IFN-. gamma.and signals from T cells. Like other inflammatory cytokines, IL-12 production is tightly controlled by positive and negative regulatory mechanisms. Different cytokines such as IFN-gamma, TNF-beta, GM-CSF enhance the ability of cells to produce IL-12, IFN-gamma enhances the transcription of genes encoding p40 and p35, which have a particularly pronounced effect on heterodimer production, and IFN-gamma forms a positive feedback mechanism with the ability of IL-12 production in inflammatory and Th1 responses.

In the anti-tumor immunity of the body, tumors can escape from immunity through immune tolerance, antigen modulation, immunosuppressive factors and the like. Researches show that the tumor patients have the characteristics of reduced DC number and functional defects, and the number and the function of tumor tissues and DC infiltrated around the tumor tissues have close relation with the occurrence, the development, the metastasis and the prognosis of tumors. The tumor densely infiltrated by the DC has high differentiation degree and better prognosis; whereas tumors that are mildly infiltrated by DCs are often associated with low differentiation and malignant progression. Tumor cells have high-level Fas expression, can induce apoptosis of lymphocytes expressed by FasL, and can secrete immunosuppressive cytokines such as TGF-beta, IL-10 and the like, so that the antigen presenting capability is reduced, and immune attack is avoided.

In recent years, it has become clear that the immune system does recognize tumor antigens, but despite the presence of tumor antigens, T cells are assured to remain quiescent. There is a hypothesis that: antigen presenting cells in the patient, which fail to correctly recognize the tumor antigen, present it to T lymphocytes, causing a tumor-specific immune response. Therefore, how to activate immune cells with immune tolerance and induce tumor-specific immune response becomes a key point in the current tumor immunization research.

Disclosure of Invention

The invention provides an engineered immune cell which is used for solving at least part of technical problems in the prior art. The invention discovers that the capacity of antigen presenting cells for activating CD3+ immune cells can be greatly improved by modifying the antigen presenting cells through an engineering means so that the antigen presenting cells secrete soluble CD80 fragments while excessively expressing recombinant interleukin-12. The present invention has been accomplished based at least in part on this finding. Specifically, the present invention includes the following.

In a first aspect of the invention, there is provided an engineered antigen presenting cell which is obtained by introducing a foreign gene into a wild-type antigen presenting cell by engineering means to overexpress recombinant interleukin-12 and soluble CD 80.

The engineered antigen presenting cell according to the present invention, wherein the exogenous gene comprises a gene encoding recombinant interleukin-12 and a gene encoding soluble CD 80.

The engineered antigen presenting cell of the invention, wherein the recombinant interleukin-12 is a fusion protein of two subunits of p40 and p35, and the gene sequence coding the recombinant interleukin-12 is shown as SEQ ID No.1 or has more than 95% homology with the gene sequence and is derived from the same species.

The engineered antigen presenting cell of the invention, wherein the soluble CD80 comprises an extracellular region of CD80, and the gene sequence encoding the soluble CD80 is shown in SEQ ID No.2 or has more than 95% homology with the soluble CD80 and is derived from the same species.

An engineered antigen presenting cell according to the invention, further comprising an antigen or a nucleic acid encoding said antigen.

An engineered antigen presenting cell according to the invention, wherein said antigen is the GPC3 antigen.

An engineered antigen presenting cell according to the invention, wherein it is a dendritic cell.

In a second aspect of the invention, there is provided an immunomodulatory composition comprising an engineered antigen presenting cell of the first aspect and/or a secretion thereof.

In a third aspect of the invention, there is provided a method for activating a CD3+ immune cell, comprising the step of contacting an engineered immune cell of the first aspect or an immunomodulatory composition of the second aspect with an immune-tolerant CD3+ immune cell.

The method for activating a CD3+ immune cell, wherein the CD3+ immune cell is an in vitro cell.

The invention finds that the soluble CD80 can greatly enhance the ability of IL-12 to activate CD3+ cells on the basis of a large number of screening experiments, and the two can play a synergistic effect, promote the continuous amplification of CD3+ immune cells such as T cells, generate more lymphocytes with anti-tumor activity and improve the treatment of tumors.

