CN110478474B

CN110478474B - Immunomodulator, vaccine, cell and application

Info

Publication number: CN110478474B
Application number: CN201910771185.9A
Authority: CN
Inventors: 蒋俊; 林鑫; 其他发明人请求不公开姓名
Original assignee: Qichensheng Biotechnology (zhuhai) Co Ltd
Current assignee: Qichensheng Biotechnology (zhuhai) Co Ltd
Priority date: 2019-08-21
Filing date: 2019-08-21
Publication date: 2020-06-02
Anticipated expiration: 2039-08-21
Also published as: CN110478474A

Abstract

The invention discloses an immunomodulator, a vaccine, a cell and application. The immunomodulators of the present invention comprise (a) IL-15 or a nucleic acid encoding therefor, capable of binding to its receptor; (b) IL-15 receptor alpha or a nucleic acid encoding same; and (c) a polypeptide comprising the extracellular domain of a TGF beta receptor 3 protein or a nucleic acid encoding the same. The immunomodulator can be combined with TGF-beta on one hand, and relieve the inhibiting effect of TGF-beta on T cell function and antigen presentation of DC cells; on the other hand, IL-15-IL-15Ra can regulate the activation and proliferation of T cells and NK cells, can also promote the T cell sensitizing capability of DC cells, induces stronger anti-tumor specific T cell response, and shows stronger anti-tumor effect than the traditional DC vaccine.

Description

Immunomodulator, vaccine, cell and application

Technical Field

The present invention relates to pharmaceutical products, in particular to immunomodulators, vaccines, cells and methods of increasing the proportion of TNF-a + and/or IFN-r + cells in a lymphocyte population.

Background

The immune response of the body is first to capture antigen by Antigen Presenting Cells (APC), processed and processed to present antigen information to lymphocytes, and then to initiate a series of specific immune responses. Dendritic Cells (DCs) are currently considered to be the most powerful APCs, and are characterized by their ability to stimulate the proliferation and activation of naive T cells, a central link in the initiation, regulation and maintenance of specific immune responses. 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 DC number and function defects, the number and the function of tumor tissues and DC infiltration around the tumor tissues are closely related to the occurrence, development, metastasis and prognosis of tumors, the tumors with dense DC infiltration have high differentiation degree and better prognosis, while tumors with mild DC infiltration are 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- β and IL-10, 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 that T cells remain quiescent despite the presence of tumor antigens. From this viewpoint: 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. In recent years, increasing the number of antigen presenting cells, and improving the ability of antigen presenting cells, especially DC cells, to take up, transport, present antigen, and stimulate T cells, are a major focus in the current tumor immunization research.

TGF- β (TGF- β) is a multifunctional cytokine that can affect cell growth, differentiation, apoptosis, etc. in addition, TGF- β has important immunomodulatory effects TGF- β can effectively inhibit T cell function and antigen presenting ability of DC cells recent studies have demonstrated the direct role of TGF- β in upregulating plasma cell-like DC immunomodulatory enzyme indoleamine 2, 3-dioxygenase (IDO) expression and resulting in long-term T cell tolerance.

TGFBR1/2 belongs to the transmembrane type receptor serine/threonine kinase family, which contains a serine/threonine kinase domain intracellularly, after binding to TGFBR1/2 heterodimers, transforming growth factor- β activates downstream signaling molecules and further activates signaling, the intracellular segment of TGFBR3 does not contain a kinase active region and does not directly participate in signaling, primarily regulates the binding of TGF- β to signal receptors, also known as co-receptors.

In recent years, the DC tumor vaccine has better application prospect in the prevention and treatment of tumors. DC-related vaccines have been studied extensively in prostate cancer. For example, Sipuleucel-T directly transfects the prostatic cancer acid phosphatase antigen to DC to excite the organism to generate specific anti-tumor reaction. The IMPACT study showed that the median survival time (25.8 months) was 4.1 months higher in the Sipuleucel-T group than in the control group (21.7 months), although the objective efficacy evaluation showed limited efficacy (< 5%). In 4 months 2010, the U.S. FDA approved Sipuleucel-T for the treatment of refractory prostate cancer (CRPC) that is asymptomatic or asymptomatic, metastatic castration therapy ineffective.

