CN110563851A

CN110563851A - Fusion protein with immunoregulation function, pharmaceutical composition, cell and application

Info

Publication number: CN110563851A
Application number: CN201910871223.8A
Authority: CN
Inventors: 蒋俊; 祝道成; 吴斐然; 林鑫
Original assignee: Qichensheng Biotechnology (zhuhai) Co Ltd
Current assignee: Qichensheng Biotechnology (zhuhai) Co Ltd
Priority date: 2019-08-20
Filing date: 2019-09-16
Publication date: 2019-12-13
Anticipated expiration: 2039-09-16
Also published as: CN110563851B

Abstract

The invention discloses a fusion protein with an immunoregulation function, a pharmaceutical composition, a cell and application. The fusion protein of the present invention comprises a soluble PD-1-derived a fragment capable of binding to CD28 and a soluble CD 80-derived B fragment capable of binding to CTLA-4. The fusion protein can be combined with PD-L1 and PD-L2, and can also be combined with CTLA-4 to block an inhibitory signal path mediated by the CTLA-4; meanwhile, the compound can be combined with CD28 on the surface of T cells to provide a costimulatory signal for T cell activation, and the proliferation of the T cells and the secretion of cytokines are induced.

Description

Fusion protein with immunoregulation function, pharmaceutical composition, cell and application

Technical Field

The invention relates to a medical product, in particular to an immunopotentiator, a pharmaceutical composition, a cell and application thereof.

Background

Traditionally, it was thought that tumor cells express tumor-specific antigens and are presented on the surface of tumor cells by MHC complexes, whereas anti-tumor T cells are not fully activated. Based on this, tumor cell immunotherapy is mainly to stimulate the immune system by vaccination or adoptive cell immunotherapy, thereby eliciting an immune response.

In recent years, it has become clear that the immune system does recognize tumor antigens, but despite the presence of tumor antigens, T cells are assured to remain quiescent. Based on this phenomenon, there is a hypothesis that: there are inhibitory mechanisms that limit or turn off the specificity of the anti-tumor immune response. When a negative regulatory T cell surface molecule is up-regulated in the expression of activated T cells, its activity is attenuated, resulting in less effective tumor cell killing. These inhibitory molecules are called negative co-stimulatory molecules. These proteins, also known as immunodetection point proteins, play a role in multiple pathways, such as attenuation of early activation signals, competition with positive costimulatory molecules and direct inhibition by antigen presenting cells. The members of the protein family are programmed death factor-1 (PD-1) and ligands thereof B7-H1/PD-L1 and B7-DC/PD-L2 and CTLA-4/CD80 or CD 86.

CD28 is a cell surface glycoprotein that is constitutively expressed on the surface of most mature T cells and thymocytes. CTLA-4 receptors are detectable only in T cells 2-3 days after activation, but are not expressed in resting T cells. The primary ligands for CD28 and CTLA-4 molecules are CD80 and CD86 expressed on the surface of Antigen Presenting Cells (APCs). The biological rationale for the presence of at least two receptors (CD28 and CTLA-4) and two ligands (CD80 and CD86) is currently unknown. Current studies indicate that CD80 inhibits T cell activation by binding to CTLA-4 on the T cell surface; meanwhile, CD80 can also provide a costimulatory signal for T cell activation by binding with CD28 on the surface of T cells, and induce the proliferation of T cells and the secretion of cytokines.

PD-1 is expressed on activated T cells, B cells, and on monocytes that are involved in mediating the balance of immune activation and tolerance. Its main role is to limit T cell activity around inflammation and to limit autoimmunity in the inflammatory response to infection. The basis for this regulation is that the ligands PD-L1 and PD-L2 of the PD-1 molecule are upregulated in response to a variety of proinflammatory factors and can bind to PD-1 molecules on activated T cells in inflamed tissues, inhibiting T cell function, thereby limiting the immune response.

In addition, it was found that PD-L1 expression is often upregulated in a variety of different tumors, which results in the development of immune escape by inhibiting local anti-tumor T cell responses by binding to PD-1 on tumor-infiltrating lymphocytes. Researches show that nearly more than half of tumor infiltration CD8 positive T cells in cervical cancer and liver cancer express PD-1 molecules, and the combination of the PD-L1 molecules expressed on the surface of tumor cells can lead to the exhaustion and apoptosis of the T cells.