Drawings

FIG. 1 shows the results of CD 8T cell immune responses in experiments in which DC cells were transfected with mRNA encoding each component protein of the composition of the present invention and GPC3 antigen mRNA, and then primed in vitro with T cells. In each column set of FIG. 1, from left to right, the proportion of CD8 IFN-. gamma. +, CD8 IFN-. gamma. +, TNF-. alpha. +, and CD8 TNF-. alpha. +, to total CD 8T cells, respectively.

FIG. 2 shows the results of CD 4T cell immune responses in experiments in which DC cells were transfected with mRNA encoding each component protein of the composition of the present invention and GPC3 antigen mRNA, and then primed in vitro with T cells. In each column set of FIG. 2, from left to right, the proportion of CD4 IFN-. gamma. +, CD4 IFN-. gamma. +, TNF-. alpha. +, and CD4 TNF-. alpha. +, to total CD 4T cells, respectively.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

[ engineered immune cells ]

In a first aspect of the invention, an engineered antigen presenting cell is provided that can be used as an immunomodulatory composition to activate an immune cell in conjunction with a corresponding antigen. The engineered antigen presenting cell is obtained by introducing exogenous genes into a wild-type antigen presenting cell through an engineering means so as to over-express recombinant interleukin-12 and soluble CD 80.

In the present invention, wild-type antigen presenting cells generally refer to natural antigen presenting cells isolated from the body of a subject. The subject is generally a mammal, and examples thereof include rats, mice, pigs, rabbits, and primates. The subject is also preferably a human.

In the present invention, the foreign gene refers to a gene derived from the outside of the wild-type antigen presenting cell, and includes a gene derived from a different subject or the same source as the wild-type antigen presenting cell, and preferably, the wild-type antigen presenting cell and the foreign gene are derived from the same source, for example, both of human origin.

In the present invention, the foreign gene includes a gene encoding recombinant interleukin-12 and a gene encoding soluble CD 80. IL-12 is known to be a heterodimer p70 consisting of two chains, p35 and p40, with two subunits linked together by disulfide bonds. In nature, the amount of p40 far exceeds p 70. Excess p40 forms a p40 homodimer that is biologically inactive, but binds to IL-12R β and competes with p70 for the IL-12 receptor (IL-12R) to antagonize its biological effects. Based on this, the present invention preferably uses a fusion protein of two subunits, p40 and p 35. The production of unwanted p40 in nature is avoided by this fusion protein, with unexpected beneficial effects.

In the invention, the recombinant interleukin-12 has similar functions of natural IL-12, can proliferate activated T cells and increase the cytotoxic activity of the activated T cells, induce NK cells and T cells to generate gamma-interferon (IFN-gamma), regulate the development of Th1/Th2 cells and promote the differentiation of the cells to Th1 cells. In the present invention, recombinant IL-12 also induces cytokines in CTL immune responses, initiating cell-mediated immune responses. The recombinant IL-12 can not only activate innate immune cells, promote APC functions, particularly DC maturation, and improve antigen presentation efficiency. In addition, the recombinant IL-12 also cooperates with the IL-18 to promote the activation of memory T cells, so that the infected cells can be rapidly eliminated, and simultaneously, the secretion of other cytokines is promoted, the virus replication is inhibited, and the immune function of other cells is regulated.

In certain embodiments, the invention of the recombinant IL-12 by SEQ ID No.1 shows the sequence of the gene coding, the recombinant IL-12 produced by this lacks IL-12p35 signal peptide sequence. It will be readily appreciated by those skilled in the art that the sequence shown in SEQ ID No.1 is exemplary only and that modifications may be made to this sequence without affecting the achievement of the objects of the invention. Such modifications include the use of biased codons to optimize SEQ ID No. 1. Therefore, the present invention can also use a sequence having 95% or more, preferably 97% or more, more preferably 99% or more homology with SEQ ID No.1 and derived from the same species.

In the present invention, the recombinant IL-12 amino acid sequence is not particularly limited, its examples include SEQ ID No.3 shows the sequence, or with its homology of 95% or more, preferably 97% or more, more preferably 99% or more and derived from the same species sequence.

