CN110804588B - Composition for synergistically improving properties of T lymphocytes and use thereof - Google Patents

Abstract

The invention discloses a composition for synergistically improving T lymphocyte properties and application thereof. The composition of the present invention comprises the following components (a) to (d): (a) IL-15, a mutant or functional fragment thereof, or a nucleic acid encoding same; (b) IL-15 receptor alpha, a mutant or functional fragment thereof, or a nucleic acid encoding same; (c) IL-12, a mutant or functional fragment thereof, or a nucleic acid encoding same; and (d) an inhibitor of TGF- β activity. The compositions of the invention are capable of synergistically enhancing immune responses of a T lymphocyte nature.

Description

Composition for synergistically improving properties of T lymphocytes and use thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a composition for synergistically improving properties of T lymphocytes and application thereof.

Background

In the anti-tumor immunity of the body, the cellular immunity mediated by T cells plays an important role. Research finds that the tumor patients have the characteristics of reduced Dendritic Cell (DC) quantity and functional defects, and the quantity and the function of tumor tissues and the DC infiltrated around the tumor tissues have close relation with the occurrence, the development, the metastasis and the prognosis of tumors. The tumor with dense DC infiltration has high differentiation degree and better prognosis, while the tumor with mild DC infiltration is often accompanied with low differentiation degree 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, there have been a number of reports of tumor treatment using antigen-loaded DC vaccines, and from the data reported so far, DC vaccines appear to represent a new and very promising approach for improved tumor immunotherapy. However, the use of DC vaccines alone often does not result in the desired improvement in immunotherapeutic effects and does not result in satisfactory clinical results. Current clinical trials indicate that the response rate of DC therapeutic vaccines rarely exceeds 15%, the overall response rate is low, and the use of DC vaccines alone generally does not result in the expected improvement of immunotherapeutic effects and does not result in satisfactory clinical results.

Similarly, in preclinical models that have been reported, IL-15 has been shown to enhance the anti-tumor immunity of CD8+ T cells. Two types of IL-15 superagonists are currently available, one method being the binding of IL-15 to IL-15 Ra-Fc (R & D Systems) to generate a complex in vitro, called IL-15 SA. Another IL-15 superagonist complex, called ALT-803.

In addition, studies targeting TGF- β signaling pathways for tumor therapy are of great interest, and a variety of approaches to block TGF- β signaling pathways have been introduced in preclinical or clinical trials. For example, 1) through using TGF-beta antibody, TGF-beta binding protein or antisense TGF-beta mRNA, neutralize TGF-beta's effects, achieve the goal of inhibiting tumor invasion and metastasis, there are anti-human TGF-beta 1 monoclonal antibody CAT-192 and anti-human TGF-beta 2 monoclonal antibody CAT-152, but because the member of TGF-beta family has different degree of rising in the tumor, the antibody blocking effect of the single anti-some monomer is not as effective as the antibody of the whole blocking TGF-beta family, there has been two TGF-beta monoclonal antibodies combined and applied report at present. 2) By preventing TGF-beta ligand from combining with receptor on cell membrane, soluble human TGF-beta receptor is recombined by genetic engineering method, so that the soluble human TGF-beta receptor and receptor on cell membrane compete for ligand to achieve the effect of blocking signal path. 3) The activity of TGFBR1 is blocked by directly destroying the catalytic activity of a receptor, such as competitive inhibitors SBI-14352, NPC-30345, LY364947 and the like of an ATP binding site of TGFBR1 kinase, so that the TGFBR2 cannot continuously conduct effective signals after being combined with a ligand, and the aim of enabling TGFBR2 to be vacant is fulfilled. 4) By interfering a TGF-beta receptor downstream signal transduction system, mainly aiming at Smad protein family members, a polypeptide or polypeptide-like drug is applied to simulate Smads transcription complex, and a downstream signal path is activated, so that the aim of inhibiting the growth of tumor cells is fulfilled.

Like IL-15, DC vaccines directed against the TGF- β signaling pathway also typically do not achieve the desired immunotherapeutic effect. There is still a need to develop more efficient immunotherapeutic regimens.

Disclosure of Invention

To solve at least part of the technical problems of the prior art, the present invention provides a composition that can be used in combination with a DC vaccine. The invention finds that the synergistic effect of each component can be exerted by simultaneously regulating a plurality of pathways and further using TGF-beta binding peptide and regulatory peptide to enhance the blockage of TGF-beta signal pathway, thereby greatly improving the immunity of T lymphocytes. The present invention has been accomplished, at least in part, based on this. Specifically, the present invention includes the following.

In a first aspect of the present invention, there is provided a composition for synergistically improving T lymphocyte properties, which comprises the following components (a) to (d):

(a) IL-15, a mutant or functional fragment thereof, or a nucleic acid encoding same;

(b) IL-15 receptor alpha, a mutant or functional fragment thereof, or a nucleic acid encoding same;

(c) IL-12, a mutant or functional fragment thereof, or a nucleic acid encoding same; and

(d) inhibitors of TGF-beta activity.

In certain embodiments, the T lymphocyte properties comprise: the nature of IFN- γ and/or TNF- α expression in CD 8T lymphocytes; and the expression profile of IFN-. gamma.and/or TNF-. alpha.in CD 4T lymphocytes.

In certain embodiments, the inhibitor of TGF- β activity comprises a TGF- β binding peptide, or a nucleic acid encoding the same, and a TGF- β regulatory peptide, or a nucleic acid encoding the same.

In certain embodiments, the TGF- β binding peptide is an extracellular domain recombinant peptide of TGFBR 2.

In certain embodiments, the TGF- β binding peptide is an extracellular domain recombinant peptide of TGFBR 2. The TGF-beta regulatory peptide comprises a sequence of one of (a) to (c) below:

(a) an amino acid sequence shown as SEQ ID No. 8;

(b) an amino acid sequence having a homology of 95% or more with (a) having the same species origin;

(c) a sequence obtained by amino acid mutation on the basis of (a) and still retaining the original activity.

In a second aspect of the invention, there is provided an immune cell for synergistically improving T lymphocyte properties, comprising within said immune cell a composition according to the first aspect.

In certain embodiments, the immune cell further comprises an antigen or a nucleic acid encoding the antigen. Preferably, the antigen is AKR1B10 or a nucleic acid encoding same.

