CN110917342B - Immunomodulatory compositions and uses thereof - Google Patents

Abstract

The invention discloses an immunoregulation composition and application thereof. The composition of the present invention comprises the following components (a) to (e) or nucleic acids encoding them: (a) IL-15, a mutant or functional fragment thereof; (b) IL-15 receptor alpha, a mutant or functional fragment thereof; (c) IL-12, a mutant or functional fragment thereof; (d) TGFBR2, a mutant or functional fragment thereof and (e) PD1-CD80 fusion protein, a mutant or functional fragment thereof. The composition of the invention can synergistically improve the immune response of T lymphocytes.

Description

Immunomodulatory compositions and uses thereof

Technical Field

The invention relates to the fields of immunology and medicine, in particular to an immunomodulatory composition and application thereof.

Background

Traditionally, tumor cell immunotherapy has been mainly to stimulate the immune system by vaccination or adoptive cell immunotherapy, thereby eliciting an immune response. This approach is based on the assumption that: tumor cells express tumor-specific antigens and are presented on the surface of tumor cells by the Major Histocompatibility Complex (MHC), whereas anti-tumor T cells are not fully activated. Therefore, to address this problem, attempts have been made primarily to increase recognition of these antigens by stimulating key positive co-immune and innate immune pathways, such as CD28, CD40L (CD154) and various TLR receptors, or inhibiting negative immunosuppressive pathways, such as CTLA-4 receptor, PD-1 receptor.

The existing research shows that the expression level of CD80 in cancer tissues is obviously lower than that in cancer-surrounding tissues and normal tissues, and the CD80 is usually expressed in a cytoplasm region and a cell nucleus, so that the tumor-specific T cell activation is difficult to induce, which is an important reason for immune monitoring of tumor escape. Dendritic Cells (DCs) are the most potent Antigen Presenting Cells (APCs) known at present, express abundant CD80 on their surface, take up tumor antigens in vitro and in vivo, directly activate naive T lymphocytes, and promote the production of T helper cells (Th) and Cytotoxic T Lymphocytes (CTLs), thereby playing an important role in tumor immunity.

A large number of researches find that the expression of CD80 on the membrane of infiltrating dendritic cells (TIDCs) in tumor tissues and lymph nodes in the tumor tissues is obviously inhibited, so that the DCs cannot effectively induce anti-tumor immunity to cause local immune function to be low, and the escape of tumor cells is caused.

Additional studies have shown that tumors can suppress immune functions in the body by secreting transforming growth factor-beta (TGF β), such as: TGF β promotes tumor growth by blocking DC maturation, leading to an increased number of immature DCs, which in turn can lead to an immunosuppressive state of loss of T cell function.

In recent years, the DC tumor vaccine has better application prospect in the prevention and treatment of tumors. DC is the most potent APC known at present, and expresses abundant CD80 on its surface, and can take up tumor antigens in vitro and in vivo, directly activate naive T lymphocytes, and promote the generation of T helper cells (Th) and Cytotoxic T Lymphocytes (CTL), thereby playing an important role in tumor immunity. 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 Sipuleu cel-T for the treatment of refractory prostate cancer (CRPC) that is asymptomatic or mildly symptomatic metastatic castration therapy ineffective.

However, since Sipuleucel-T has been marketed in poor quantities, the original manufacturer, Dandelion Corporation, declared a failure. And several other large DC cell-based tumor vaccines failed clinically in three stages. For example, phase III studies by merck corporation, termed tecemotide as targeted MUC1 in 2013, were two-fold declared unsuccessful, and phase III studies by glatiramer smick corporation, GSK1572932A (targeted MAGEA3) were also discontinued in clinical phase III, data indicating that the use of DC vaccines alone generally did not result in the expected improvement in immunotherapeutic effects and did not result in satisfactory clinical results. This may be due to the influence of various immunosuppressive factors, resulting in DC vaccines that do not induce the production of sufficient anti-tumor T cells, leading to clinical failure.