In gliomas, the autologous tumor lysate loaded DC vaccine DC-VAX-L is still undergoing phase III clinical trials. There are tumor vaccines in which RNA and CD40L mRNA from autologous kidney tumors are electroporated into DCs, and reports of combinations with the targeted drug sunitinib are also currently in clinical trials. Recent studies have found that DC vaccines significantly prolong patient survival.

There are 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. The current clinical experiment shows that the response rate of DC therapeutic vaccine rarely exceeds 15%, and the overall response rate is low.

Disclosure of Invention

In order to solve at least part of the technical problems in the prior art, the invention discovers an immunomodulator which can synergistically enhance or improve the immunity of a vaccine after intensive research. 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 an immunomodulator comprising three components (a) to (c):

(a) IL-15 or a nucleic acid encoding same capable of binding to its receptor;

(b) IL-15 receptor alpha or a nucleic acid encoding same; and

(c) a polypeptide containing the extracellular domain of TGF beta receptor 3 protein or a nucleic acid encoding the same.

In certain embodiments, the (a) is a nucleic acid encoding IL-15 having the sequence shown in SEQ ID NO. 1.

In certain embodiments, said (b) is a nucleic acid encoding IL-15 receptor alpha having the sequence shown in SEQ ID NO. 2.

In certain embodiments, the (a) and (b) are linked by a chemical bond to form a molecule.

In certain embodiments, the polypeptide of component (c) further comprises an Fc fragment of an immunoglobulin.

In certain embodiments, the (c) is a nucleic acid encoding a polypeptide having the sequence shown in SEQ ID NO. 3.

In a second aspect of the invention, there is provided a vaccine comprising at least one antigen or nucleic acid encoding the same, and an immunomodulatory agent of the first aspect.

In certain embodiments, the antigen is a tumor antigen or a pathogen antigen.

In a third aspect of the invention, there is provided a cell comprising (a ') a nucleic acid encoding IL-15, (b ') a nucleic acid encoding IL-15 receptor alpha, and (c ') a nucleic acid encoding a polypeptide comprising the extracellular domain of TGFbeta receptor 3 protein.

In a fourth aspect of the invention, there is provided a method of increasing the proportion of TNF-a + and/or IFN-r + cells in a population of lymphocytes, comprising the step of allowing an immunomodulatory agent of the first aspect or a vaccine of the second aspect to act on said population of lymphocytes.

The immunomodulator can be combined with TGF-beta on one hand, and relieve the inhibiting effect of TGF-beta on T cell function and antigen presentation of DC cells; on the other hand, IL-15-IL-15Ra can regulate the activation and proliferation of T cells and NK cells, can also promote the T cell sensitizing capability of DC cells, induces stronger anti-tumor specific T cell response, and shows stronger anti-tumor effect than the traditional DC vaccine.

The immune regulator components of the invention have synergistic effect, greatly enhance the immunity of the vaccine, can provide protective immunity from pathogen infection, and particularly can be used for enhancing the effect of preventing and/or treating various tumors.

Drawings

Figure 1CD8 positive T cell response results. In each column group, from left to right, CD8IFN-r + TNF-a +, CD8TNF-a + are sequentially arranged.

Figure 2CD4 positive T cell response results.

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. Unless otherwise indicated, "%" is percent by weight.

In the present invention, the term "antigen" refers to a substance that can be recognized by the immune system and is capable of eliciting an antigen-specific immune response by forming antibodies or/and antigen-specific T cells. In general, an antigen can be a protein or polypeptide that comprises at least one antigenic epitope and can be presented by the Major Histocompatibility Complex (MHC) 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 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, there is a modified guanosine nucleotide known as a 5 ' cap structure, and at the 3 ' end there is an adenosine nucleotide (i.e., poly-A tail) structure of about 200-300 bases in length, and at the 5 ' and 3 ' ends UTR sequences such as those of human beta-globin.

[ immunomodulators ]

In a first aspect of the present invention, there is provided an immunomodulator comprising at least three components (a) to (c):

(a) IL-15 or a nucleic acid encoding same;

(b) IL-15 receptor alpha or a nucleic acid encoding same; and

In the present invention, the ratio of the three components (a), (b) and (c) is not particularly limited, but the molar ratio of the components (a) and (b) is generally 0.9 to 1.1:1, preferably 1: 1. In general, the molar ratio of the components (a) and (c) is 0.5 to 1.5:1, preferably 0.6 to 1.4:1, more preferably 1: 1.