In a third aspect of the invention, a method is provided for increasing the expression level of IFN- γ and/or TNF- α in CD 4T lymphocytes.

In a fourth aspect of the invention, there is provided a method for increasing the expression level of IFN- γ and/or TNF- α in CD 8T lymphocytes, comprising the step of contacting the composition of the first aspect with CD 8T lymphocytes.

The compositions of the invention are useful as immunomodulating compositions that prime T cells with DC vaccines to generate specific cytotoxic T cells. Preferably, the TGF-beta binding peptide and the regulatory peptide are used, and the affinity difference of the TGF-beta binding peptide and the regulatory peptide to different TGF-beta molecules is utilized, so that TGF-beta 1, TGF-beta 2 and TGF-beta 3 molecules in a tumor microenvironment are better bound, the inhibition effect of the TGF-beta molecules on immune cells is relieved, and the function of killing tumors is released. Furthermore, by utilizing the capability of IL15 and IL12 in regulating T cell activation and proliferation, the multi-channel synergistic effect promotes DC vaccine to induce and generate specific cytotoxic T cells to continuously expand, generate more lymphocytes with anti-tumor activity and improve the tumor treatment effect of the DC vaccine.

Drawings

FIG. 1 shows the results of CD 8T cell immune response in experiments in which DC cells were transfected with mRNA encoding proteins of the components of the composition of the present invention and antigen mRNA, and then primed in vitro with T cells. In each column group of FIG. 1, from left to right, the proportion of CD8 IFN-gamma +, CD8 IFN-gamma +, TNF-alpha +, CD8 TNF-alpha + cells to total CD 8T cells is shown.

FIG. 2 shows the results of CD 4T cell immune response in experiments in which DC cells were transfected with mRNA encoding proteins of the components of the composition of the present invention and 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 +, CD4 TNF-alpha + cells 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.

In the present invention, the term "mutant" refers to a protein which has a mutation compared to the naturally occurring protein or the wild-type protein, but which is substantially homologous and still retains the original activity. The mutation may be, for example, deletion, insertion or substitution of one or more amino acids, or a combination thereof. Such mutations may be naturally occurring mutations or artificially introduced mutations. In case of mutations to amino acid substitutions, conservative amino acid substitutions are preferred, i.e. one residue is substituted by another with similar properties. Typical substitutions are within the group of aliphatic amino acids, within the group of amino acids containing aliphatic hydroxyl side chains, within the group of amino acids containing acidic groups, within the group of amino acids containing amide derivatives, within the group of amino acids containing basic groups or between amino acids containing aromatic groups.

In the present invention, "substantial homology" means a degree of identity of 60% or more, preferably 80% or more, and more preferably 90% or more with the subject sequence. For example, 95% or more, 97% or more, and most preferably 99% or more.

In the present invention, the term "functional fragment" refers to a protein consisting of a portion of contiguous amino acids derived from a naturally occurring protein or a wild-type protein and still retaining the original activity.

In the present invention, the term "nucleic acid" includes deoxyribonucleic acid (i.e., DNA) and ribonucleic acid (i.e., RNA). In the case of RNA, various optimizations of the nucleic acid molecule can be performed based on the known multiple natural degradation pathways of RNA in order to prevent instability of RNA and degradation of multiple pathways. For example, the terminal structure is crucial for the stability of mRNA. For example, at the 5 ' end of a naturally occurring mRNA, a modified guanosine nucleotide is present, referred to as a 5 ' cap structure, and at the 3 ' end there is a stretch of adenosine nucleotides (i.e., poly-A tails) of about 50-300 bases in length (e.g., 50-100, 60-90, etc.), and at the 5 ' and 3 ' ends UTR sequences, such as those of human beta-globin.

[ composition ]

In a first aspect of the invention, there is provided a composition, sometimes also referred to herein simply as "the composition of the invention", for synergistically improving T lymphocyte properties. The composition of the present invention comprises the following components (a) to (d): (a) IL-15, a mutant or functional fragment thereof, or a nucleic acid encoding same; (b) IL-15 receptor alpha, a mutant or functional fragment thereof, or a nucleic acid encoding same; (c) IL-12, a mutant or functional fragment thereof, or a nucleic acid encoding same; and (d) an inhibitor of TGF- β activity.

In the present invention, IL-15(interleukin-15) refers to a naturally occurring or wild-type interleukin-15, including different splice variants and naturally occurring variants. The IL-15 may be any species of IL-15. For example, a mouse, rat, guinea pig, rabbit, cow, goat, sheep, horse, pig, dog, cat or monkey, preferably a human.

The IL-15 of the present invention is a cytokine with a structure similar to that of IL-2, and is widely expressed in various cells and tissues, such as monocytes, macrophages, DC cells, fibroblasts and the like. The IL-15 of the invention can be combined with IL-15 receptor alpha, activate downstream JAK1 and JAK3, lead to the phosphorylation of downstream STAT3 and STAT5 and the activation of a signal channel, induce the phosphorylation of BCL2, a MAP kinase channel, LCK and SYK, and lead to the proliferation and the maturation of cells. In addition, IL-15 can regulate the activation and proliferation of T cells and NK cells, and can maintain the survival of memory T cells in the absence of antigen stimulation. It has been demonstrated that IL-15 inhibits apoptosis in rodent lymphocytes by inducing BCL2L1/BCL-x (L). Similarly, it has also been found in the present invention that Il-15 inhibits apoptosis of T lymphocytes in humans by inducing BCL2 and/or BCL-xL.

In the present invention, the amino acid sequence of IL-15 is not particularly limited, and examples thereof include the sequence shown in SEQ ID No.1, 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, the IL-15 receptor alpha (IL-15R alpha) is an alpha chain among three chains of alpha, beta and gamma of the IL-15 receptor, and is an essential subunit forming IL-15R alpha beta gamma with high specificity and high affinity, and the amino acid sequence of the IL-15 receptor alpha is not particularly limited as long as it has a function of binding to IL-15, and examples thereof include the sequence shown in SEQ ID No.3, and a sequence having a homology of 95% or more, preferably 97% or more, more preferably 99% or more, and derived from the same species.