Disclosure of Invention

The inventor of the invention carries out intensive research in the research and development field of immunomodulators, and based on the discovery of a high-throughput screening technology, the inventor can exert the synergistic effect of each component, relieve the immunosuppression of T lymphocytes and greatly improve the activity of antigen presenting cells by regulating each pathway by using IL12 and Il5/IL15Ra and further enhancing the blocking of other signal pathways by using TGFBR2 and PD1-CD 80. The present invention has been accomplished based at least in part on the above findings. Specifically, the present invention includes the following.

In a first aspect of the present invention, there is provided an immunomodulatory composition comprising the following (a) to (e):

(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;

(d) TGFBR2, a mutant or functional fragment thereof, or a nucleic acid encoding thereof; and

(e) PD1-CD80 fusion protein, a mutant or functional fragment thereof, or a nucleic acid encoding the same.

In certain embodiments, the sequence of TGFBR2 is one of the following (a ') to (c'):

(a') the amino acid sequence shown as SEQ ID No. 6;

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

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

In certain embodiments, the compositions of (a) and (b) are capable of binding to form a complex, and the compositions comprise an Fc domain.

In certain embodiments, (a) component comprises the sequence shown in SEQ ID No.1, (b) component comprises the sequence shown in SEQ ID No.3, (c) component comprises the sequence shown in SEQ ID No.4, and (e) component comprises the sequence shown in SEQ ID No. 8.

In certain embodiments, the nucleic acids encoding components (a) and (b) are the same nucleic acid, preferably having the sequence shown in SEQ ID No.2, the nucleic acid encoding component (c) has the sequence shown in SEQ ID No.5, the nucleic acid encoding component (d) has the sequence shown in SEQ ID No.7, and the nucleic acid encoding component (e) has the sequence shown in SEQ ID No. 9.

In a second aspect of the invention, there is provided an engineered dendritic cell capable of being used to modulate T cell function, comprising within the engineered dendritic cell a composition according to the first aspect.

In certain embodiments, the antigen presenting cell further comprises an antigen or a nucleic acid expressing the antigen.

In certain embodiments, the antigen is a GPC3 antigen.

In a third aspect of the invention, there is provided a method of producing an engineered dendritic cell, comprising the step of including a composition according to the first aspect in a dendritic cell, thereby obtaining the engineered dendritic cell.

In a fourth aspect of the invention, there is provided a method for modulating T cell function comprising the step of contacting a composition according to the first aspect or a dendritic cell according to the second aspect with a T lymphocyte, preferably in vitro, to thereby improve its properties.

The invention uses IL15, IL15R alpha and IL12 to regulate the activation and proliferation capacity of T cells, promotes DC cells to induce and generate specific cytotoxic T cells to continuously expand, generates more lymphocytes with anti-tumor activity, and improves the effect of treating tumors, such as DC vaccine on treating tumors. Furthermore, the TGFBR2 is specifically combined with TGF-beta in a tumor microenvironment, the inhibition effect of TGF-beta on DC cells and T cells is blocked, the blockage of TGF beta on DC maturation is relieved, the immunosuppression state of T cell function loss is relieved, PD-L1/2 is blocked from being combined with PD-1 on the surface of the T cells through PD1-CD80 protein, the inhibition on the T cells is further relieved, and the tumor killing function of the T cells is released. Experiments prove that the activation of IL15 and IL15R alpha channels, the blocking of TGF-beta channels and the combination with PD1-CD80 can generate multi-channel synergistic effect, and the effect of each component is greatly improved.

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, the proportion of CD8 IFN-gamma +, CD8 TNF-alpha +, CD8 IFN-gamma + and TNF-alpha + cells to total CD 8T cells is from left to right.

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 ratio of CD4 IFN-. gamma. +, CD4 TNF-. alpha. +, CD4 IFN-. gamma. +, and TNF-. alpha. +, to total CD 4T cells, respectively.