Component (a)

IL-15 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, and IL-15 activates downstream JAK1 and JAK3 by binding to IL-15 receptor α, leads to phosphorylation of downstream STAT3 and STAT5 and activation of signal pathways, induces phosphorylation of BCL2, MAP kinase pathway, Lck and syk, and leads to proliferation and maturation of cells.

The IL-15 of the present invention is capable of regulating the activation and proliferation of T cells and NK cells and of maintaining the survival of memory T cells in the absence of antigenic stimulation. In rodent lymphocytes, IL-15 inhibits apoptosis by inducing BCL2L1/BCL-x (L). Similarly, it has also been found that Il-15 inhibits apoptosis of T lymphocytes by inducing BCL2 and/or BCL-xL in humans.

IL-15 of the invention includes proteins having a full-length amino acid sequence or nucleic acids encoding same, and also includes truncated fragments of IL-15 or nucleic acids encoding same that are capable of binding to its receptor. That is, the IL-15 of the present invention comprises at least a sequence of amino acids that bind to a receptor. In certain embodiments, component (a) of the present invention is a nucleic acid having the sequence shown in SEQ ID NO. 1.

Component (b)

As described above, IL-15 receptor α of the present invention binds to its IL-15 to activate downstream JAK1, JAK3, leading to phosphorylation of downstream STAT3 and STAT5 and activation of the signaling pathway, inducing phosphorylation of BCL2, MAP kinase pathway, Lck and syk, leading to proliferation and maturation of cells, thus IL-15 receptor alpha of the present invention comprises at least an amino acid sequence that binds to IL-15.

Component (c)

Component (c) of the present invention is a polypeptide comprising the extracellular domain of a TGF beta receptor 3 protein or a nucleic acid encoding the same, and is useful for inhibiting the TGF- β signaling pathway.

TGFBR3 expression levels are found to be significantly down-regulated compared to patient paracancerous normal tissue in the early stages of many tumors, such as breast cancer.

In the present invention, in order to prolong the half-life and improve the stability of the fusion protein, it is preferable that the component (c) is a polypeptide comprising the extracellular domain of TGFbeta receptor 3 protein and an Fc fragment of immunoglobulin. To reduce renal clearance, it is preferred to use longer Fc fragments to increase molecular volume. On the other hand, however, if the molecular size is too large, the activity of soluble A and B fragments 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 100-300AA, preferably 150-300 AA. Preferably, the Fc fragment has at least the sequence shown in SEQ ID NO. 5. More preferably, the Fc fragment has the sequence shown in SEQ ID NO 6. Also preferably, the sequence of the Fc fragment is shown in SEQ ID NO 6.

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 Fc fragment of the present invention is encoded by a nucleic acid comprising SEQ ID NO. 4.

In certain embodiments, component (c) is a nucleic acid comprising the sequence shown in SEQ ID NO. 3, or a protein encoded thereby.

Although the components (a), (b) and (c) are described above, respectively, it is known to those skilled in the art that the three components are not limited in the form in which they exist as long as they can synergistically enhance immunity. For example, in the case where the three components are nucleic acids, the components (a), (b) and (c) may exist in the form of three different nucleic acid molecules, or may exist in the form of one molecule of any two or more nucleic acids. In this case, the nucleic acid of the same molecule can encode two or more proteins independently present at the same time. As an illustrative example, this can be achieved by linking, for example, a ribosome entry site (IRES) between two adjacent genes. Alternatively, this can also be achieved by linking nucleic acid sequences encoding self-cleaving polypeptide sequences between two adjacent genes. For example, the nucleic acid of the invention may be a nucleic acid encoding both IL-15 and IL-15Ra proteins.

[ vaccine ]

In a second aspect of the invention, there is provided a vaccine capable of providing one or more antigens (preferably immunogens) for use in the prevention or treatment of at least one condition or symptom in a patient. The vaccine of the invention comprises at least one antigen or nucleic acid encoding the same, and an immunomodulator according to the first aspect of the invention.

The antigen in the vaccine of the present invention comprises at least one epitope, and the nucleic acid encoding the antigen may be DNA or RNA. Vaccines may also include cells expressing the antigen, such as DC cells or PBMC cells. The antigen or immunogen may be derived from any material suitable for vaccination.