In certain embodiments, components (a) and (b), i.e., IL-15 and IL-15 receptor alpha, of the compositions of the invention are each a separate protein (i.e., not in the state of a fusion protein), and the molar ratio of components (a) and (b) in the compositions of the invention is preferably (0.9-1.3):1, more preferably 1: 1.

In certain embodiments, where the compositions of the invention comprise nucleic acids, the nucleic acids of the invention may be nucleic acids encoding multiple proteins simultaneously, or a combination of nucleic acids encoding different proteins separately, with each nucleic acid encoding only one protein. Preferably, the nucleic acid of the invention is a nucleic acid encoding two or more proteins simultaneously. As used herein, "simultaneously encode" means that the same nucleic acid molecule can encode more than two proteins. In this case two or more proteins may be present in a fused form, but more preferably the same nucleic acid molecule encodes for the production of two or more proteins which are present separately. In the case where two or more proteins are produced simultaneously from the same nucleic acid molecule, the production can be achieved by linking two adjacent genes to each other, for example, by a ribosome entry site (IRES). Alternatively, this can also be achieved by linking nucleic acid sequences encoding self-cleaving polypeptide sequences between two adjacent genes. As an illustrative example, the nucleic acid of the invention may be a nucleic acid encoding both IL-15 and IL-15 Ra. Examples of such nucleic acids include, but are not limited to, the nucleic acid set forth in SEQ ID No.2, which encodes for the production of both the IL-15 protein and the IL-15Ra protein separately.

In the present invention, 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 is known to induce IFN- γ production, which is also required in the in vivo immune response to bacterial or parasitic infections. The invention can be used for the immune response of tumors by combining the compounds with components such as IL-15 and the like. The possible reason is that, firstly, IL12 can promote the differentiation of CD4+ Th0 cells into Th1 cells, and the activity of LAK cells can be induced by the cooperation of sub-dose IL-2. Secondly, IL12 can induce cytotoxic activity of CTL and NK cells and promote secretion of cytokines such as IFN-gamma, TNF-alpha and GM-CSF. Again, IL12 was able to promote expression of NK cells and IL-2r α, TNF receptors and CD56 molecules, enhancing antibody-dependent cellular cytotoxicity (ADCC) effects on tumor cells.

In the present invention, the preferred IL-12 contains the amino acid 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. Preferably, the IL-12, its mutant or its functional fragment of the invention is composed of SEQ ID No.5 shows the sequence of the nucleic acid coding.

TGF-beta in the compositions of the invention is a multifunctional cytokine that not only affects cell growth, differentiation and apoptosis, but also has important immunomodulatory effects. TGF- β can effectively inhibit T cell function and antigen presenting capacity of DC cells, for example recent studies have elucidated a direct role of TGF- β in the expression of the upregulating plasma cell-like DC (pdc) immunomodulatory enzyme indoleamine 2, 3-dioxygenase (IDO), and lead to long-term T cell tolerance. IDO inhibits the activity of effector T cells and promotes Treg differentiation and activation by catalyzing the degradation of the essential amino acid tryptophan.

In certain embodiments, inhibitors of TGF- β activity of the present invention include TGF- β binding peptides or nucleic acids encoding the same, and TGF- β regulatory peptides or nucleic acids encoding the same. TGF-. beta.signals by binding to the transforming growth factor-beta receptor (TGFBR) complex. TGF-beta binding peptides of the invention bind to TGF-beta, particularly TGF-beta 2, and block signal transduction thereof.

TGFBR1/2 is known to belong to the transmembrane type receptor serine/threonine kinase family, and intracellular contains serine/threonine kinase domain, and after transforming growth factor-beta is combined with TGFBR1/2 heterodimer, downstream signal molecules are activated, and then signal transduction is activated. TGF-beta binding peptides in the present invention are recombinant peptides that are capable of binding TGF-beta, particularly TGF-beta 2, but are incapable of exerting or transducing downstream signals. Preferably, the TGF- β binding peptide is a recombinant peptide comprising the extracellular domain of TGFBR 2. The TGF- β binding peptide of the present invention preferably has an amino acid sequence represented by SEQ ID No.6, or a sequence that has 95% or more, preferably 97% or more, more preferably 99% or more homology thereto and is derived from the same species. Preferably, the binding peptide of the invention is encoded by a nucleic acid comprising the sequence shown in SEQ ID No. 7.

TGF-beta regulatory peptides of the invention are useful for modulating the binding of TGF-beta to its signaling receptor, preferably for increasing or promoting binding of both. Preferably, the TGF- β modulating peptide comprises the sequence of one of (a) to (c) below:

(a) an amino acid sequence shown as SEQ ID No. 8;

(b) an amino acid sequence having a homology of 95% or more with (a) having the same species origin;

(c) the sequence obtained by amino acid mutation on the basis of (a).

Preferably, a TGF- β regulatory peptide of the present invention is encoded by a nucleic acid comprising the sequence shown in SEQ ID No. 9. More preferably, the sequence of the nucleic acid encoding a TGF- β regulatory peptide of the invention is shown in SEQ ID No. 9.

In certain embodiments, the TGFF-beta binding peptide and TGFF-beta modulating peptide of the compositions of the invention each further comprise an Fc fragment. It is noted that the Fc fragment in TGFF-beta binding peptides and TGF-beta regulatory peptides is preferably a longer Fc fragment, thereby increasing molecular size. In general, too large a molecular volume may affect the stability and activity of the composition. Therefore, the length or size of the Fc fragment in the present invention affects achievement of the object of the present invention. For the purposes of the present invention, the length of the Fc fragment is generally 150-350AA, preferably 180-300 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 mutations of "YTE", i.e. methionine (Met, M), serine (Ser, S) and threonine (Thr, T) at positions 252, 254 and 256 are replaced by tyrosine (Tyr, Y), T and glutamic acid (Glu, E), respectively, thereby obtaining a fusion protein with a longer half-life. For another example, by genetic engineering and modification of disulfide bonds of an Fc fragment, Fc fusion proteins can be aggregated into multimeric complexes, thereby obtaining fusion proteins with superior stability. In certain embodiments, the amino acid sequences of the TGFB-beta binding peptides and TGF-beta regulatory peptides of the present invention are sequences comprising SEQ ID No.6 and SEQ ID No.8, respectively.