The CD80 of the present invention belongs to immunoglobulin superfamily (IgSF), and exists in the form of oligomer in most antigen presenting cells, such as dendritic cells, activated T cells, B cells, macrophages, etc. CD80 is known to be an important costimulatory molecule on APC expression, and is a positive factor that has important promoiety regulatory effects on antigen-induced initiation of T cell activation, proliferation and effector function generation after binding to T cell CD 28. Under wild conditions, CD80 bound to the surface of cells as a membrane protein. The present inventors have found that in contrast to this, soluble CD80 is secreted outside the cell and, as a soluble fragment, synergistically enhances the ability of IL-12 to activate immune cells. The reason for this may be that soluble CD80, on the one hand, due to its increased freedom of solubility, binds more readily to its receptor, e.g. CD28, thereby enhancing the transduction of downstream signals. On the other hand, CD80 is used as a downstream factor of IL-12, and when the downstream factor is overproduced, the feedback inhibition is generated so as to negatively regulate the production or activation of IL-12, while the invention is not influenced by the negative regulation by simultaneously overexpressing recombinant IL-12 and soluble CD80, so that the capability of activating immune cells can be greatly enhanced.

In the present invention, soluble CD80 is a fragment comprising the extracellular domain of CD 80. Preferably, further comprising other fragments. Examples of other fragments include, but are not limited to, Fc fragments, which are useful for extending the half-life and improving stability of the protein. It is preferred to use longer Fc fragments to increase molecular volume. However, if the molecular size is too large, the activity of soluble CD80 may be affected. Thus, the length or size of the Fc fragment influences the achievement of the object of the present invention. For the purposes of the present invention, the length of the Fc fragment is generally 105-315AA, preferably 120-315 AA. The Fc fragment of the present invention may comprise a fragment naturally occurring in an immunoglobulin, and may further comprise a mutant Fc fragment modified by known genetic engineering means to obtain more superior performance. For example, the Fc fragment contains 3 mutated fragments of "YTE", i.e. methionine (Met, M), serine (Ser, S) and threonine (Thr, T) of 252, 254 and 256 are replaced by tyrosine (Tyr, Y), T and glutamic acid (Glu, E), respectively, thereby obtaining soluble proteins with longer half-lives. For another example, by genetic engineering and modification of disulfide bonds of an Fc fragment, Fc proteins can be aggregated into multimeric complexes, thereby obtaining soluble proteins with superior stability.

In certain embodiments, the soluble CD80 of the invention is encoded by the sequence shown in SEQ ID No.2 or a sequence having more than 95%, preferably more than 96%, more preferably more than 98%, even more preferably more than 99% homology thereto. In the present invention, the amino acid sequence of soluble CD80 is not particularly limited, and examples thereof include the sequence shown in SEQ ID No.4, or a sequence having 95% or more, preferably 97% or more, more preferably 99% or more homology thereto and derived from the same species.

In the present invention, an engineered cell refers to a cell that has been artificially modified. Herein, the antigen presenting cell refers to a cell having the functions of taking up, processing and transmitting antigen information in the body, presenting the antigen to immune cells and assisting and regulating T cells, B cells recognizing the antigen and inducing immune response. Examples include, but are not limited to, macrophages, dendritic cells, and syndactylic cells, pancreatic cells, and B cells. Preferably, the immune cells of the invention are dendritic cells, more preferably human dendritic cells. The dendritic cells of the present invention may be mature dendritic cells or immature dendritic cells. It should be noted that the dendritic cells are obtained by in vitro induction culture, i.e., Peripheral Blood Mononuclear Cells (PBMCs), which are induced to become DC cells under stimulation of various types of culture media and various types of cytokines. In specific embodiments, the culture medium used for in vitro culture includes AIM-V medium, iDC medium and mDC medium, and examples of cytokines used for in vitro induction culture include, but are not limited to, granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4.

Preferably, the antigen presenting cell of the invention further comprises an antigen or a precursor capable of producing an antigen, and the composition of the first aspect. "antigen" refers to a substance that is recognized by the immune system and is capable of eliciting an antigen-specific immune response through the formation of antibodies or/and antigen-specific T cells. In general, an antigen can be a protein or polypeptide that contains at least one antigenic epitope, is captured by an APC and can be presented on the surface of a T cell. In the present invention, the antigen may be a product of translation of mRNA or a product of transcription and translation of DNA. In certain embodiments, the antigen of the invention is a GPC3 antigen that is closely associated with the development and progression of primary liver cancer.