The vaccine of the present invention may be any type of vaccine, examples of which include, but are not limited to, tumor vaccines, infectious disease vaccines. The present invention preferably uses a tumor vaccine as a vaccine.

In certain embodiments, the vaccines of the present invention comprise a nucleic acid encoding an antigen. The nucleic acid of the present invention may be one or more. Each nucleic acid can encode at least one antigenic open reading frame.

In one embodiment, the antigenic nucleic acid molecule encodes an immunogenic peptide fragment of a bacterial, viral, fungal, or other pathogen. Wherein the pathogens include, but are not limited to, human hepatitis viruses including HAV, HBV, HCV, cytomegalovirus CMV, human immunodeficiency virus HIV, EB virus, dengue virus, Human Papilloma Virus (HPV), respiratory syncytial virus, rhinovirus, human T-lymphotropic virus type I (HTLV-1), influenza, Bovine Leukemia Virus (BLV), pertussis, polio, measles, mumps, rubella, smallpox, shingles, anthrax, tetanus, rotavirus, rabies, fowl pox, meningococcus, anthrax, encephalitis, pneumococcus, streptococcus, staphylococcus, Neisseria, Escherichia coli, Shigella, leishmania, respiratory syncytial virus, parainfluenza, adenovirus, varicella, flavivirus, Mycobacterium tuberculosis, malaria, and the like.

In one embodiment, the antigenic nucleic acid molecule encodes a tumor antigen. In this case, the tumor antigen may be expressed on the surface, cytoplasm, or nucleus of the tumor cell. The tumor antigen may also be selected from proteins that are overexpressed in tumor cells compared to normal cells. Tumor antigens can be further divided into tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs). TAA is a class of antigenic molecules that are present in both tumor and normal cells, examples of which include: embryonic proteins, glycoprotein antigens, squamous cell antigens, and the like. TAA is not specific to tumor cells, but can be synthesized in trace amounts by normal cells, and is highly expressed when tumor cells proliferate. TSA refers to a novel antigen that is expressed only on the surface of tumor cells and not on normal cells. Such antigens may be present in tumors of the same tissue type in different individuals, e.g., melanoma specific antigens encoded by human malignant melanoma genes, may be present in melanoma cells in different individuals, but not expressed by normal melanocytes. TSA can also be common to tumors of different histological types, for example, mutated Ras gene products can be commonly found in lung cancer, digestive tract tumors, etc., but because its amino acid sequence is inconsistent with the expression product of normal proto-oncogene Ras, it can be recognized by the immune system of the body to stimulate the immune response of the body. In general, such antigens resulting from mutations are referred to as neoantigens (neoantigens). These antigens are all recognized by cytotoxic T lymphocytes and cells presenting the antigen can be killed by T lymphocytes.

In certain embodiments, the vaccines of the present invention comprise an antigen as a polypeptide. The antigen of the present invention is preferably a tumor antigen, which may be selected from at least one of the group consisting of: TDO, MAGEC, HMOX, WT, LY6, AIM, IDO, CHI3L, IL13RA, LCK, GFAP, KIF20, CNTN, MUC, PEG, TNC, SOX, IGF2BP, S100A, AKAP, TTK, CHI3L, PTHHLH, CDC, PMEL, TOP2, PTTG, NRCAM, HMMR, MUC, LY6, SOX, FOSL, PRAME, FOLR, BIRC, KIF2, ITGAV, ART, PROM, CT, S100A, PPIB, S100A, STAT, EPBP, MLANA, KAAG, KLK, NT5, PTPRZ, SPAG, MET, RGS, CSPG, D1LG, MUC, CD274, PSCA, FABP, PLIN, KR, GCCR, TPCC, ACAGS, SARG, NLGN4X, SART3, PRKDC, FOXP3, HBEGF, PIK3R1, SLC1A3, PCNA, KIF1C, BSG, ATP2A3, SPAG9, RPSA, NFYC, LRRC8A, IQGAP1, LY6E, TRIOBP, ART1, BAGE, BIRC7, CA9, CCDC54, DCT, IDO2, MAGED4, SOX2, SYCP1, TYR, T4, BAGE2, BORIS, CALR3, CSAG 3, CTAG 13, CTAGE 3, FMR 3, GAGE3, GASESSGES 3, GASSGESSGENFR 3, GASENG 3, MAGSAGE 3, MAGE 3, MAGSAGESSGETFAS 3, MAGSSGEN 3, MAGSAGE 3, MAGSSGEN 3, MAGE 3, MAGSAGE 3, MAGSSGET 3, MAG 3, MAGSAGE 3, MAG 3, MAGE 3, MAG 3, MAGE 3, MAG 3, MAGE 363672, MAGE 3, MAG 3, MAGE 3, MAG 363672, MAGE 3, MAGE 3636363672, MAGE 36363672, MAGE 3636363636363636.