Recently, several immunodetection point inhibitor drugs targeting the PD-1 receptor and its ligand PD-L1 have been approved for sale. The immune checkpoint inhibitors have good clinical efficacy in patients with various tumors such as melanoma, renal cancer, colorectal cancer, non-small cell lung cancer, liver cancer and the like. However, many current clinical research data indicate that the clinical response rate of using an immune checkpoint inhibitor such as PD-1/PD-L1 antibody and CTLA-4 antibody is low, such that only about 15% of patients with PD-1 antibody can receive clinical benefit in liver cancer patients; in a plurality of clinical experiments for treating melanoma by using the CTLA-4 antibody, the effective rate is between 5 and 22 percent by using the CTLA-4 monoclonal antibody ipilimumab alone or in combination.

Studies have reported that transfection of dendritic cells with siRNAs for PD-1 ligands PD-L1 and PD-L2 inhibited the expression of PD-L1 and PD-L2, and dendritic cell vaccines were prepared by loading dendritic cells with a minor histocompatibility antigen (MiHA). These MiHA-loaded PD-L1/L2 silenced DCs showed highly efficient activation and amplification of MiHA-specific T cell function: the function of MiHA-specific CD8+ T cells can be obviously enhanced; compared to MiHA-loaded DCs that were not silenced for PD-L1/L2, the expanded MiHA-specific CD8+ T cells increased 14.4-fold during one week of in vitro stimulation and 20-fold after two weeks of in vitro stimulation (A.B. van der Waart et al, 2015. cancer Immunol Immunother (2015)64: 645-654).

Similarly, a dendritic cell vaccine prepared by silencing PD-L expression of DC cells with siRNA-lipid-nanoparticle complexes targeting PD-L1/L2 and loading the DC with antigen mRNA or polypeptide can effectively increase helper T cells (Th1 and Th2 cells) and antigen-specific CD8+ T cell responses from allogeneic stem cell transplanted tumor patients (Mieke W.H.Roeven et al, J Immunother 2015; 38: 145-154).

Various clinical data show that the single immune checkpoint inhibitor can effectively act in various cancer species, but the single immune checkpoint inhibitor has the problem of low response rate.

Disclosure of Invention

In order to solve at least part of technical problems in the prior art, the invention finds that a fusion protein obtained by fusing proteins with different inhibitory functions can greatly stimulate anti-tumor immune response in a tumor patient 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 a fusion protein having an immunomodulatory function, comprising a soluble a fragment derived from PD-1 capable of binding to CD28, and a soluble B fragment derived from CD80 capable of binding to CTLA-4.

Preferably, the soluble A fragment is further capable of binding to PD-L1/2.

Preferably, the soluble a fragment and the soluble B fragment are linked by a C fragment derived from an immunoglobulin.

Preferably, the fusion protein has the structure A-C-B, or B-C-A.

In a second aspect of the invention, there is provided a nucleic acid comprising a sequence encoding the fusion protein of the first aspect.

Preferably, the nucleic acid of the invention has the sequence shown in SEQ ID NO. 4.

In a third aspect of the invention, there is provided a pharmaceutical composition comprising a fusion protein according to the first aspect or a nucleic acid according to the second aspect, and an antigen or a nucleic acid encoding said antigen.

In a fourth aspect of the invention, there is provided a cell comprising a nucleic acid according to the second aspect and capable of producing a fusion protein according to the first aspect, or a derivative thereof.

Preferably, the cells of the invention are PBMC cells and/or DC cells.

In a fifth 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 a fusion protein according to the first aspect or a nucleic acid according to the second aspect to act on a population of lymphocytes.

The fusion protein can be combined with PD-L1 and PD-L2, and can also be combined with CTLA-4 to block an inhibitory signal path mediated by the CTLA-4; meanwhile, the compound can be combined with CD28 on the surface of T cells to provide a costimulatory signal for T cell activation, and the proliferation of the T cells and the secretion of cytokines are induced. The fusion protein has the immune enhancement effect far greater than that of a single PD-1 inhibitor or that of a CTLA-4 inhibitor, and shows a synergistic effect.