[ immune modulating composition ]

In a second aspect of the invention, there is provided an immunomodulatory composition comprising an engineered cell of the first aspect and/or a secretion thereof, capable of providing one or more antigens (preferably an oncogene) to a subject in need thereof, and modulating (in particular enhancing) the immunity of the antigen, thereby alleviating, delaying or curing the condition or disorder in the subject. The pharmaceutical compositions of the invention have enhanced, increased or boosted immunocompetence as compared to a drug that provides the antigen alone, or as compared to a composition that provides the antigen and IL-12 or CD 80. The immunomodulatory compositions of the invention may optionally further comprise other ingredients, such as pharmaceutically acceptable carriers. Examples of suitable pharmaceutically acceptable carriers include, but are not limited to: 1) dulbecco phosphate buffered saline, pH about 7.4, with or without about 1mg/ml to 25mg/ml human serum albumin; 2) 0.9% saline (0.9% w/v sodium chloride), and 3) 5% (w/v) glucose; antioxidants such as tryptamine and stabilizers such as Tween20 may also be included.

[ method for activating CD3+ immune cells ]

In a third aspect of the invention, there is provided a method for activating a CD3+ immune cell, comprising the step of contacting an engineered immune cell of the first aspect or an immunomodulatory composition of the second aspect with a CD3+ immune cell. Wherein the CD3+ immune cells are preferably immune tolerant CD3+ immune cells. Herein, CD3+ immune cells refer to cells comprising CD3 on the surface, preferably CD3+ T cells

In an exemplary method of activating CD3+ immune cells, it comprises the steps of:

(1) preparing a nucleic acid construct, wherein the nucleic acid construct can be one or more, comprising a nucleic acid that expresses recombinant interleukin-12, a nucleic acid that expresses soluble CD80, and optionally a nucleic acid that expresses an antigen;

(2) performing in vitro transcription to obtain a corresponding ribonucleic acid molecule;

(3) performing in vitro induction culture to obtain antigen presenting cells for expressing the ribonucleic acid molecules of step (2);

(4) introducing the ribonucleic acid molecule of the step (2) into the antigen presenting cell of the step (3) through transfection and expressing, thereby obtaining an engineered immune cell; and

(5) the engineered immune cells are contacted or co-cultured with CD3+ immune cells.

Preparation example 1

This preparation example was used to prepare DNA and mRNA encoding an antigen and serving as a precursor of an interleukin complex

1. Preparation of DNA and mRNA constructs

DNA sequences encoding IL12 p70 and soluble CD80 mRNA were constructed separately and used for subsequent in vitro transcription reactions. The coding sequence is followed by a segment of polyadenylation. The DNA sequence information is shown in Table 1 below.

In addition, a coding sequence of human tumor antigen GPC3 for in vitro sensitization is constructed, the coding sequence of GPC3 of the invention consists of a sequence shown in SEQ ID No.5, and the amino acid sequence consists of a sequence shown in SEQ ID No. 6. The sequence of GPC3 can be obtained from the Genebank database. The antigen disclosed in CN107583042A was used in this example.

TABLE-1 Gene sequence Listing

Name (R)	Serial number
		IL-12	SEQ ID No.1
CD80	SEQ ID No.2
		GPC3	SEQ ID No.5

2. In vitro transcription

First, the corresponding DNA plasmid obtained by the preparation was linearized with a restriction enzyme, and mRNA was prepared by in vitro transcription using T7 RNA polymerase using the linearized plasmid as a template. The prepared mRNA was then purified by lithium chloride precipitation.

Example 1

In vitro induction culture of DC cells

Aseptically extracting venous blood 50ml of hepatocellular carcinoma patient, separating peripheral blood mononuclear cells with lymphocyte separation medium in ultraclean bench, adding mononuclear cells into AIM-V culture medium, placing at 37 deg.C and 5% CO₂Incubation in an incubator allows monocytes to adhere. After 2h, nonadherent cells were removed, adherent cells were added to iDC medium (GM-CSF at a final concentration of 800U/mL and IL-4 at a final concentration of 500U/mL in AIM-V medium), and the mixture was placed at 37 ℃ with 5% CO₂Culturing in an incubator for 6 days. Half of the cell culture medium was transferred to a centrifuge tube, and 500g of the medium was centrifuged to collect cells, the supernatant was removed, and an equal volume of fresh mDC medium (configuration of mDC fresh medium: GM-CSF at a final concentration of 1600U/mL and IL-4, TNF- α (5ng/mL), IL-1 β (5ng/mL), IL-6(150ng/mL) and prostaglandin E2(PGE2) (1. mu.g/mL) were added to AIM-V medium), and after resuspension of the cells, the cells were added to a flask and cultured for 8-18 hours to induce maturation of the DC cells.