In the initial research on the biological functions of TGF-beta, the TGF-beta is found to play an important role in regulating the growth, differentiation and immune functions of cells mainly in the aspects of inflammation, tissue repair, embryonic development and the like. In general, TGF-. beta.s act as stimulators for cells of mesenchymal origin, but as inhibitors for cells of epithelial or neuroectodermal origin. The details are as follows.

a. Inhibition of proliferation of immunocompetent cells: inhibit colony formation of mouse hematopoietic precursor cells and bone marrow long-term culture (LTBMC) induced by IL-3, GM-CSF, M-CSF, and reduce responsiveness of megakaryocytes to IL-3T and CSF; inhibiting thymocyte proliferation induced by ConA or ConA combined with IL-2 and IL-6; inhibiting mitogen, alloantigen-stimulated T cell proliferation or IL-2 dependent T cell growth; inhibit IL-2 dependent B cell proliferation following SAC stimulation.

b. Modulation of cell phenotype: the expression of IL-2-induced T cell IL-2R, TfR and TLiSA1 activation antigen is inhibited, and no influence on CD3 expression is observed; inhibiting IFN-gamma to induce the expression of MHC II antigen of melanoma cells.

c. Inhibition of lymphocyte differentiation: inhibiting IL-2 and BCDF dependent B cells from secreting IgM and promoting B cells to secrete Ig type to be converted into IgA and IgE; inhibition of CTL, NK and LAK function in Mixed Lymphocyte Cultures (MLC), which inhibition is reversed by TNF-alpha (mouse MIC) or IL-2 (human MLC); inhibiting NK activity in PBMCs and responsiveness of NK cells to TNF- α; the inhibition of ConA and IL-2 and IL-6 synergistically induces the activity of MHC non-restricted killer cells in thymus of mice.

d. Inhibition of cytokine production: such as inhibiting IFN-. gamma.and TNF-. alpha.production in PBMCs.

e. Other regulatory effects: the growth of fibroblasts, osteoblasts and Schwann cells is promoted, and the generation of human fibroblast IL-6 is promoted by TGF-beta 1 and TGF-beta 2, wherein the mechanism of the generation is probably through the regulation of IL-6 gene transcription; inhibiting the growth of epithelial cells, osteoclasts, endothelial cells and the formation of fat, cardiac and skeletal muscle, TGF- β antagonizes certain biological functions of EGF; promoting the expression of extracellular matrix (ECM) such as collagen and fibronectin, inhibiting the degradation of ECM, playing an important role in the morphogenesis, proliferation and differentiation processes of cells, and being beneficial to embryonic development and cell repair. Animal in vivo experiments show that local injection of TGF-beta can promote wound healing and typical granulation tissue formation; chemoattractants for monocytes and fibroblasts, but not for the production of colloidal particles and oxides; inhibiting the adhesion of lymphocytes to endothelial cells; promoting the release of histamine from basophils.

Tgf- β 1 and proto-oncogene expression: TGF-. beta.1 induces expression of c-sis but inhibits expression of c-myc, either in connection with the different functions of the affected cell types and TGF-. beta.s. For example, TGF-. beta.induces c-sis gene expression in fibroblasts and is associated with promoting growth in soft agar, while inhibition of growth of epithelial keratinocyte is associated with inhibiting c-myc gene expression. TGF-. beta.s are very similar in most biological roles, but may differ greatly in some roles, e.g., TGF-. beta.2 inhibits the growth of vascular endothelial cells and hematopoietic progenitor cells by only 1% of the other factors of TGF-. beta.s.

The invention discovers that the combination of the TGF-beta activity inhibitor and other components can have synergistic effect by a high-throughput screening means, thereby greatly improving the immune effect. T lymphocyte properties may be improved by the compositions of the invention, the improved properties including the expression properties of IFN-gamma and/or TNF-alpha in CD 8T lymphocytes and the expression properties of IFN-gamma and/or TNF-alpha in CD 4T lymphocytes.

Preferably, the molar ratio of component (a) to (b) to (c) to (d) in the composition of the invention is preferably 1:1 (0.9-1.3) to (0.9-1.3), more preferably 1:1 (0.95-1.2) to (0.95-1.2).

[ immune cells ]

In a second aspect of the invention, an immune cell is provided that is an artificially engineered cell. Herein, the immune cell refers to a cell having functions of taking up, processing and transmitting antigen information in the body, presenting the antigen to the immune cell and assisting and regulating T cells, B cells recognizing the antigen and inducing an 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 antigen presenting cells. More preferably, dendritic cells of human origin. The dendritic cells of the present invention may be mature dendritic cells or immature dendritic cells.

The immune cells of the invention are preferably in vitro cells. The in vitro cells of the invention may be introduced into the body. The in vitro cells may be autologous cells or allogeneic cells.

In certain embodiments, the immune cells of the invention further comprise an antigen or a nucleic acid encoding the same. "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 Antigen Presenting Cell (APC), and can be presented to 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 antigens of the invention are hepatocellular carcinoma antigens, such as AKR1B 10.

[ method for improving T lymphocyte Properties ]

In a third aspect of the invention, there is provided a method of improving T lymphocyte properties, sometimes also referred to herein simply as "the improvement method of the invention", comprising the step of contacting a composition according to the first aspect or an immune cell according to the second aspect with CD 4T lymphocytes and CD 8T lymphocytes, respectively, in certain embodiments, the improved T lymphocyte properties comprise: the nature of IFN- γ and/or TNF- α expression in CD 8T lymphocytes and the nature of IFN- γ and/or TNF- α expression in CD 4T lymphocytes.

In the improved methods of the invention, T lymphocytes generally refer to cells in vitro, including T lymphocytes cultured in vitro.

Example 1

This example is the preparation of DNA and mRNA encoding antigens and immunodetection Point inhibitors

1. Preparation of DNA and mRNA constructs

DNA sequences for the production of mrnas encoding IL12, IL15, IL15R α and TGF- β binding and TGF- β regulatory peptides as described in the present invention were constructed separately and used for subsequent in vitro transcription reactions. These are followed by a segment of polyadenylation. The DNA sequence information is shown in Table 1 below.

In addition, the coding sequence of human tumor antigen AKR1B10 for in vitro sensitization was constructed.