Drawings

FIG. 1 expression level of fusion protein in supernatant of cells of each experimental group after 24 hours of transfection of mRNA. In each column, the left side is the mDC control and the right side is the mDC-mRNA experimental group.

FIG. 2 shows mDC cells transfected with the fusion protein of the present invention, which show no difference in phenotype compared to untransfected mDC cells.

FIG. 3 shows that mDC cells co-transfected with the fusion protein of the present invention and mRNA of PP65 antigen and mRNA of control protein, after co-culture with PBMC, detected specific immune responses against PP 65. As shown in FIG. 3, the proportion of cells that elicited specific immune responses was higher than that of the other groups after co-transfection with the fusion protein of the present invention. Wherein, TNF-a + CD4, IFN-r + CD4, TNF-a + CD8 and IFN-r + CD8 are respectively arranged in each group of columns from the left side to the right side.

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 order to enhance the adaptability of the host cell, preference is preferably given to codon-biased engineering of at least part of the nucleic acids. 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.

[ fusion protein ]

In a first aspect of the present invention, there is provided a fusion protein having an immunomodulatory function, comprising a soluble a fragment derived from PD-1 capable of binding to CD28, and a soluble B fragment derived from CD80 capable of binding to CTLA-4. Preferably, the soluble a fragment and the soluble B fragment are linked by a chemical bond or a chemical group. Optionally, further comprises an immunoglobulin-derived C fragment positioned between the soluble A fragment and the soluble B fragment for linking the two.

In the present invention, the order of ligation between the soluble A fragment and the soluble B fragment is not limited, and the A fragment-B fragment (i.e., A-B) may be used, or the B fragment-A fragment (i.e., B-A) may be used. In the case of containing the C fragment, the order of linkage among the soluble A fragment, the soluble B fragment and the C fragment is also not particularly limited, and may be an A fragment-C fragment-B fragment (i.e., A-C-B), a B fragment-C fragment-A fragment (i.e., B-C-A), or an A fragment-B fragment-C fragment (i.e., A-B-C) or a B fragment-A fragment-C fragment (i.e., B-A-C). Here, "-" denotes a chemical bond or a chemical group.

In the present invention, the soluble a fragment, the soluble B fragment and the C fragment are generally derived from mammals or humans, and the three may be derived from the same species or different species. Preferably, the three fragments are of the same origin as the subject to be administered. For example, in the case of a human being as a subject, it is preferable that the soluble a fragment, the soluble B fragment and the C fragment are all derived from a human being.

Soluble A fragment

The soluble a fragment of the invention is a truncated portion derived from the full-length sequence of PD-1, which truncated portion comprises at least the amino acid portion that binds to CD28, preferably also the amino acid portion that binds to PD-L1 and/or PD-L2 (abbreviated PD-L1/2). Herein, an "amino acid moiety" may be a sequence of a plurality of consecutive amino acids linked by peptide bonds, or a combination of a plurality of spaced or partially spaced amino acids that play a critical role in binding to CD28 or binding to PD-L1/2. The amino acid moiety that binds to CD28 and the amino acid moiety that binds to PD-L1/2 may not share any amino acids between them, preferably at least one amino acid is shared between the two. In certain embodiments, a soluble A fragment of the invention is encoded by the nucleic acid shown as SEQ ID NO. 1. Preferably, the soluble A fragment of the invention has the sequence shown in SEQ ID NO. 5. More preferably, the sequence of the soluble A fragment of the invention is shown in SEQ ID NO 5. The soluble A fragment not only co-stimulates signals of T cell activation, but also can silence PD-L1/L2, so that DC shows high-efficiency activation.

Soluble B fragment

The soluble B fragment of the present invention refers to a truncated portion derived from the full-length sequence of the CD80 protein, which truncated portion comprises at least the portion of amino acids that bind to CD 80. Like the soluble a fragment, the amino acid portion herein can be a plurality of contiguous amino acid sequences linked by peptide bonds, or a combination of a plurality of spaced or partially spaced amino acids that play a critical role in binding to CD 80. Soluble B fragments are capable of blocking CTLA-4 mediated inhibitory signaling pathways.