Detailed Description

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

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

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

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 tail) of about 150-300 bases in length, e.g., 150-200, 220-270, etc., 5 ' and 3 ' UTR sequences, such as those of human beta-globin.

[ composition ]

In a first aspect of the present invention, there is provided an immunomodulatory composition, sometimes also referred to herein simply as "the composition of the invention", comprising the following (a) to (e): (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; (d) TGFBR2, a mutant or functional fragment thereof, or a nucleic acid encoding thereof; and (e) a PD1-CD80 fusion protein, a mutant or functional fragment thereof, or a nucleic acid encoding the same.

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.

In the present invention, it is preferred that IL-15 is a recombinant protein, for example, a recombinant protein having a sequence shown in SEQ ID No. 1. The invention finds that the recombinant protein still has a structure similar to IL-2, and can be combined with an IL-15 receptor alpha, activate downstream JAK1 and JAK3, lead to phosphorylation of downstream STAT3 and STAT5 and activation of a signal path, induce phosphorylation of BCL2, a MAP kinase path, LCK and SYK, and lead to proliferation and maturation of cells. In addition, such IL-15 can regulate T cell and NK cell activation and proliferation, can maintain the absence of antigen stimulation under memory T cell survival. Likewise, it has also been found in the present invention that such 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 may be a sequence having a homology of 95% or more, preferably 97% or more, more preferably 99% or more with the same species, in addition to the sequence shown in SEQ ID No. 1. Preferably, the IL-15 of the invention consists of the amino acid sequence shown in SEQ ID No. 1.

In the present invention, the IL-15 receptor alpha (IL-15R alpha) is an alpha chain of three chains of alpha, beta and gamma of the IL-15 receptor, and is an essential subunit for forming high-specificity and high-affinity IL-15R alpha beta gamma. The present inventors have found that downstream signal transduction can be achieved by activation of the IL-15 pathway only through overexpression of the alpha chain without the need for the other two subunits. The amino acid sequence of the IL-15 receptor alpha in the present invention is not particularly limited as long as it has a function of binding to IL-15, and examples thereof include a sequence represented by SEQ ID No.3, or a sequence having a homology of 95% or more, preferably 97% or more, more preferably 99% or more and derived from the same species. Preferably, the IL-15 receptor alpha of the present invention consists of the amino acid sequence shown in SEQ ID No. 3.

In a particular embodiment, components (a) and (b), i.e., IL-15 and IL-15 receptor alpha, of the compositions of the invention are separate proteins that bind to each other to form a complex. 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, a nucleic acid of the invention can be a nucleic acid that encodes both IL-15 and IL-15R α. Examples of such nucleic acids include, but are not limited to, the nucleic acid set forth in SEQ ID NO.2, which encodes for production of the IL-15 protein and the IL-15R α protein separately.

IL-12(interleukin-12) is known to be a dimeric form of two subunits in the body and functions as a dimer. Wherein, the two subunits are p35 and p40, which are expressed by two different genes respectively, and are connected together through a disulfide bond to form a dimer after obtaining the two subunits. In addition, in nature, the amount of p40 far exceeds p 70. Excess p40 forms a p40 homodimer that is biologically inactive, but binds to IL-12R β and competes with p70 for the IL-12 receptor (IL-12R) to antagonize its biological effects. In the present invention, IL-12 is preferably a fusion protein of two subunits, p40 and p 35. The production of unwanted p40 in nature is avoided by this fusion protein, with unexpected beneficial effects. More preferably, the fusion protein lacks the signal peptide of the p35 subunit. In certain embodiments, the IL-12 of the invention comprises the amino acid sequence shown in SEQ ID No.4, or a sequence that is more than 95%, preferably more than 97%, more preferably more than 99% homologous thereto and derived from the same species. Preferably, the IL-12 of the invention by SEQ ID No.5 shows the sequence of nucleic acid encoding.