TABLE-1 DNA sequence Listing

Name (R)	Serial number
		IL-15\IL-15Rα	SEQ ID NO.2
IL-12	SEQ ID NO.5
		TGF- β binding peptides	SEQ ID NO.7
TGF- β regulatory peptides	SEQ ID NO.9

2. In vitro transcription

First, the corresponding DNA plasmid prepared was linearized using restriction enzymes, 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 2

This example is used to investigate the effect of the compositions of the invention on T cell responses.

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 centrifuged at 500gThe cells were harvested, the supernatant 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 prostagladin E2(PGE2) (1. mu.g/mL) added to AIM-V medium) was added to the medium, and after resuspension of the cells, the cells were cultured in a flask for 8-18 hours to induce maturation of the DC cells.

2. Transfection of DC cells with compositions

On the day of transfection, DC cells were digested into cell suspensions using non-enzymatic cell digestion reagents, centrifuged, washed twice with PBS, resuspended in PBS, and adjusted to a cell density of 25-30 × 10⁶DCs/ml. According to each 10⁶DC cells were transfected at a ratio of 10. mu.g mRNA, mixing the DC cells with mRNA combinations of antigen mRNA and different proteins (IL15/IL15R α, IL12, TGF- β binding peptide and TGF- β regulatory peptide), adding the cell-mRNA mixture to an electric rotor, transfecting the antigen mRNA into the DC cells using an ECM630 electric rotor, re-suspending the cells after the electric conversion in cytokine-free 1640 medium, adjusting the cell density to 2 × 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 mRNA encoding AKR1B10 antigen (AKR1B10 control group)

3) mRNA encoding AKR1B10 antigen and mRNA of IL12 (IL12 group)

4) mRNA encoding AKR1B10 antigen and mRNA of IL15/IL15R alpha (group IL 15)

5) mRNA encoding AKR1B10 antigen and mRNA encoding TGF-. beta.regulatory peptide (regulatory peptide group)

6) mRNA encoding AKR1B10 antigen and mRNA encoding TGF-beta binding peptide (TGFBR2 group)

7) mRNA encoding AKR1B10 antigen and mRNA of IL12+ IL15/IL15Ra + TGF-beta binding peptide + T GF-beta regulatory peptide (IL12+ IL15/IL15Ra + TGFBR2+ TGF-beta regulatory peptide group)

3. Peripheral Blood Mononuclear Cells (PBMC) revived overnight at 2 × 10⁶The cells were seeded in 96-well plates at a concentration of one ml, and each well was seeded with 100. mu.l of cells to stimulate T lymphocytesAnd (6) alive. The test grouping case is: PBMC control group without DC cells, groups co-cultured with the nine grouped DC cells in the previous step and PBMC cells, 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, 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.

Recovering the cryopreserved DC cells loaded with the prepared antigen, counting the cells by trypan blue staining, resuspending the cells in RPMI containing IL-7 and IL-2 cytokines, and adjusting the cell concentration to 2 × 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 FIGS. 1 and 2, the use of mRNA of the protein of the present invention can induce an anti-tumor specific immune response in T lymphocytes having a stronger anti-tumor specific immune response. The present invention has a synergistic immune enhancing effect compared to drugs using one modulator alone.

In addition, the composition (IL12+ IL15/IL15Ra + TGF-beta binding peptide + TGF-beta regulatory peptide) can remarkably improve the T cell sensitizing capacity of DC loaded with AKR1B10 antigen. Traditional DC vaccines simply load DC cells with mRNA or polypeptide encoding the antigen, which are then infused back into the patient for treatment. It has been reported in the literature that this method can sensitize lymphocytes to elicit an antigen-specific immune response, but only a very small number of cells are activated, resulting in a very low proportion of immune responses, whereas the ability to activate an immune response is greatly enhanced using DC cells co-transfected with antigen mRNA using the composition of the present invention.

As shown in FIG. 1, the ratios of IFN-. gamma.positive, IFN-. gamma.and TNF-. alpha.double-positive and TNF-. alpha.positive cells to CD 8T cells were 0.25%, 0.25% and 0.76% in the AKR1B10 control group, while the ratios were 6.15%, 1.42% and 2.52% in the experimental group using the composition of the present invention, which were increased by 23.6 times, 4.68 times and 2.32 times, respectively.

Similarly, as shown in fig. 2, the ratios of IFN- γ positive, IFN- γ and TNF- α double positive and TNF- α positive cells to CD 4T cells were 1.23%, 0.36% and 1.22% in the AKR1B10 control group, respectively, whereas the ratios were 21.46%, 4.96% and 6.69% in the experimental group using the composition of the present invention, which were increased by 17.8-fold, 9.78-fold and 2.04-fold, respectively.

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.

Sequence listing

<110> Qichensheng Biotechnology (Zhuhai) Co., Ltd

<120> composition for synergistically improving T lymphocyte properties and use thereof

<141>2019-11-22

<160>9

<170>SIPOSequenceListing 1.0

<210>1

<211>162

<212>PRT

<213> human (Homo sapiens)

<400>1

Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr

1 5 10 15

Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His

20 25 30

Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala

35 40 45

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

50 55 60

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

65 70 75 80

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

85 90 95

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

100 105 110

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

115 120 125

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

130 135 140

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

145 150 155 160

Thr Ser

<210>2

<211>1951

<212>DNA

<213> human (Homo sapiens)

<400>2

atgagaattt cgaaaccaca tttgagaagt atttccatcc agtgctactt gtgtttactt 60

ctaaacagtc attttctaac tgaagctggc attcatgtct tcattttggg ctgtttcagt 120

gcagggcttc ctaaaacaga agccaactgg gtgaatgtaa taagtgattt gaaaaaaatt 180

gaagatctta ttcaatctat gcatattgat gctactttat atacggaaag tgatgttcac 240

cccagttgca aagtaacagc aatgaagtgc tttctcttgg agttacaagt tatttcactt 300

gagtccggag atgcaagtat tcatgataca gtagaaaatc tgatcatcct agcaaacaac 360

agtttgtctt ctaatgggaa tgtaacagaa tctggatgca aagaatgtga ggaactggag 420

gaaaaaaata ttaaagaatt tttgcagagt tttgtacata ttgtccaaat gttcatcaac 480

acttcttgag cggccgccgc ccgccccacg acccgcagcg cccgaccgaa aggagcgcac 540

gaccccatca tccaattccg cccccccccc ctaacgttac tggccgaagc cgcttggaat 600

aaggccggtg tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg 660

tgagggcccg gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc 720

tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt 780

cttgaagaca aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg 840

acaggtgcct ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac 900

cccagtgcca cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg 960

tattcaacaa ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg 1020

ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt aaaaaacgtc taggcccccc 1080