Furthermore, tumor antigens may also include individual tumor-specific neoantigens which are produced by genetic mutations in tumor cells. The mutated gene may be any gene in a cell, and the expression product thereof may be expressed on the cell surface or inside the cell.

The vaccines of the present invention may be administered by methods known in the art. Preferably, delivery to the host cell is by in vivo delivery. In one embodiment, the nucleic acid molecule is introduced into a subject in need thereof by a viral vector such as adenovirus (AdV), adeno-associated virus (AAV), retrovirus, lentivirus, herpes simplex virus, and the like, into a pharmaceutical composition of the invention. In addition, the pharmaceutical compositions of the present invention may also be introduced into a subject by transfection of liposomal nanoparticles into a host cell. In one embodiment, the pharmaceutical composition of the present invention can be introduced into the subject's own DC cells by electroporation, and the DC cells are used as vectors to be introduced into the subject. In one embodiment, the pharmaceutical composition of the present invention can be introduced into autologous PBMC cells or allogeneic PBMC cells of a subject by electroporation, and the autologous PBMC cells or allogeneic PBMC cells are used as vectors for introduction into the subject.

[ cells ]

In a third aspect of the invention, a cell is provided. The cells of the invention contain (a ') a nucleic acid encoding IL-15, (b') a nucleic acid encoding IL-15 receptor alpha, and (c) a nucleic acid encoding a polypeptide comprising the extracellular domain of a TGF beta receptor 3 protein, and these nucleic acids are capable of being expressed and/or translated within the cell, thereby enabling production of the corresponding protein.

In certain embodiments, the cells of the invention are capable of constitutively expressing the corresponding protein intracellularly. That is, the cells constantly produce proteins that act as immunomodulators. In certain embodiments, the cells of the invention transiently express a protein as an immunomodulator, or the nucleic acid within the cell is in a quiescent state, and when desired, e.g., after modulation, the nucleic acid within the cell is expressed and begins to produce the corresponding protein. The protein of the present invention may be expressed in cells or secreted outside cells.

The cell type of the present invention is not particularly limited, and is generally an immune-related cell, and examples thereof include, but are not limited to, PBMC cells and DC cells. The cell of the present invention may be one of the above-mentioned two cells, or a mixed population of the two cells.

[ method of increasing the proportion of TNF-a + and/or IFN-r + cells in a lymphocyte population ]

In a fourth aspect of the invention, there is provided a method of increasing the proportion of TNF-a + and/or IFN-r + cells in a population of lymphocytes, comprising the step of allowing an immunomodulator or vaccine of the invention to act on the population of lymphocytes.

The lymphocytes of the invention are target cells for immunomodulator or vaccine action, including cells cultured in vitro or cells in a subject. Interaction with lymphocytes can be performed in a variety of ways. Where the immunomodulator or vaccine comprises a protein, the lymphocyte may be acted on by contacting the protein directly with the lymphocyte. Where the immunomodulator or vaccine comprises a nucleic acid, the nucleic acid may be expressed as the corresponding protein, and the resulting protein then contacted with the lymphocyte. For example, the nucleic acid is first expressed in the expression cell, and then the expression cell is contacted with a lymphocyte as a target cell. The expression cells herein include PBMC cells and/or DC cells.

Example 1

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

1. Preparation of DNA and mRNA constructs

A DNA sequence encoding human tumor antigen GPC3 for in vitro sensitization, and DNA sequences encoding IL-15, IL-15Ra and TGFBR3mRNA were constructed separately and used for the subsequent in vitro transcription reaction. Following the coding sequence, a segment of polyadenylation was used to prepare the construct. The coding nucleic acid sequence of IL-15 is shown in SEQ ID NO. 1, the coding nucleic acid sequence of IL-15Ra is shown in SEQ ID NO. 2, and the coding nucleic acid sequence of TGFBR3 is shown in SEQ ID NO. 3.