TGFBR2 of the invention is a truncated protein of the naturally occurring TGF-beta receptor 2, preferably the extracellular domain of the receptor, further preferably also including an Fc fragment. The invention uses TGFBR2 specificity to combine with TGF-beta in a tumor microenvironment to block the inhibiting effect of TGF-beta on DC cells and T cells.

In certain embodiments, TGFBR2 of the invention comprises the sequence of one of the following (a ') to (c'):

(a') the amino acid sequence shown as SEQ ID No. 6;

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

(c ') a sequence obtained by amino acid mutation based on (a').

Preferably, the sequence of TGFBR2 of the invention is the sequence shown in SEQ ID No. 6. The TGFBR2 of the present invention is encoded by the nucleic acid shown in SEQ ID No.7 or a sequence having a homology of 95% or more, preferably 96% or more, more preferably 98% or more, and still more preferably 99% or more.

In the invention, PD1-CD80 is a fusion protein of PD1 or a fragment thereof and CD80 or a fragment thereof. Preferably, the fragment of PD1 is the extracellular domain thereof, and the fragment of CD80 is the extracellular domain thereof. In the present invention, CD80 belongs to immunoglobulin superfamily (IgSF), and as an important co-stimulatory molecule expressed on APC, CD80 has important initial regulatory effects on antigen-induced initial T cell activation, proliferation and effector function generation after binding to T cell CD28, and is a positive factor. PD-1 belongs to a member of the CD28 superfamily, and transmits a co-inhibitory signal to a T Cell Receptor (TCR) upon binding to ligand programmed deaTh ligand 1 (PD-L1) or programmed deaTh ligand 2 (PD-L2). PD-1 is expressed primarily on the surface of activated T cells, but also on B cells, Treg cells, NK cells and MDSCs. The ligands PD-L1 and PD-L2 are mainly expressed in a plurality of inflammatory cells such as macrophages and monocytes. In addition to immune cells, tumor cells may also express PD-1 ligands on their surface, such as melanoma, renal cell carcinoma, bladder cancer, and non-small cell lung cancer. Activation of T cells requires two signals, the first from specific binding of TCR and antigen, and the second from activation of helper molecules such as CD80/CD 28. When PD-1 located on the surface of T cells is combined with ligand, tyrosine in the tyrosine activation motif domain of an immune receptor of the PD-1 cytoplasmic region is phosphorylated, protein tyrosine phosphatase containing two SH structures is recruited, zeta chain related protein kinase 70 and phosphatidylinositol 3 kinase downstream of TCR and CD28 are dephosphorylated, and activation of T cells is blocked. PD-1 may be inducibly expressed by inflammatory mediators, such as interferon gamma (IFN-. gamma.), causing transcription of PD-1 by T cells.

Simultaneously, stimulation of IFN- γ also upregulates cell surface expression of PD-L1. In the tumor microenvironment, T cells are activated and expanded into effector T cells after recognizing tumor antigens, kill tumor cells specifically and secrete a large amount of inflammatory factors IFN-gamma. Activated T cells begin to express PD-1, and long-term antigen stimulation causes the T cells to over-express PD-1, so that the T cells are disabled. Most tumor cells escape from the attack of immune cells in this way. Therefore, the blocking agent is used for blocking the interaction between PD-1 and PD-L1, and the activity of T cells and the capability of killing tumor cells can be restored, which is the theoretical basis of the immunotherapy of the current clinical therapy by using the PD-1/PD-L1 antibody.

In an exemplary embodiment, the PD1-CD80 fusion protein comprises the sequence shown in SEQ ID No.8, 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 PD1-CD80 fusion protein of the invention is encoded by a nucleic acid comprising the sequence shown in SEQ ID No.9 or a sequence having a homology of 95% or more, preferably 96% or more, more preferably 98% or more, even more preferably 99% or more.

In certain embodiments, TGFBR2 and PD1-CD80 of the compositions of the invention further comprise an Fc fragment. Preferably, the Fc fragment is a longer Fc fragment. In general, too large a molecular volume may affect the stability and activity of the composition. 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 200-350AA, preferably 220-300 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 mutated fragments 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), 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.