2. Transfection of DC cells

On the day of transfection, digestion with non-enzymatic cellsThe reagent is used for digesting DC cells into cell suspension, washing the cells twice by PBS after centrifugation, re-suspending the cells by PBS, and adjusting the cell density to be 25-30 multiplied by 10⁶DCs/ml. According to each 10⁶DC cells were transfected with a proportion of 10. mu.g mRNA, the combination of DC cells and antigen mRNA with mRNA for the different proteins IL12 and CD80 were mixed, the cell-mRNA mixture was added to an electric rotor, and antigen mRNA was transfected into DC cells using an ECM630 electrotransfer. The cells after the electroporation were resuspended in a cytokine-free 1640 medium, and the cell density was adjusted to 2X 10⁵DCs/ml, placed at 37 ℃ in 5% CO₂The cultivation was continued in the cell incubator for 6 hours. In this experiment, the mRNA combinations used were as follows:

1) control without any mRNA (mDC control group)

2) Only the mRNA encoding the GPC3 antigen (GPC3 control group)

3) mRNA encoding GPC3 antigen and mRNA of IL-12p70 (IL12 group)

4) mRNA encoding GPC3 antigen and mRNA of CD80 (group CD 80)

5) mRNA encoding GPC3 antigen was compared with mRNA encoding IL-12p70 and CD80 (Experimental group)

3. Peripheral Blood Mononuclear Cells (PBMC) revived overnight at 2X 10⁶The cells were seeded in 96-well plates at a concentration of one ml and 100. mu.l of cells were seeded per well for activation of T lymphocytes. The test grouping case is: a PBMC control group without DC cells, a group co-cultured with the PBMC cells with the six groups of DC cells in the previous step, respectively; according to grouping conditions, DC cells loaded with corresponding mRNA are added into different wells, and the ratio of PBMC to DC is 10: 1; the cells were cultured at 37 ℃ for 10-12 days.

4. Intracellular cytokine assays were performed 10-12 days after co-culture.

5-8h before collecting cells, mixing cultured T cells, and adjusting cell density to 2 × 10⁶Each well was inoculated into a 96-well plate at a volume of 100. mu.l per well, and incubated at 37 ℃ in an incubator. The positive control was PMA (50ng/ml) + ionomycin (1. mu.g/ml), and the negative control contained suspension cells only.

Antigen-loaded DC cells were prepared as target cells. Antigen-loaded cryopreserved DC cells that have been prepared are thawed and trypan usedBlue staining to count cells, resuspending the cells in complete medium with RPMI containing IL-7 and IL-2 cytokines, and adjusting the cell concentration to 2X 10⁵Mu.l of cells were added per well.

Add Monensin or 3. mu.g/ml Brefeldin A to the cell culture medium to a final concentration of 2. mu.M, mix well. Monensin and Brefeldin A are used as protein transport blockers, and intracellular staining detection is carried out after the time in cell sap is not more than 12h and 4-6 h.

5. The cells were removed, transferred to corresponding flow tubes, stained with fluorescently labeled antibodies to CD3, CD4, and CD8, fixed and permeabilized, and stained intracellularly with fluorescently labeled antibodies to TNF- α and IFN- γ.

6. The ratio of TNF-alpha + and IFN-gamma + cells in lymphocytes was measured by flow cytometry.

As shown in the results of FIG. 1 and FIG. 2, the composition provided by the invention can significantly improve the proportion of IFN-gamma positive cells and the proportion of TNF-alpha positive cells in the CD 8T cell subset, and the proportion of TNF-alpha positive cells in the CD 4T cell subset, and especially has synergistic immune cell activation effect compared with the single use of IL-12 and CD 80.

Compared with the group transfected with GPC3 antigen alone, the proportion of TNF-alpha positive CD 4T cells was 0.44%, whereas in the experimental group, the proportion of TNF-alpha positive CD 4T cells was 1.08%, which was 1.45-fold higher. Similarly, the proportion of IFN- γ positive CD 8T cells in the GPC3 group was 0.062% and TNF- α positive CD 8T cells was 0.372%, which were 0.266% and 1.016%, respectively, in the experimental group, and increased by 3.29-fold and 1.73-fold, respectively, showing better initial CD4 and CD 8T cell activation capacity.