2. In vitro transcription

Firstly, linearizing the corresponding DNA plasmid prepared in the step 1 by using a speI endonuclease, and preparing mRNA by using T7RNA polymerase in vitro transcription by using the linearized plasmid as a template. The prepared mRNA was then purified by lithium chloride precipitation.

Example 2

This example investigates the Effect of immunomodulators on T cell response

In vitro induction culture of DC cells

Aseptically extracting healthy human venous blood 50ml, 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% CO₂Transferring half of the cell culture medium into a centrifuge tube, centrifuging at 500g to collect cells, removing supernatant, adding an equal volume of fresh mDC culture medium according to the formula of 1600U/mL GM-CSF and 1000U/mL IL-4, TNF-a (5ng/mL), IL-1 β (5ng/mL), IL-6(150ng/mL) and prostaglandin E2(PGE2) (1ug/mL), resuspending the cells, adding the resuspended cells into a culture flask, culturing for 8-18 hours, and inducing the maturation of DC cells.

2. Transfection of DC cells with immunosuppressant 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 25-30X 10⁶DCs/ml. According to each 10⁶Transfection of DC cells with 10ug mRNA, mixing DC cells with antigen mRNA in combination with different fusion protein (IL-15, IL-15Ra and TGFBR3-Fc) mRNA, adding the cell-mRNA mixture to an electric rotor, and transfecting the antigen mRNA into the DC cells using an ECM630 electric rotor. 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:

controls without any mRNA addition

Adding only mRNA encoding GPC3 or AKR1B10 antigen

mRNA encoding antigen and mRNA for IL-15/IL-15Ra

mRNA encoding antigen and mRNA of TGFBR3-Fc

mRNA encoding antigen and mRNA for IL-15/IL-15Ra + TGFBR3-Fc

3. Peripheral Blood Mononuclear Cells (PBMC) revived overnight at 2X 10⁶The T lymphocytes were activated by seeding in 96-well plates at a concentration of one ml, and 100ul of cells were seeded per well. The test grouping case is: a PBMC control group without DC cells, a group co-cultured with the five divided 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 to 12 days.

4. And detecting intracellular cytokines 10-12 days after co-culture.

4.1 mix cultured T cells evenly 5-8h before cell collection, adjust cell density to 2X 10⁶Each well was inoculated into a 96-well plate at 100. mu.l per well, and incubated at 37 ℃ in an incubator. The positive control is PMA (50ng/ml) + ionomycin (1ug/ml), and the negative control contains only suspension cells.

4.2 antigen-loaded DC cells were prepared as target cells. The prepared antigen-loaded cryopreserved DC cells were recovered and counted by trypan blue staining, the cells were resuspended by complete culture in RPMI containing IL-7 and IL-2 cytokines and adjusted to a cell concentration of 2X 10⁵Mu.l of cells were added per well.

4.3 adding monensin with the final concentration of 2 mu M or brefeldin A with the final concentration of 3 mu g/ml into the cell culture solution, and fully and uniformly mixing; (Monensin and Brefeldin A as protein transport blockers, in the cytosol should not exceed 12h), after 4-6 hours, intracellular staining was detected.

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-a and IFN-r.

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

As shown in FIGS. 1 and 2, the groups transfected with the immunopotentiators of the present invention were the most potent in sensitizing CD 8-positive T cells, and caused a cell response specific to the GPC3 antigen that was stronger than those of TGFBR3, IL-15/IL-15Ra alone or GPC3 antigen alone.

Only using GPC3 antigen to sensitize DC cells, and after 12 days of culture, 0.66% of CD8 positive T cells are positive for IFN-r; 1.53% of CD4 positive T cells appeared IFN-r positive; in the group transfected with immunopotentiator and GPC3 antigen, 2.75% of CD8 positive T cells were positive for IFN-r; 1.42% of CD4 positive T cells appeared IFN-r positive and 1.2% of CD8T cells were both IFN-r and TNF-a positive, this type of cells was only 1.4% in the group transfected with GPC3 antigen alone and only 0.51% and 0.1% in the group transfected with antigen and IL-15/IL-15Ra or antigen and TGFBR3, respectively.