gaaccacggg gacgtggttt tcctttgaaa aacacgatga taatatggcc acaacgtcga 1140

cgccaccatg gctcctagga gagccagagg gtgtaggaca ctgggactgc cagctctgct 1200

gctgctgctg ctgctgagac ctccagctac aaggggaatc acctgccctc ctcctatgag 1260

cgtggagcac gccgacattt gggtgaagag ctacagcctg tacagccggg agcgctacat 1320

ttgcaacagc ggcttcaaga ggaaggccgg aacaagctct ctcaccgagt gcgtgctgaa 1380

caaggccacc aacgtggccc attggacaac ccctagcctg aagtgcatca gggacccagc 1440

actggtgcac cagagaccag ctcctcctag cacagtgacc acagccggag tgacacctca 1500

gccagaaagc ctgagcccta gcggaaaaga accagccgcc tctagcccca gcagcaataa 1560

taccgccgcc acaacagccg ctattgtgcc aggaagccag ctgatgccta gcaagagccc 1620

tagcaccggc acaacagaga tcagcagcca cgagagcagc cacggaacac ctagccagac 1680

cacagccaag aattgggagc tgaccgccag cgccagccac cagcctccag gagtgtaccc 1740

tcagggacac agcgatacca ccgtggccat ctctaccagc acagtgctgc tgtgcggact 1800

gtcagctgtg tccctgctgg cttgctacct gaagagcaga cagacccctc ctctggccag 1860

cgtggaaatg gaggctatgg aggccctgcc agtgacttgg ggaacctcta gcagagacga 1920

ggacctggag aattgcagcc accacctgta g 1951

<210>3

<211>267

<212>PRT

<213> human (Homo sapiens)

<400>3

Met Ala Pro Arg Arg Ala Arg Gly Cys Arg Thr Leu Gly Leu Pro Ala

1 5 10 15

Leu Leu Leu Leu Leu Leu Leu Arg Pro Pro Ala Thr Arg Gly Ile Thr

20 25 30

Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser

35 40 45

Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys

50 55 60

Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala

65 70 7580

Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp

85 90 95

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

100 105 110

Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly Lys Glu

115 120 125

Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr Thr Ala

130 135 140

Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro Ser Thr

145 150 155 160

Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr Pro Ser

165 170 175

Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser His Gln

180 185 190

Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr Val Ala Ile

195 200 205

Ser Thr Ser Thr Val Leu Leu Cys Gly Leu Ser Ala Val Ser Leu Leu

210 215 220

Ala Cys Tyr Leu Lys Ser Arg Gln Thr Pro Pro Leu Ala Ser Val Glu

225 230 235240

Met Glu Ala Met Glu Ala Leu Pro Val Thr Trp Gly Thr Ser Ser Arg

245 250 255

Asp Glu Asp Leu Glu Asn Cys Ser His His Leu

260 265

<210>4

<211>603

<212>PRT

<213> human (Homo sapiens)

<400>4

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

420425 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

580585 590

Ser Trp Ser Glu Trp Ala Ser Val Pro Cys Ser

595 600

<210>5

<211>1812

<212>DNA

<213> human (Homo sapiens)

<400>5

atgtggcccc ctgggtcagc ctcccagcca ccgccctcac ctgccgcggc cacaggtctg 60

catccagcgg ctcgccctgt gtccctgcag tgccggctca gcatgtgtcc agcgcgcagc 120

ctcctccttg tggctaccct ggtcctcctg gaccacctca gtttggccag aaacctcccc 180

gtggccactc cagacccagg aatgttccca tgccttcacc actcccaaaa cctgctgagg 240

gccgtcagca acatgctcca gaaggccaga caaactctag aattttaccc ttgcacttct 300

gaagagattg atcatgaaga tatcacaaaa gataaaacca gcacagtgga ggcctgttta 360

ccattggaat taaccaagaa tgagagttgc ctaaattcca gagagacctc tttcataact 420

aatgggagtt gcctggcctc cagaaagacc tcttttatga tggccctgtg ccttagtagt 480

atttatgaag acttgaagat gtaccaggtg gagttcaaga ccatgaatgc aaagcttctg 540

atggatccta agaggcagat ctttctagat caaaacatgc tggcagttat tgatgagctg 600

atgcaggccc tgaatttcaa cagtgagact gtgccacaaa aatcctccct tgaagaaccg 660

gatttttata aaactaaaat caagctctgc atacttcttc atgctttcag aattcgggca 720

gtgactattg atagagtgat gagctatctg aatgcttccg gatccggagc caccaacttc 780

agcctgctga agcaggccgg cgacgtggag gagaaccccg gccccatgtg tcaccagcag 840

ttggtcatct cttggttttc cctggttttt ctggcatctc ccctcgtggc catatgggaa 900

ctgaagaaag atgtttatgt cgtagaattg gattggtatc cggatgcccc tggagaaatg 960

gtggtcctca cctgtgacac ccctgaagaa gatggtatca cctggacctt ggaccagagc 1020

agtgaggtct taggctctgg caaaaccctg accatccaag tcaaagagtt tggagatgct 1080

ggccagtaca cctgtcacaa aggaggcgag gttctaagcc attcgctcct gctgcttcac 1140

aaaaaggaag atggaatttg gtccactgat attttaaagg accagaaaga acccaaaaat 1200

aagacctttc taagatgcga ggccaagaat tattctggac gtttcacctg ctggtggctg 1260

acgacaatca gtactgattt gacattcagt gtcaaaagca gcagaggctc ttctgacccc 1320

caaggggtga cgtgcggagc tgctacactc tctgcagaga gagtcagagg ggacaacaag 1380

gagtatgagt actcagtgga gtgccaggag gacagtgcct gcccagctgc tgaggagagt 1440

ctgcccattg aggtcatggt ggatgccgtt cacaagctca agtatgaaaa ctacaccagc 1500

agcttcttca tcagggacat catcaaacct gacccaccca agaacttgca gctgaagcca 1560

ttaaagaatt ctcggcaggt ggaggtcagc tgggagtacc ctgacacctg gagtactcca 1620

cattcctact tctccctgac attctgcgtt caggtccagg gcaagagcaa gagagaaaag 1680

aaagatagag tcttcacgga caagacctca gccacggtca tctgccgcaa aaatgccagc 1740

attagcgtgc gggcccagga ccgctactat agctcatctt ggagcgaatg ggcatctgtg 1800

ccctgcagtt ag 1812

<210>6

<211>391

<212>PRT

<213> human (Homo sapiens)