[ dendritic cells for regulating T cell function ]

In a second aspect of the invention, there is provided a dendritic cell which is a human engineered cell. Herein, the dendritic cell (or dendritic cell) of the present invention is preferably a human dendritic cell. The dendritic cells of the present invention may be mature dendritic cells or immature dendritic cells. It should be noted that the dendritic cells are obtained by in vitro induction culture, i.e., Peripheral Blood Mononuclear Cells (PBMCs), which are induced to become DC cells under stimulation of various types of culture media and various types of cytokines. In certain embodiments, the culture medium used to perform the in vitro culture includes AIM-V medium, iDC medium, and mDC medium, and the cytokines used to perform the in vitro induction culture include granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4.

Preferably, the dendritic cell of the present invention further comprises 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 antigen of the invention is a hepatocellular carcinoma antigen, such as the GPC3 antigen.

[ preparation method of engineered Tremella Fuciformis cells ]

In a third aspect of the invention, there is provided a method of producing an engineered dendritic cell for use in relieving loss of T cell function while enhancing proliferation and activity thereof, comprising introducing into a host cell a composition according to the first aspect, thereby engineering the dendritic cell.

[ method for regulating T cell function ]

In a fourth aspect of the invention, there is provided a method for modulating T cell function comprising the step of contacting a composition according to the first aspect or an immune cell according to the second aspect with said T lymphocyte to thereby improve a property thereof.

In a preferred embodiment, the method of the invention further comprises the step of constructing an antigen-producing nucleic acid.

In certain embodiments, the methods of the invention comprise preparing a DNA plasmid comprising the IL-15, IL-15 receptor alpha, IL-12, TGFBR2, and PD1-CD80 fusion proteins and antigen-corresponding. Then, the in vitro transcription process is carried out, firstly, the plasmid is linearized by using restriction endonuclease, and the linearized plasmid is used as a template to prepare ribonucleic acid molecule by in vitro transcription by using T7 RNA polymerase. Finally, the in vitro induction culture and transfection expression process of the antigen presenting cells are carried out.

Preparation example

This preparation is a preparation of DNA and mRNA encoding an antigen and an immunodetection site inhibitor

1. Preparation of DNA and mRNA constructs

DNA sequences encoding IL12, IL15, IL15R a, and TGFBR2 and PD1-CD80 mRNAs of the present invention were constructed separately and used for subsequent in vitro transcription reactions. The coding sequence is followed by a segment of polyadenylation. The DNA sequence information is shown in Table 1 below.

In addition, a coding sequence of human tumor antigen GPC3 for in vitro sensitization is constructed, the coding sequence of GPC3 of the invention consists of a sequence shown in SEQ ID No.10, and the amino acid sequence consists of a sequence shown in SEQ ID No. 11. Additional sequences of antigens are available from Genebank databases.

TABLE-1 DNA sequence Listing

Name (R)	Serial number
		IL-15\IL-15Rα	SEQ ID No.2
IL-12	SEQ ID No.5
		TGFBR2	SEQ ID No.7
PD1-CD80	SEQ ID No.9
		GPC3	SEQ ID No.10

2. In vitro transcription

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

Example 1

This example is used to study 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 500g of the medium was centrifuged to collect cells, the supernatant was removed, and an equal volume of fresh mDC medium (configuration of mDC fresh medium: GM-CSF at a final concentration of 1600U/mL and IL-4, TNF- α (5ng/mL), IL-1 β (5ng/mL), IL-6(150ng/mL) and prostaglandin E2(PGE2) (1. mu.g/mL) were added to AIM-V medium), and after resuspension of the cells, the cells were added to a flask and cultured for 8-18 hours to induce maturation of the DC cells.