Preferably, the soluble B fragment comprises the extracellular domain of CD 80. In certain embodiments, the soluble B fragment of the invention is encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO. 2. Preferably, the soluble B fragment of the invention has the sequence shown in SEQ ID NO 6. More preferably, the sequence of the soluble B fragment of the invention is shown in SEQ ID NO 6.

C fragment

The C fragment of the present invention is a portion derived from an immunoglobulin, preferably an Fc fragment. The Fc fragment is useful for extending the half-life and improving stability of the fusion protein. 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 from 100 to 300AA, preferably from 150 to 300 AA.

The Fc fragment of the present invention may comprise a fragment naturally occurring in an immunoglobulin, and may further comprise a mutant Fc fragment modified by known genetic engineering means to obtain more superior performance. For example, the Fc fragment contains 3 mutations of "YTE", i.e. methionine (Met, M), serine (Ser, S) and threonine (Thr, T) at positions 252, 254 and 256 are replaced by tyrosine (Tyr, Y), T and glutamic acid (Glu, E), respectively, thereby obtaining a fusion protein with a longer half-life. For another example, by genetic engineering and modification of disulfide bonds of an Fc fragment, Fc fusion proteins can be aggregated into multimeric complexes, thereby obtaining fusion proteins with superior stability. In certain embodiments, the Fc fragment of the invention is a polypeptide consisting of a sequence comprising seq id NO: 3.

[ nucleic acid ]

In a second aspect of the invention, there is provided a nucleic acid comprising a sequence encoding a fusion protein according to the first aspect of the invention.

In certain embodiments, the nucleic acid sequences of the present invention comprise the sequence shown as SEQ ID NO. 1, the sequence shown as SEQ ID NO. 2, and the sequence shown as SEQ ID NO. 3. Preferably, the sequences shown in SEQ ID NO 1-3 are linked by covalent bonds to form an open reading frame. The sequence of the sequence shown in SEQ ID NO 1-3 is not particularly limited, and the sequence from the 5 'end to the 3' end of the nucleic acid may be SEQ ID NO 1, SEQ ID NO 3 and SEQ ID NO 2, or SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 1. In an exemplary embodiment, the nucleic acid of the invention has the sequence shown in SEQ ID NO. 4.

In certain embodiments, the nucleic acids of the invention further comprise a gene regulatory element operably linked to the nucleic acid molecule described above. Examples of such gene regulatory elements include, but are not limited to, promoters, enhancers, leader peptide genes, and the like.

[ pharmaceutical composition ]

In a third aspect of the invention, there is provided a pharmaceutical composition capable of providing one or more antigens (preferably immunogens) to a subject in need thereof with a means for alleviating, delaying or curing a condition or disorder in the subject. The pharmaceutical composition of the present invention has an enhanced, increased or potentiated immune capacity as compared to a drug that provides the antigen alone. The pharmaceutical composition of the invention comprises at least one antigen or a nucleic acid encoding the antigen, and the fusion protein of the first aspect or the nucleic acid of the second aspect. Optionally, other ingredients, such as pharmaceutically acceptable carriers, may also be included.

As described above, the fusion protein or its encoding nucleic acid included in the pharmaceutical composition of the present invention has been described in detail and will not be described herein again. The antigen of the present invention is described in detail below.

The antigen of the invention may be a nucleic acid encoding at least one antigen open reading frame. The nucleic acid of the present invention may be one or more. 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 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, an antigen of the invention comprises at least one polypeptide. The polypeptides herein comprise at least one antigenic epitope. 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, CALR 2, CSAG2, CTAG 12, CTAGE 2, FMR 2, GAGE2, GASES 2, GASESSGES 2, GCAST 2, GCAGEN 2, GCE 2, GCAST 2, GCE 2, GCAGEN 2, GCE 2, GCAST 2, GCE 2, GCT 2, GCE 36363636363672, GCE 2, GCE 363636363672, GCE 2, GCE 36363672, GCE 363636363636363672, GCE 3636363672, GCE 36363672, GCE 2, GCE 36363672, GCE 2, 363672, GCE 363636363636363636363672, GCE 2, GCE 2, 36363636363636363672, GCE 363672, GCE 2, GCE 36363672, GCE 363636363672, 363636363636363636363636363636363672, 3636363636363636363636363636363636.