<400>6

Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu

1 5 10 15

Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Val

20 25 30

Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro

35 40 45

Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln

50 55 60

Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro

65 70 75 80

Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr

85 90 95

Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile

100 105 110

Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys

115 120 125

Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn

130 135 140

Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser AsnPro Asp Glu

145 150 155 160

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

165 170 175

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

245 250 255

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

260 265 270

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

275 280 285

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

290 295 300

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

305 310 315 320

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

325 330 335

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

340 345 350

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

355 360 365

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

370 375 380

Leu Ser Leu Ser Pro Gly Lys

385 390

<210>7

<211>1176

<212>DNA

<213> human (Homo sapiens)

<400>7

atgggtcggg ggctgctcag gggcctgtgg ccgctgcaca tcgtcctgtg gacgcgtatc 60

gccagcacga tcccaccgca cgttcagaag tcggttaata acgacatgat agtcactgac 120

aacaacggtg cagtcaagtt tccacaactg tgtaaatttt gtgatgtgag attttccacc 180

tgtgacaacc agaaatcctg catgagcaac tgcagcatca cctccatctg tgagaagcca 240

caggaagtct gtgtggctgt atggagaaag aatgacgaga acataacact agagacagtt 300

tgccatgacc ccaagctccc ctaccatgac tttattctgg aagatgctgc ttctccaaag 360

tgcattatga aggaaaaaaa aaagcctggt gagactttct tcatgtgttc ctgtagctct 420

gatgagtgca atgacaacat catcttctca gaagaatata acaccagcaa tcctgacgag 480

cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 540

ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 600

cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 660

tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 720

aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 780

aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 840

tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 900

gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 960

atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1020

gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1080

tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1140

acgcagaaga gcctctccct gtctccgggt aaatga 1176

<210>8

<211>878

<212>PRT

<213> human (Homo sapiens)