2. Transfection of DC cells 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 25-30X 10⁶DCs/ml. According to each 10⁶DC cells were transfected with a proportion of 10. mu.g mRNA,the DC cells and the antigenic mRNA were mixed with mRNA combinations of the different proteins IL15/IL15R α, IL12, PD1-CD80 and TGFBR2, the cell-mRNA mixture was added to an electric rotor, and the antigenic mRNA was transfected into the DC cells using an ECM630 electric converter. The cells after the electroporation were resuspended in a cytokine-free 1640 medium, and the cell density was adjusted to 2X 10⁵DCs/ml, placed at 37 ℃ in 5% CO₂The cultivation was continued in the cell incubator for 6 hours. In this experiment, the mRNA combinations used were as follows:

1) control without any mRNA (mDC control group)

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

3) mRNA encoding GPC3 antigen and mRNA encoding IL12 (IL12 group)

4) mRNA encoding GPC3 antigen and mRNA of IL15/IL15R α (IL15 group)

5) mRNA encoding GPC3 antigen and mRNA encoding PD1-CD80 (group PD1-CD 80)

6) mRNA encoding GPC3 antigen and mRNA of TGFBR2 (TGFBR2 group)

7) mRNA encoding GPC3 antigen and mRNA for IL12+ IL15/IL15R α + TGFBR2+ PD1-CD80 (Experimental group)

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

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

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

Antigen-loaded DC cells were prepared as target cells. Resuscitating prepared antigen-loaded cryopreservedDC cells were counted by trypan blue staining, cells were resuspended in complete medium with RPMI containing IL-7 and IL-2 cytokines and adjusted to a cell concentration of 2X 10⁵Mu.l of cells were added per well.

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

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

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

The results obtained are shown in FIGS. 1 and 2.

In fig. 1 and 2, only a small fraction of CD4 as well as CD 8T cell responses were elicited using only mRNA-loaded DC cells encoding the GPC3 antigen. After the mRNAs encoding IL12, IL15/IL15Ra, TGFBR2 or PD1-Fc-CD80 were used, the CD 4T cell response or the CD 8T cell response was significantly improved compared to the group without these mRNAs.

In the experimental group using the combination of the invention (IL12+ IL15/IL15Ra + TGFBR2+ PD1-Fc-CD80), the CD4 and CD 8T cell responses were significantly improved, both compared with the group using GPC3 antigen mRNA alone (GPC3 control group) and compared with the group using a portion of the components of the composition of the invention. Wherein, the proportion of IFN-gamma positive CD 8T cells is 27.67 percent, which is 15.16 times higher than that of 1.71 percent of GPC3 control group. Compared with other groups, the proportion of IFN-gamma positive CD 8T cells is 2.56 times that of the other groups with the best effect (IL12 group, the proportion of IFN-gamma positive CD 8T cells is 10.8%). Similarly, the proportion of TNF-alpha positive CD 8T cells was 4.58 times higher than that of GPC3 group; the proportion of TNF-alpha and IFN-gamma double positive CD 8T cells in the experimental group is 4.44%, which is 9.83 times higher than that in the GPC3 group. In CD 4T cells, the proportion of IFN-gamma positive cells in the experimental group is improved by 4.07 times compared with that in the GPC3 group. The proportion of TNF-alpha positive cells was 1.33 times that of the GPC3 group. The proportion of TNF-alpha and IFN-gamma double positive CD 4T cells in the experimental group is 3.14 percent, which is 4.32 times higher than that of 0.59 alpha percent of GPC3 group.