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 antigens of the invention are also represented by cells, such as DC cells or PBMC cells, containing nucleic acids encoding the antigens.

In addition to the above-described fusion protein or its encoding nucleic acid and antigen or its encoding nucleic acid, the pharmaceutical composition of the present invention may further comprise other components. Examples of such other ingredients include pharmaceutically acceptable carriers, which are well known in the art and which can be determined by one of ordinary skill in the art to meet clinical criteria. Pharmaceutically acceptable carriers include diluents and excipients.

Examples of suitable pharmaceutically acceptable carriers include, but are not limited to: (1) dulbecco phosphate buffered saline, pH about 7.4, with or without about 1mg/ml to 25mg/ml human serum albumin; (2) 0.9% saline (0.9% w/v sodium chloride), and (3) 5% (w/v) glucose; antioxidants such as tryptamine and stabilizers such as Tween20 may also be included.

The pharmaceutical composition of the present invention may be in any suitable dosage form. For example, injections, suspensions, emulsifiers and the like. The pharmaceutical composition of the present invention can be administered into the body by known means. For example, delivery into the tissue of interest by intramuscular injection, optionally via intravenous, transdermal, intranasal, oral, mucosal, or other delivery methods. Such administration may be via a single dose or multiple doses. It will be understood by those skilled in the art that the actual dosage to be administered herein may vary greatly depending on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.

Preferably, the pharmaceutical composition of the invention is delivered to a host cell in vivo. 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 or derivatives thereof ]

In a fourth aspect of the invention, there is provided a cell or derivative thereof. The cell of the invention comprises a nucleic acid according to the second aspect and the nucleic acid is capable of being expressed and/or translated within the cell, thereby enabling the production of a fusion protein according to the first aspect of the invention.

In certain embodiments, the cells of the invention are capable of constitutively expressing the fusion protein intracellularly. That is, the cell is constantly producing the fusion protein. In certain embodiments, the cells of the invention are transiently expressing the fusion protein, or the nucleic acid within the cell is in a quiescent state, and when desired, e.g., after regulation, the nucleic acid within the cell is expressed and fusion protein production is initiated. The fusion protein of the present invention may be expressed intracellularly or extracellularly.

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.

The cell derivative of the present invention refers to a mixture containing at least a fusion protein derived from the cell of the present invention, and examples thereof include, but are not limited to, cell lysates, cell secretions, cell extracts, isolates, and the like.

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

In a fifth 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 a fusion protein or nucleic acid of the invention to act on the population of lymphocytes.

The lymphocytes of the invention are fusion protein or nucleic acid target cells, including cells cultured in vitro or cells in a subject. CD4+ cells and/or CD8+ cells are preferred. The action of the fusion protein or nucleic acid with lymphocytes can be performed in various ways. The fusion protein can be made to act on lymphocytes by directly contacting the lymphocytes with the fusion protein. It is also possible to express the nucleic acid as the corresponding protein and then to bring the resulting protein into contact with lymphocytes. 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 fusion proteins

1. Preparation of DNA and mRNA constructs

The DNA sequence for the mRNA encoding the CMV virus pp65 protein (sequence shown in SEQ ID NO: 7), the DNA sequence for the mRNA encoding the sPD-1-Fc, sCD80-Fc and the sPD-1-Fc-CD80 fusion proteins, and for the subsequent in vitro transcription reaction were constructed. Constructs were made by introducing high GC sequences to stabilize the synthetic mRNA by codon optimization, followed by 3' UTR sequences derived from human beta-globin, followed by a polyadenylation segment. The specific sequence is shown in Table 1.

TABLE-1

Name (R)	Nucleic acid sequence number
		sPD-1	SEQ ID NO：1
sCD80	SEQ ID NO：2
		Fc	SEQ ID NO：3
PD1-Fc-CD80	SEQ ID NO：4

2. In vitro transcription

The corresponding DNA plasmid obtained by preparation is firstly linearized by using the speI endonuclease, and mRNA is prepared by in vitro transcription by using T7RNA polymerase by using the linearized plasmid as a template. The prepared mRNA was then purified by lithium chloride precipitation.