While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Many modifications and variations may be made to the exemplary embodiments of the present description without departing from the scope or spirit of the present invention. The scope of the claims is to be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.

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Met Trp Pro Pro Gly Ser Ala Ser Gln Pro Pro Pro Ser Pro Ala Ala

1 5 10 15

Ala Thr Gly Leu His Pro Ala Ala Arg Pro Val Ser Leu Gln Cys Arg

20 25 30

Leu Ser Met Cys Pro Ala Arg Ser Leu Leu Leu Val Ala Thr Leu Val

35 40 45

Leu Leu Asp His Leu Ser Leu Ala Arg Asn Leu Pro Val Ala Thr Pro

50 55 60

Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg

65 70 75 80

Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr

85 90 95

Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys

100 105 110

Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu

115 120 125

Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys

130 135 140

Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser

145 150 155 160

Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn

165 170 175

Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn

180 185 190

Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser

195 200 205

Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys

210 215 220

Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala

225 230 235 240

Val Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser Gly Ser Gly

245 250 255

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

260 265 270

Pro Gly Pro Met Cys His Gln Gln Leu Val Ile Ser Trp Phe Ser Leu

275 280 285

Val Phe Leu Ala Ser Pro Leu Val Ala Ile Trp Glu Leu Lys Lys Asp

290 295 300

Val Tyr Val Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met

305 310 315 320

Val Val Leu Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr

325 330 335

Leu Asp Gln Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile

340 345 350

Gln Val Lys Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly

355 360 365

Gly Glu Val Leu Ser His Ser Leu Leu Leu Leu His Lys Lys Glu Asp

370 375 380

Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn

385 390 395 400

Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr

405 410 415

Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys

420 425 430

Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala

435 440 445

Thr Leu Ser Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr

450 455 460

Ser Val Glu Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser

465 470 475 480

Leu Pro Ile Glu Val Met Val Asp Ala Val His Lys Leu Lys Tyr Glu

485 490 495

Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro

500 505 510

Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu

515 520 525

Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe

530 535 540

Ser Leu Thr Phe Cys Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys

545 550 555 560

Lys Asp Arg Val Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg

565 570 575

Lys Asn Ala Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser

580 585 590

Ser Trp Ser Glu Trp Ala Ser Val Pro Cys Ser

595 600

<210> 4

<211> 474

<212> PRT

<213> human (Homo sapiens)

<400> 4

Met Gly His Thr Arg Arg Gln Gly Thr Ser Pro Ser Lys Cys Pro Tyr

1 5 10 15

Leu Asn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu Ser His Phe Cys

20 25 30

Ser Gly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu

35 40 45

Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile

50 55 60

Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp

65 70 75 80

Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr

85 90 95

Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly

100 105 110

Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg

115 120 125

Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr

130 135 140

Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile

145 150 155 160

Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu

165 170 175

Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp

180 185 190

Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met

195 200 205

Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg

210 215 220

Val Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro

225 230 235 240

Asp Asn Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

355 360 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

370 375 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385 390 395 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

420 425 430

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465 470

<210> 5

<211> 1985

<212> RNA

<213> human (Homo sapiens)