<400>8

Met Thr Ser His Tyr Val Ile Ala Ile Phe Ala Leu Met Ser Ser Cys

1 5 10 15

Leu Ala Thr Ala Gly Pro Glu Pro Gly Ala Leu Cys Glu Leu Ser Pro

20 25 30

Val Ser Ala Ser His Pro Val Gln Ala Leu Met Glu Ser Phe Thr Val

35 40 45

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

50 55 60

His Val Leu Asn Leu Arg Thr Ala Gly Gln Gly Pro Gly Gln Leu Gln

65 70 75 80

Arg Glu Val Thr Leu His Leu Asn Pro Ile Ser Ser Val His Ile His

85 90 95

His Lys Ser Val Val Phe Leu Leu Asn Ser Pro His Pro Leu Val Trp

100 105 110

His Leu Lys Thr Glu Arg Leu Ala Thr Gly Val Ser Arg Leu Phe Leu

115 120 125

Val Ser Glu Gly Ser Val Val Gln Phe Ser Ser Ala Asn Phe Ser Leu

130 135 140

Thr Ala Glu Thr Glu Glu Arg Asn Phe Pro His Gly Asn Glu His Leu

145 150 155 160

Leu Asn Trp Ala Arg Lys Glu Tyr Gly Ala Val Thr Ser Phe Thr Glu

165 170 175

Leu Lys Ile Ala Arg Asn Ile Tyr Ile Lys Val Gly Glu Asp Gln Val

180 185 190

Phe Pro Pro Lys Cys Asn Ile Gly Lys Asn Phe Leu Ser Leu Asn Tyr

195 200 205

Leu Ala Glu Tyr Leu Gln Pro Lys Ala Ala Glu Gly Cys Val Met Ser

210 215 220

Ser Gln Pro Gln Asn Glu Glu Val His Ile Ile Glu Leu Ile Thr Pro

225 230 235 240

Asn Ser Asn Pro Tyr Ser Ala Phe Gln Val Asp Ile Thr Ile Asp Ile

245 250 255

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

260 265 270

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

275 280 285

Lys Gly Ser Leu Lys Ile Ile Ala Pro Asn Ser Ile Gly Phe Gly Lys

290 295 300

Glu Ser Glu Arg Ser Met Thr Met Thr Lys Ser Ile Arg Asp Asp Ile

305 310 315 320

Pro Ser Thr Gln Gly Asn Leu Val Lys Trp Ala Leu Asp Asn Gly Tyr

325 330 335

Ser Pro Ile Thr Ser Tyr Thr Met Ala Pro Val Ala Asn Arg Phe His

340 345 350

Leu Arg Leu Glu Asn Asn Ala Glu Glu Met Gly Asp Glu Glu Val His

355 360 365

Thr Ile Pro Pro Glu Leu Arg Ile Leu Leu Asp Pro Gly Ala Leu Pro

370 375 380

Ala Leu Gln Asn Pro Pro Ile Arg Gly Gly Glu Gly Gln Asn Gly Gly

385 390 395 400

Leu Pro Phe Pro Phe Pro Asp Ile Ser Arg Arg Val Trp Asn Glu Glu

405 410 415

Gly Glu Asp Gly Leu Pro Arg Pro Lys Asp Pro Val Ile Pro Ser Ile

420 425 430

Gln Leu Phe Pro Gly Leu Arg Glu Pro Glu Glu Val Gln Gly Ser Val

435 440 445

Asp Ile Ala Leu Ser Val Lys Cys Asp Asn Glu Lys Met Ile Val Ala

450 455 460

Val Glu Lys Asp Ser Phe Gln Ala Ser Gly Tyr Ser Gly Met Asp Val

465 470 475 480

Thr Leu Leu Asp Pro Thr Cys Lys Ala Lys Met Asn Gly Thr His Phe

485 490 495

Val Leu Glu Ser Pro Leu Asn Gly Cys Gly Thr Arg Pro Arg Trp Ser

500 505 510

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

515 520 525

Ala Leu Gly Asp Ser Ser Gly Trp Pro Asp Gly Tyr Glu Asp Leu Glu

530 535 540

Ser Gly Asp Asn Gly Phe Pro Gly Asp Met Asp Glu Gly Asp Ala Ser

545 550 555 560

Leu Phe Thr Arg Pro Glu Ile Val Val Phe Asn Cys Ser Leu Gln Gln

565 570 575

Val Arg Asn Pro Ser Ser Phe Gln Glu Gln Pro His Gly Asn Ile Thr

580 585 590

Phe Asn Met Glu Leu Tyr Asn Thr Asp Leu Phe Leu Val Pro Ser Gln

595 600 605

Gly Val Phe Ser Val Pro Glu Asn Gly His Val Tyr Val Glu Val Ser

610 615 620

Val Thr Lys Ala Glu Gln Glu Leu Gly Phe Ala Ile Gln Thr Cys Phe

625 630 635 640

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

645 650 655

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

660 665 670

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

675 680 685

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

690 695 700

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

705 710 715 720

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

725 730 735

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

740 745 750

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

755 760 765

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

770 775 780

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

785 790 795 800

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

805 810 815

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

820 825 830

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

835 840 845

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

850 855 860

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

865 870 875

<210>9

<211>2637

<212>DNA

<213> human (Homo sapiens)

<400>9

atgaccagcc actacgtgat cgccatcttc gccctgatga gcagctgtct ggccacagca 60

ggaccagagc caggcgccct gtgtgaactc agcccagtgt ccgcttctca tccagtgcag 120

gccctgatgg agagcttcac agtgctgagc ggctgcgcca gcagaggcac aacaggactg 180

cctcaggagg tgcacgtgct gaacctgaga accgcaggac agggaccagg acagctgcag 240

agggaagtga ccctgcacct gaaccccatc agcagcgtgc acatccacca caagagcgtg 300

gtgttcctgc tgaacagccc tcacccactg gtctggcacc tgaagaccga gagactggct 360

acaggcgtgt ccagactgtt cctggtgtcc gaaggcagcg tggtgcagtt tagcagcgct 420

aacttcagcc tgaccgccga aaccgaggag agaaacttcc cccacggcaa cgagcacctg 480

ctgaattggg ccaggaagga gtacggagcc gtgaccagct tcaccgagct gaagatcgcc 540

cggaacatct acatcaaggt cggcgaggac caggtgttcc cacccaagtg caacatcggc 600

aagaacttcc tgagcctgaa ctacctggcc gagtatctgc agcctaaagc cgcagagggc 660

tgcgtgatgt ctagccagcc ccagaacgag gaggtgcaca tcatcgagct gatcaccccc 720

aacagcaacc cctacagcgc cttccaggtg gacatcacca tcgacatccg gcctagccag 780

gaggatctgg aggtcgtgaa gaacctgatc ctgatcctca agtgcaagaa gagcgtgaat 840

tgggtcatca agagcttcga cgtgaagggc agcctgaaga tcatcgcccc caacagcatc 900

ggctttggca aagagagcga gcggagcatg accatgacca agagcatccg ggacgacatc 960

ccctctacac agggcaacct cgtcaagtgg gcactggata acggctacag ccctatcacc 1020

agctacacca tggccccagt ggccaacaga ttccacctgc ggctggagaa caacgccgaa 1080

gagatgggcg acgaggaagt gcacaccatc cctcccgagc tgagaatcct gctggacccc 1140

ggcgccctgc cagctctgca gaatcctcct attagaggcg gcgagggaca gaacggagga 1200

ctgcctttcc ctttccccga catcagcagg agagtgtgga acgaggaggg cgaagacgga 1260

ctgcctagac ctaaggaccc cgtgatccct agcatccagc tgttcccagg cctgagagag 1320

ccagaggaag tgcagggaag cgtggacatc gctctgagcg tcaagtgcga caacgagaag 1380

atgatcgtgg ccgtggagaa ggacagcttc caggctagcg gatacagcgg aatggacgtg 1440

accctgctgg accctacttg caaggccaag atgaacggca cccacttcgt gctggagtcc 1500

cccctgaacg gttgcggcac aagacctagg tggagcgctc tggacggagt ggtgtactac 1560

aactccatcg tgatccaggt gcccgctctg ggagattcta gcggttggcc agacggctac 1620

gaggatctgg agagcggaga caacggcttc ccaggcgata tggacgaggg agacgcttct 1680

ctgttcacca ggcccgagat cgtggtgttc aattgcagcc tgcagcaggt ccgcaaccct 1740

tctagcttcc aggagcagcc tcacggcaac atcaccttca acatggagct gtacaacacc 1800

gacctgttcc tggtgccatc acagggagtg ttcagcgtgc ccgagaacgg acacgtgtac 1860

gtggaggtgt ccgtgaccaa ggcagaacag gagctgggct tcgccatcca gacttgcttc 1920

atcagcccct acagcaacga gcccaaatct tgtgacaaaa ctcacacatg cccaccgtgc 1980

ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac 2040

accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 2100

gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 2160

aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 2220

caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 2280

gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 2340

accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc 2400

aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 2460

aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag 2520

ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 2580

gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaatga 2637

Claims (1)

1. An in vitro culture method for increasing the proportion of IFN- γ positive cells to TNF-a positive cells in CD 4T cells, comprising the step of contacting DC cells in vitro with T lymphocytes cultured in vitro, wherein said DC cells comprise a composition consisting of four components (a) to (d) below and AKR1B10 antigen or a nucleic acid encoding the same:

(a) IL-15 or its coding nucleic acid with sequence shown in SEQ ID No. 1;

(b) IL-15 receptor alpha with the sequence shown as SEQ ID No.3 or coding nucleic acid thereof;

(c) IL-12 or its coding nucleic acid with the sequence shown in SEQ ID No. 4; and

(d) a TGF-beta activity inhibitor comprising a TGF-beta binding peptide having a sequence shown in SEQ ID No.6 or a nucleic acid encoding the same, and a TGF-beta regulatory peptide having a sequence shown in SEQ ID No.8 or a nucleic acid encoding the same;

wherein (a) and (b) are in the case of proteins, separate proteins rather than a fusion protein state.

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