Example 2

This example serves to verify the expression level of the fusion protein mRNA described in the present invention in dendritic cells and the effect on dendritic cell phenotype.

In vitro induction culture of DC cells

Aseptically extracting 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% CO₂Culturing in an incubator for 6 days. Half of the cell culture medium was transferred to a centrifuge tube, cells were collected by centrifugation at 500g, the supernatant was removed, and an equal volume of fresh mDC medium was added, the formulation of which was as follows: 1600U/mLGM-CSF and 1000U/mL IL-4, TNF-a (5ng/mL), IL-1 beta (5ng/mL), IL-6(150ng/mL) and prostaglandin E2(PGE2) (1ug/mL), after resuspension of the cells, were added to the flask and cultured for 8-18 hours to induce DC cell maturation.

2. DC cells transfected with the fusion protein of the invention

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 into 4ug mRNA ratio, mixing DC cells with soluble protein (sPD-1-Fc, sCD80-Fc or sPD-1-Fc-CD80) mRNA, adding the cell-mRNA mixture to the cuvette, and using ECM630 electrotransfer instrument, antigen mRNA was transfected into DC cells. The cells after the electroporation were resuspended in cytokine-free AIM-V medium and the cell density was adjusted to 1X 10⁶DCs/ml, 200ul per well, into 96 well cell culture plates, placed at 37 ℃ in 5% CO₂And continuing culturing in the cell culture box. GFP mRNA was transfected into DC cells under the same conditions as the control group.

3. Determination of transfection efficiency

After 24 hours of transfection, the proportion of DC cells expressing green fluorescent protein in all DC cells was analyzed by flow cytometry, and the transfection efficiency of DC cells after 24 hours of transfection was more than 50%.

4. Detection of fusion protein expression in cell supernatant

After 24 hours of transfection, cell supernatants were collected, and the concentrations of each fusion protein in the supernatants were measured using ELISA kits. The results of the detection are shown in FIG. 1. As a result, it can be seen that the mRNA encoding the soluble protein of the present invention can express a higher level of the target protein in DC cells.

Identification of DC cell phenotype

Using direct immunofluorescence labeling, transfected DC cells were centrifuged and the cells were resuspended in FACS buffer (2% FBS in PBS) at a cell concentration of 1X 10⁶cells/ml, 100ul of transfected DC cell suspension was added to the flow cell tube, and 5ul of the corresponding antibodies CD80, CD83, CD86, and the corresponding isotype control were added, respectively. Staining at 4 ℃ for 30min in the dark. 3ml of FACS Buffer was added to each tube to wash the cells, the supernatant was discarded, 500ul of FACS Buffer was added, and expression of CD80, CD83, and CD86 was detected by flow analysis. As shown in fig. 2, the DC cell surface molecules CD80, CD83, CD86 transfected with the fusion protein of the present invention were stably expressed without significant difference compared to untransfected DC cells.

Example 3

This example is used to study the Effect of immunomodulator compositions on T cell response

In vitro induction culture of DC cells

Aseptically extracting 50ml of human venous blood, separating peripheral blood mononuclear cells with lymphocyte separation solution in a superclean bench, and separating mononuclear cellsAdding the nuclear 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 and adherent cells were cultured in iDC medium (GM-CSF was added to AIM-V medium to a final concentration of 800U/mL and IL-4 was added to 500U/mL) at 37 ℃ in a 5% CO2 incubator for 6 days. Half of the cell culture medium was transferred to a centrifuge tube, centrifuged at 500g to collect cells, the supernatant was removed, an equal volume of fresh mDC medium was added, formulated as 1600U/mL GM-CSF and 1000U/mL IL-4, TNF-a (5ng/mL), IL-1 β (5ng/mL), IL-6(150ng/mL) and prostaglandinE2(PGE2) (1ug/mL), 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 fusion proteins

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 10ug of mRNA, the DC cells were mixed with antigen mRNA in combination with different fusion protein (sPD-1-Fc, sCD80-Fc or sPD-1-Fc-CD80) mRNA, the cell-mRNA mixture was added to an electric rotor, and the antigen mRNA was transfected 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:

1) Controls without any mRNA addition

2) Adding only mRNA encoding pp65 antigen (pp65mRNA)

3) mRNA encoding pp65 and sPD-1-Fc

4) mRNA encoding pp65 and sCD80-Fc

5) mRNA encoding pp65 and mRNA of sPD-1-Fc-CD80

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: PBMC control group without DC cells, one of the above was usedThe group of five groups of DC cells co-cultured with PBMC cells in step (ii) and the positive control group using the immunostimulant PMA/Ionomycin; according to grouping conditions, adding DC cells loaded with corresponding mRNA into different holes, wherein the ratio of MNC to DC is 10: 1; the concentration of PMA/Ionomycin in the positive control is 50ng/ml and 1 ug/ml; culturing 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 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.

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.

Adding 2 mu M monensin or 3 mu g/ml Brefeldin A into cell culture solution, mixing well to block protein transport, wherein the time of the substances in the cell culture solution should not exceed 12h, and performing intracellular staining detection after 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-a and IFN-r.

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

As shown in FIG. 3, the use of the mRNA fusion protein of the present invention can induce an anti-tumor specific immune response in T lymphocytes, which have a stronger anti-tumor specific immune response. The present invention has a synergistic immune enhancing effect compared to drugs using one modulator alone.

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> fusion protein with immunoregulation function, pharmaceutical composition, cell and application