<400> 5

gagaccggcc ucgagcagcu gaagcuuccu gcaggucgac ucuagagcca ccaugagggc 60

ccugugggug cugggccucu gcugcguccu gcugaccuuc gggucgguca gagcugacga 120

ugaaguugau gugcagccuc cuccuccucc uccagacgcu acaugucacc agguccgcuc 180

cuucuuccag aggcugcagc caggacucaa gugggugcca gagacaccag ugccaggaag 240

cgaucugcag gucugucugc cuaagggccc uaccuguugc ucccggaaga uggaggagaa 300

guaccagcug accgccaggc ugaacaugga acagcugcug cagagcgcca gcauggagcu 360

gaaguuccug aucauccaga acgccgccgu guuccaggag gccuucgaga ucgucgugcg 420

gcacgccaag aacuacacca acgccauguu caagaacaac uaccccagcc ugacaccuca 480

ggccuuugag uucguggggg aguucuucac cgacgugucu cuguacaucc ugggcagcga 540

caucaacgug gacgacaugg ugaacgagcu guucgacagc cuguuccccg ugaucuacac 600

ccagcugaug aacccaggcc ugccagauag cgcucuggau aucaacgagu gccugagggg 660

agccagaaga gaccugaagg uguucggcaa cuuccccaag cugaucauga cccagguguc 720

caagagccug caggucacca ggaucuuccu gcaggcccug aaccugggca ucgaggucau 780

caacaccacc gaccaccuga aguucagcaa ggauugcggc cggaugcuca cccgcaugug 840

guauuguagc uauugccagg gccugaugau ggugaagccu ugcggcggcu auugcaacgu 900

cgugaugcag gguuguaugg ccggcguggu ggagaucgac aaguauuggc gggaguacau 960

ccugagccug gaggagcugg ugaacggcau guaccggauc uacgacaugg agaacgugcu 1020

gcugggccug uucuccacca uccacgacag cauccaguac gugcagaaga acgccggcaa 1080

gcugacaacc accaucggaa agcucugcgc ccacucucag cagaggcagu acagaagcgc 1140

cuacuacccc gaggaccugu ucaucgacaa gaaggugcug aagguggccc acguggaaca 1200

cgaagagaca cugagcagcc ggaggagaga gcugauccag aagcugaagu ccuucaucuc 1260

cuucuacagc gcccugccag gcuacauuug cagccacagc ccaguggccg agaacgacac 1320

ccucuguugg aacggccagg agcuggugga gagauacucu cagaaggccg ccaggaacgg 1380

caugaagaac caguucaacc ugcacgagcu gaagaugaag ggcccagagc cagugguguc 1440

ccagaucauc gacaagcuga agcacaucaa ccagcugcug cggaccauga gcaugccuaa 1500

gggcagggug cuggacaaga accuggacga ggagggcuuc gagucaggag auugcggcga 1560

cgacgaagac gaguguauug gcggaagcgg cgacggcaug aucaagguca agaaccagcu 1620

gcgguuccug gccgaacugg ccuacgaucu ggacguggac gacgcuccag gcaauucuca 1680

gcaggccaca ccuaaggaca acgagaucag caccuuccac aaccugggca acgugcacuc 1740

uccucugaag cugcugacca gcauggccau uagcgucguc ugcuucuucu uccuggugca 1800

ucugaucccc aucgcugugg guggugcccu ggcggggcug guccucaucg uccucaucgc 1860

cuaccucguc ggcaggaaga ggagucacgc aggcuaccag acuaucuagg aauucuuaau 1920

uaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1980

aaaaa 1985

<210> 6

<211> 618

<212> PRT

<213> human (Homo sapiens)