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> Beijing Qichen Biotech Ltd

<120> immunomodulatory compositions and uses thereof

<141> 2019-12-17

<160> 11

<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 75 80

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

225 230 235 240

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

Ser Trp Ser Glu Trp Ala Ser Val Pro Cys Ser

595 600

<210> 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 Asn Pro 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 Ser Asp

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> 622

<212> PRT

<213> human (Homo sapiens)

<400> 8

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Glu Pro Lys Ser Cys Asp Lys Thr His

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp Gln Lys

435 440 445

Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp Met Asn Ile Trp

450 455 460

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

465 470 475 480

Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly Thr Tyr Glu Cys

485 490 495

Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His Leu Ala

500 505 510

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

515 520 525

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

530 535 540

Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn Gly Glu

545 550 555 560

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

565 570 575

Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr Asn His

580 585 590

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

595 600 605

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

610 615 620

<210> 9

<211> 1869

<212> DNA

<213> human (Homo sapiens)

<400> 9

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca agagcccaaa tcttgtgaca aaactcacac atgcccaccg 540

tgcccagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 600

gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 660

gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 720

acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 780

ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 840

ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 900

tacaccctgc ccccatcccg ggatgagctg accaagaacc aggtcagcct gacctgcctg 960

gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 1020

aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc 1080

aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 1140

catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaagag 1200

cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagttatcca cgtgaccaag 1260

gaagtgaaag aagtggcaac gctgtcctgt ggtcacaatg tttctgttga agagctggca 1320

caaactcgca tctactggca aaaggagaag aaaatggtgc tgactatgat gtctggggac 1380

atgaatatat ggcccgagta caagaaccgg accatctttg atatcactaa taacctctcc 1440

attgtgatcc tggctctgcg cccatctgac gagggcacat acgagtgtgt tgttctgaag 1500

tatgaaaaag acgctttcaa gcgggaacac ctggctgaag tgacgttatc agtcaaagct 1560

gacttcccta cacctagtat atctgacttt gaaattccaa cttctaatat tagaaggata 1620

atttgctcaa cctctggagg ttttccagag cctcacctct cctggttgga aaatggagaa 1680

gaattaaatg ccatcaacac aacagtttcc caagatcctg aaactgagct ctatgctgtt 1740

agcagcaaac tggatttcaa tatgacaacc aaccacagct tcatgtgtct catcaagtat 1800

ggacatttaa gagtgaatca gaccttcaac tggaatacaa ccaagcaaga gcattttcct 1860

gataactga 1869

<210> 10

<211> 1985

<212> RNA

<213> human (Homo sapiens)