<130> BH1900215-1

<141> 2019-09-16

<150> 201910767836.7

<151> 2019-08-20

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 501

<212> DNA

<213> Homo sapiens

<400> 1

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 a 501

<210> 2

<211> 627

<212> DNA

<213> Homo sapiens

<400> 2

gttatccacg tgaccaagga agtgaaagaa gtggcaacgc tgtcctgtgg tcacaatgtt 60

tctgttgaag agctggcaca aactcgcatc tactggcaaa aggagaagaa aatggtgctg 120

actatgatgt ctggggacat gaatatatgg cccgagtaca agaaccggac catctttgat 180

atcactaata acctctccat tgtgatcctg gctctgcgcc catctgacga gggcacatac 240

gagtgtgttg ttctgaagta tgaaaaagac gctttcaagc gggaacacct ggctgaagtg 300

acgttatcag tcaaagctga cttccctaca cctagtatat ctgactttga aattccaact 360

tctaatatta gaaggataat ttgctcaacc tctggaggtt ttccagagcc tcacctctcc 420

tggttggaaa atggagaaga attaaatgcc atcaacacaa cagtttccca agatcctgaa 480

actgagctct atgctgttag cagcaaactg gatttcaata tgacaaccaa ccacagcttc 540

atgtgtctca tcaagtatgg acatttaaga gtgaatcaga ccttcaactg gaatacaacc 600

aagcaagagc attttcctga taactga 627

<210> 3

<211> 651

<212> DNA

<213> Homo sapiens

<400> 3

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> 4

<211> 1869

<212> DNA

<213> Homo sapiens

<400> 4

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> 5

<211> 167

<212> PRT

<213> Homo sapiens

<400> 5

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

165

<210> 6

<211> 208

<212> PRT

<213> Homo sapiens

<400> 6

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

1 5 10 15

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

20 25 30

Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp Met Asn

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

<210> 7

<211> 1872

<212> DNA

<213> Cytomegalo virus

<400> 7

atgagggccc tgtgggtgct gggcctctgc tgcgtcctgc tgaccttcgg gtcggtcaga 60

gctgacgatg aagttgatgt gatggagagc cggggccggc ggtgccccga aatgatcagc 120

gtgctgggac caatttccgg ccatgtgctc aaagccgtgt ttagccgggg agacactccc 180

gtcctgcccc atgaaaccag actgctccag accggcatcc acgtgagagt gagccaaccc 240

tccctgatcc tcgtcagcca atacaccccc gacagcacac cctgccatcg gggcgacaat 300

cagctgcagg tgcagcacac ttatttcacc ggaagcgagg tggagaacgt cagcgtgaac 360

gtgcacaacc ccaccgggcg gagcatttgc ccctcccagg agcccatgtc catctacgtg 420

tacgccctgc ccctgaagat gctgaacatc ccatccatca acgtgcacca ctatccttcc 480

gccgccgaac ggaagcatcg gcacctcccc gtcgccgacg ccgtgatcca cgccagcggc 540

aagcaaatgt ggcaggcccg gctgaccgtg agcggcctgg cctggacccg gcagcaaaac 600

cagtggaagg agcctgacgt gtactacaca agcgccttcg tcttcccaac caaggacgtc 660

gcactgcggc acgtggtgtg cgcccacgag ctcgtctgtt ccatggaaaa caccagagcc 720

accaaaatgc aggtgattgg cgaccagtac gtgaaggtgt acctggagtc cttctgcgag 780

gacgtgccct ccgggaagct cttcatgcac gtgaccctgg gcagcgacgt ggaggaggac 840

ctcaccatga cccggaaccc ccagcccttt atgcggcccc acgagcggaa cggcttcacc 900

gtcctgtgtc ccaagaacat gatcatcaag cccggcaaga tcagccacat catgctggac 960

gtggctttca ccagccacga gcacttcggg ctgctctgcc ccaagtccat ccccggcctc 1020

agcatcagcg gcaatctcct catgaacggc cagcagatct tcctggaggt gcaagccatc 1080

cgggagaccg tcgagctgcg gcagtacgat cccgtggccg ccctgttctt cttcgacatc 1140

gacctgctcc tgcagcgggg cccccaatac agcgagcacc ccaccttcac cagccagtac 1200

cggattcagg gcaagctcga gtaccggcac acctgggacc ggcacgacga gggcgccgcc 1260

cagggagacg acgacgtgtg gaccagcggc tccgacagcg acgaggagct cgtgacaact 1320

gagcggaaga ccccccgggt caccggcggg ggcgccatgg ccagcgccag cacctccgcc 1380

ggccggaaac ggaagagcgc cagcagcgct accgcctgca ccgccggcgt gatgacacgg 1440

gggcggctga aggccgagag caccgtggcc cccgaggagg acaccgacga agactccgac 1500

aacgaaatcc acaaccccgc cgtcttcacc tggcccccct ggcaagccgg catcctcgcc 1560

cggaacctgg tccccatggt cgccaccgtg caggggcaaa atctgaagta ccaggagttc 1620

ttttgggacg caaacgacat ctaccggatc ttcgccgagc tggagggcgt gtggcagccc 1680

gccgcacagc ccaagagacg gcggcaccgg caggacgccc tgcccggccc atgcatcgcc 1740

agcacaccca agaagcatag aggcctgatc cccatcgctg tgggtggtgc cctggcgggg 1800

ctggtcctca tcgtcctcat cgcctacctc gtcggcagga agaggagtca cgcaggctac 1860

cagactatct ag 1872

Claims

1. A fusion protein with immunoregulatory function, comprising a soluble PD-1 derived fragment A capable of binding to CD28 and a soluble CD80 derived fragment B capable of binding to CTLA-4.

2. The immunomodulatory fusion protein of claim 1, wherein the soluble a fragment is further capable of binding to PD-L1/2.

3. The fusion protein with immunoregulatory function of claim 1, wherein the soluble A fragment and the soluble B fragment are linked by a C fragment derived from an immunoglobulin.

4. The fusion protein with immunoregulatory function according to claim 3, wherein the structure of the fusion protein is A-C-B or B-C-A.

5. A nucleic acid comprising a sequence encoding the fusion protein of any one of claims 1-4.

6. The nucleic acid of claim 5, having the sequence shown in SEQ ID NO. 4.

7. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 4 or the nucleic acid according to claim 5 or 6, and an antigen or a nucleic acid encoding said antigen.

8. A cell or derivative thereof, wherein the cell comprises a nucleic acid according to claim 5 or 6 and is capable of producing a fusion protein according to any one of claims 1-4.

9. The cell or derivative according to claim 8, wherein the cell is a PBMC cell and/or a DC cell.

10. A method of increasing the proportion of TNF-a + and/or IFN-r + cells in a population of lymphocytes, comprising the step of allowing a fusion protein according to any one of claims 1 to 4, or a nucleic acid according to claim 5 or 6, to act on the population of lymphocytes.