<400> 6

Met Arg Ala Leu Trp Val Leu Gly Leu Cys Cys Val Leu Leu Thr Phe

1 5 10 15

Gly Ser Val Arg Ala Asp Asp Glu Val Asp Val Gln Pro Pro Pro Pro

20 25 30

Pro Pro Asp Ala Thr Cys His Gln Val Arg Ser Phe Phe Gln Arg Leu

35 40 45

Gln Pro Gly Leu Lys Trp Val Pro Glu Thr Pro Val Pro Gly Ser Asp

50 55 60

Leu Gln Val Cys Leu Pro Lys Gly Pro Thr Cys Cys Ser Arg Lys Met

65 70 75 80

Glu Glu Lys Tyr Gln Leu Thr Ala Arg Leu Asn Met Glu Gln Leu Leu

85 90 95

Gln Ser Ala Ser Met Glu Leu Lys Phe Leu Ile Ile Gln Asn Ala Ala

100 105 110

Val Phe Gln Glu Ala Phe Glu Ile Val Val Arg His Ala Lys Asn Tyr

115 120 125

Thr Asn Ala Met Phe Lys Asn Asn Tyr Pro Ser Leu Thr Pro Gln Ala

130 135 140

Phe Glu Phe Val Gly Glu Phe Phe Thr Asp Val Ser Leu Tyr Ile Leu

145 150 155 160

Gly Ser Asp Ile Asn Val Asp Asp Met Val Asn Glu Leu Phe Asp Ser

165 170 175

Leu Phe Pro Val Ile Tyr Thr Gln Leu Met Asn Pro Gly Leu Pro Asp

180 185 190

Ser Ala Leu Asp Ile Asn Glu Cys Leu Arg Gly Ala Arg Arg Asp Leu

195 200 205

Lys Val Phe Gly Asn Phe Pro Lys Leu Ile Met Thr Gln Val Ser Lys

210 215 220

Ser Leu Gln Val Thr Arg Ile Phe Leu Gln Ala Leu Asn Leu Gly Ile

225 230 235 240

Glu Val Ile Asn Thr Thr Asp His Leu Lys Phe Ser Lys Asp Cys Gly

245 250 255

Arg Met Leu Thr Arg Met Trp Tyr Cys Ser Tyr Cys Gln Gly Leu Met

260 265 270

Met Val Lys Pro Cys Gly Gly Tyr Cys Asn Val Val Met Gln Gly Cys

275 280 285

Met Ala Gly Val Val Glu Ile Asp Lys Tyr Trp Arg Glu Tyr Ile Leu

290 295 300

Ser Leu Glu Glu Leu Val Asn Gly Met Tyr Arg Ile Tyr Asp Met Glu

305 310 315 320

Asn Val Leu Leu Gly Leu Phe Ser Thr Ile His Asp Ser Ile Gln Tyr

325 330 335

Val Gln Lys Asn Ala Gly Lys Leu Thr Thr Thr Ile Gly Lys Leu Cys

340 345 350

Ala His Ser Gln Gln Arg Gln Tyr Arg Ser Ala Tyr Tyr Pro Glu Asp

355 360 365

Leu Phe Ile Asp Lys Lys Val Leu Lys Val Ala His Val Glu His Glu

370 375 380

Glu Thr Leu Ser Ser Arg Arg Arg Glu Leu Ile Gln Lys Leu Lys Ser

385 390 395 400

Phe Ile Ser Phe Tyr Ser Ala Leu Pro Gly Tyr Ile Cys Ser His Ser

405 410 415

Pro Val Ala Glu Asn Asp Thr Leu Cys Trp Asn Gly Gln Glu Leu Val

420 425 430

Glu Arg Tyr Ser Gln Lys Ala Ala Arg Asn Gly Met Lys Asn Gln Phe

435 440 445

Asn Leu His Glu Leu Lys Met Lys Gly Pro Glu Pro Val Val Ser Gln

450 455 460

Ile Ile Asp Lys Leu Lys His Ile Asn Gln Leu Leu Arg Thr Met Ser

465 470 475 480

Met Pro Lys Gly Arg Val Leu Asp Lys Asn Leu Asp Glu Glu Gly Phe

485 490 495

Glu Ser Gly Asp Cys Gly Asp Asp Glu Asp Glu Cys Ile Gly Gly Ser

500 505 510

Gly Asp Gly Met Ile Lys Val Lys Asn Gln Leu Arg Phe Leu Ala Glu

515 520 525

Leu Ala Tyr Asp Leu Asp Val Asp Asp Ala Pro Gly Asn Ser Gln Gln

530 535 540

Ala Thr Pro Lys Asp Asn Glu Ile Ser Thr Phe His Asn Leu Gly Asn

545 550 555 560

Val His Ser Pro Leu Lys Leu Leu Thr Ser Met Ala Ile Ser Val Val

565 570 575

Cys Phe Phe Phe Leu Val His Leu Ile Pro Ile Ala Val Gly Gly Ala

580 585 590

Leu Ala Gly Leu Val Leu Ile Val Leu Ile Ala Tyr Leu Val Gly Arg

595 600 605

Lys Arg Ser His Ala Gly Tyr Gln Thr Ile

610 615

Claims

1. A method for increasing the proportion of TNF- α positive cells in a subpopulation of CD 8T cells in vitro, comprising the step of contacting engineered dendritic cells in vitro with an immunotolerated CD3+ immune cell;

wherein the engineered dendritic cells are obtained by introducing exogenous genes comprising a gene encoding recombinant interleukin-12, a gene encoding soluble CD80 and a gene encoding GPC3 antigen into wild-type dendritic cells by engineering means to overexpress recombinant interleukin-12, soluble CD80 and GPC3 antigen;

the recombinant interleukin-12 is a fusion protein of two subunits, namely p40 and p35, and the gene sequence for coding the recombinant interleukin-12 is shown as SEQ ID number 1, and the gene sequence for coding the soluble CD80 is shown as SEQ ID number 2.