<400> 10

gagaccggcc ucgagcagcu gaagcuuccu gcaggucgac ucuagagcca ccaugagggc 60

ccugugggug cugggccucu gcugcguccu gcugaccuuc gggucgguca gagcugacga 120

ugaaguugau gugcagccuc cuccuccucc uccagacgcu acaugucacc agguccgcuc 180

cuucuuccag aggcugcagc caggacucaa gugggugcca gagacaccag ugccaggaag 240

cgaucugcag gucugucugc cuaagggccc uaccuguugc ucccggaaga uggaggagaa 300

guaccagcug accgccaggc ugaacaugga acagcugcug cagagcgcca gcauggagcu 360

gaaguuccug aucauccaga acgccgccgu guuccaggag gccuucgaga ucgucgugcg 420

gcacgccaag aacuacacca acgccauguu caagaacaac uaccccagcc ugacaccuca 480

ggccuuugag uucguggggg aguucuucac cgacgugucu cuguacaucc ugggcagcga 540

caucaacgug gacgacaugg ugaacgagcu guucgacagc cuguuccccg ugaucuacac 600

ccagcugaug aacccaggcc ugccagauag cgcucuggau aucaacgagu gccugagggg 660

agccagaaga gaccugaagg uguucggcaa cuuccccaag cugaucauga cccagguguc 720

caagagccug caggucacca ggaucuuccu gcaggcccug aaccugggca ucgaggucau 780

caacaccacc gaccaccuga aguucagcaa ggauugcggc cggaugcuca cccgcaugug 840

guauuguagc uauugccagg gccugaugau ggugaagccu ugcggcggcu auugcaacgu 900

cgugaugcag gguuguaugg ccggcguggu ggagaucgac aaguauuggc gggaguacau 960

ccugagccug gaggagcugg ugaacggcau guaccggauc uacgacaugg agaacgugcu 1020

gcugggccug uucuccacca uccacgacag cauccaguac gugcagaaga acgccggcaa 1080

gcugacaacc accaucggaa agcucugcgc ccacucucag cagaggcagu acagaagcgc 1140

cuacuacccc gaggaccugu ucaucgacaa gaaggugcug aagguggccc acguggaaca 1200

cgaagagaca cugagcagcc ggaggagaga gcugauccag aagcugaagu ccuucaucuc 1260

cuucuacagc gcccugccag gcuacauuug cagccacagc ccaguggccg agaacgacac 1320

ccucuguugg aacggccagg agcuggugga gagauacucu cagaaggccg ccaggaacgg 1380

caugaagaac caguucaacc ugcacgagcu gaagaugaag ggcccagagc cagugguguc 1440

ccagaucauc gacaagcuga agcacaucaa ccagcugcug cggaccauga gcaugccuaa 1500

gggcagggug cuggacaaga accuggacga ggagggcuuc gagucaggag auugcggcga 1560

cgacgaagac gaguguauug gcggaagcgg cgacggcaug aucaagguca agaaccagcu 1620

gcgguuccug gccgaacugg ccuacgaucu ggacguggac gacgcuccag gcaauucuca 1680

gcaggccaca ccuaaggaca acgagaucag caccuuccac aaccugggca acgugcacuc 1740

uccucugaag cugcugacca gcauggccau uagcgucguc ugcuucuucu uccuggugca 1800

ucugaucccc aucgcugugg guggugcccu ggcggggcug guccucaucg uccucaucgc 1860

cuaccucguc ggcaggaaga ggagucacgc aggcuaccag acuaucuagg aauucuuaau 1920

uaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1980

aaaaa 1985

<210> 11

<211> 618

<212> PRT

<213> human (Homo sapiens)

<400> 11

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

Lys Arg Ser His Ala Gly Tyr Gln Thr Ile

610 615

Claims (5)

1. Use of an immunomodulatory composition and a GPC3 antigen in the manufacture of an engineered dendritic cell for increasing the proportion of IFN- γ positive CD 8T cells in T lymphocytes, wherein the immunomodulatory composition comprises the following components (a) to (e) or a nucleic acid encoding the components (a) to (e):

(a) IL-15 with the sequence shown as SEQ ID number 1;

(b) IL-15 receptor alpha with the sequence shown as SEQ ID number 3;

(c) IL-12 with the sequence shown as SEQ ID number 4;

(d) TGFBR2 with the sequence shown in SEQ ID No. 6; and

(e) PD1-CD80 fusion protein with the sequence shown in SEQ ID No. 8.

2. Use according to claim 1, wherein (a) and (b) are capable of binding to form a complex.

3. Use according to claim 1, wherein said components (a) and (b) are encoded by a nucleic acid having the sequence shown by SEQ ID No.2, said component (c) is encoded by a nucleic acid having the sequence shown by SEQ ID No.5, said component (d) is encoded by a nucleic acid having the sequence shown by SEQ ID No.7, and said component (e) is encoded by a nucleic acid having the sequence shown by SEQ ID No. 9.

4. Use according to claim 1, characterized in that said GPC3 antigen is encoded by a nucleic acid having the sequence shown in SEQ ID No. 10.

5. A method for increasing the proportion of IFN- γ positive CD 8T cells in T lymphocytes in vitro, comprising the step of contacting in vitro engineered dendritic cells with T lymphocytes, wherein:

the engineered dendritic cells are obtained by including in the dendritic cells an immunomodulatory composition comprising the following components (a) to (e) or a nucleic acid encoding the components (a) to (e):

(a) IL-15 with the sequence shown as SEQ ID number 1;

(b) IL-15 receptor alpha with the sequence shown as SEQ ID number 3;

(c) IL-12 with the sequence shown as SEQ ID number 4;

(d) TGFBR2 with the sequence shown in SEQ ID No. 6; and

(e) PD1-CD80 fusion protein with the sequence shown in SEQ ID No. 8.

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