As shown in FIG. 2, the immunopotentiator of the present invention was also capable of significantly increasing the proportion of TNF-a positive cells among CD4 positive cells. The proportion of TNF-a positive cells in the group transfected with GPC3 antigen alone was 0.97%, and in the group transfected with antigen and IL-15/IL-15Ra or antigen and TGFBR3, the proportion of TNF-a positive cells was slightly increased to 1.12% and 1.16%, respectively, whereas after the immunopotentiator of the present invention was added, the proportion of TNF-a positive cells was 2.19% which was 2.26 times that of the control group.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

Sequence listing

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

<120> immunomodulator, vaccine, cell and application

<130>BH1900069-1

<141>2019-08-20

<160>6

<170>SIPOSequenceListing 1.0

<210>1

<211>489

<212>DNA

<213>Homo sapiens

<400>1

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

acttcttga 489

<210>2

<211>804

<212>DNA

<213>Homo sapiens

<400>2

atggctccta ggagagccag agggtgtagg acactgggac tgccagctct gctgctgctg 60

ctgctgctga gacctccagc tacaagggga atcacctgcc ctcctcctat gagcgtggag 120

cacgccgaca tttgggtgaa gagctacagc ctgtacagcc gggagcgcta catttgcaac 180

agcggcttca agaggaaggc cggaacaagc tctctcaccg agtgcgtgct gaacaaggcc 240

accaacgtgg cccattggac aacccctagc ctgaagtgca tcagggaccc agcactggtg 300

caccagagac cagctcctcc tagcacagtg accacagccg gagtgacacc tcagccagaa 360

agcctgagcc ctagcggaaa agaaccagcc gcctctagcc ccagcagcaa taataccgcc 420

gccacaacag ccgctattgt gccaggaagc cagctgatgc ctagcaagag ccctagcacc 480

ggcacaacag agatcagcag ccacgagagc agccacggaa cacctagcca gaccacagcc 540

aagaattggg agctgaccgc cagcgccagc caccagcctc caggagtgta ccctcaggga 600

cacagcgata ccaccgtggc catctctacc agcacagtgc tgctgtgcgg actgtcagct 660

gtgtccctgc tggcttgcta cctgaagagc agacagaccc ctcctctggc cagcgtggaa 720

atggaggcta tggaggccct gccagtgact tggggaacct ctagcagaga cgaggacctg 780

gagaattgca gccaccacct gtag 804

<210>3

<211>2637

<212>DNA

<213>Homo sapiens

<400>3

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

<210>4

<211>651

<212>DNA

<213>Homo sapiens

<400>4

gcacctgaac tcctgggggg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 60

ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 120

cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 180

ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 240

caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 300

cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 360

ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 420

ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 480

tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 540

accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 600

gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa a 651

<210>5

<211>15

<212>PRT

<213>Homo sapiens

<400>5

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

1 5 10 15

<210>6

<211>232

<212>PRT

<213>Homo sapiens

<400>6

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

Claims

1. An immunomodulator comprising three components (a) to (c):

(a) 1 or IL-15 encoded by the nucleic acid with the sequence shown in SEQ ID NO;

(b) nucleic acid with the sequence as shown in SEQ ID NO. 2 or IL-15 receptor alpha obtained by the coding of the nucleic acid; and

(c) 3 or polypeptide containing TGF beta receptor 3 protein extracellular structural domain obtained by coding the nucleic acid with the sequence shown as SEQ ID NO.

2. The immunomodulator according to claim 1, wherein said (a) and (b) are linked by a chemical bond to form a single molecule.

3. A vaccine comprising at least one antigen or nucleic acid encoding the same and an immunomodulator according to claim 1 or 2.

4. The vaccine of claim 3, wherein the antigen is a tumor antigen or a pathogen antigen.

5. A cell comprising (a ') a nucleic acid encoding IL-15, (b ') a nucleic acid encoding IL-15 receptor alpha, and (c ') a nucleic acid encoding a polypeptide comprising the extracellular domain of a TGF beta receptor 3 protein; wherein, the sequence of (a ') is shown as SEQ ID NO. 1, the sequence of (b ') is shown as SEQ ID NO. 2, and the sequence of (c ') is shown as SEQ ID NO. 3.

6. Use of an immunomodulator according to claim 1 or 2 in the manufacture of a medicament for increasing the proportion of TNF-a + and/or IFN-r + cells in a lymphocyte population.