JP7304421B2 - CHIMERIC ANTIGEN HAVING STRENGTHENED MULTIPLE IMMUNITY FUNCTIONS BY BINDING SPECIFIC TO TARGET CELLS AND USES THEREOF - Google Patents

Description

The present invention relates to chimeric antigens that bind specifically to target cells and have enhanced multiple immune functions, and uses thereof.

"Immunity" is the ability to prevent pathogens such as bacteria and viruses from entering the body from the outside, and to recognize and resist pathogens even if they invade. Immunity includes innate immunity, which is an ability that everyone is born with, and acquired immunity, which is acquired. Immunity in the general sense refers to acquired immunity. Unlike innate immunity, which is known as non-specific immunity, acquired immunity, also called specific immunity, recognizes the type of invading pathogen, and lymphocytes and antibodies are important for the corresponding protective action. play a role. That is, B lymphocytes and T lymphocytes circulating in the body are in charge. They have receptors that recognize specific antigens on their surfaces, and when they recognize antigens, they activate humoral immunity and cell-mediated immunity, respectively.

Antigens and antibodies are involved in acquired immunity, and antigens are substances that are recognized as non-self, such as pathogens, foreign substances, and organ tissues of other species, and cause acquired immune reactions. , Antibodies are substances produced by B lymphocytes to combat specific antigens that have invaded, and bind to specific antigens to extinguish pathogenicity. In addition, since it has a function to memorize the type of pathogen that has once invaded, it can effectively deal with secondary invasion of the same pathogen. Acquired immunity is the action that occurs after pathogens completely invade the body, and is also called "secondary defense" because it works after the action of innate immunity. Acquired immunity includes cell-mediated immunity and humoral immunity. T lymphocytes and B lymphocytes have receptors that recognize specific antigens on their surfaces. Activates sexual immunity. In the former, cytotoxic T lymphocytes that recognize antigens directly attack and remove pathogen-infected cells and cancer cells. T lymphocytes stimulate B lymphocytes to differentiate and proliferate into plasma cells that secrete antibodies and memory cells that store antigens. This generation of memory cells enables a faster and stronger immune response to later re-invading pathogenic antigens than before.

When plasma cells secrete antibodies into the blood, the antibodies bind to antigens and cause an antigen-antibody reaction to remove the antigens. One antibody specifically binds to only one antigen, and this is called the specificity of the antigen-antibody reaction. By attaching to invading microorganisms, antibodies immobilize them and prevent them from entering body cells. In addition, antibody-surrounded antigens undergo a chemical chain reaction with the complement, a series of proteins found in plasma. This complement response attracts macrophages that either cause the degradation of the invading microorganisms or phagocytose the invading microorganisms. Such reactions and bindings between all kinds of antigens and antibodies are called antigen-antibody reactions.

Humoral immunity can be divided into a primary immune response and a secondary immune response in stages. During the primary immune response, when an antigen invades for the first time, lymphocytes differentiate to produce antibodies, and at this time memory cells develop. It is formed. A secondary immune response is a process in which memory cells rapidly differentiate into plasma cells and proliferate to produce a large amount of antibody when the same antigen invades again, inducing a more rapid and stronger immune response.

The adaptive immune system, composed of T and B lymphocytes, has strong anti-cancer potential along with its ability to respond to diverse tumor antigens. In addition, the immune system possesses considerable plasticity and memory components, and these attributes of the adaptive immune system may make immunotherapy a more effective method of treatment for all cancers. However, although endogenous immune responses to cancer have been observed in preclinical models and in patients, such responses are not effective and established cancers are viewed as 'self' and tolerated by the immune system. I do too. This permissive state allows tumors to exploit other mechanisms to positively subvert anti-tumor immunity, but these mechanisms have been linked to dysfunctional T-cell signaling, inhibitory Regulatory cells and endogenous "immune checkpoints" that act to protect normal tissues from collateral damage by tumors and downregulate the strength of the adaptive immune response to avoid immune destruction are exploited.

Long-term conflicts between the human immune system and chronic infections and diseases such as cancer place a high physiological burden on the host and can result in stalemate of the immune response. T-cells, exhausted and inactivated, will give the upper hand to viruses or bacteria and tumors. Based on a large molecular database, researchers have identified 9 types of exhausted T cells (Tex) that are implicated in interactions between chronic infection, autoimmunity, and cancer. When normal T cells are exhausted, they are deficient in their ability to fight bacteria and tumors. Tex expresses inhibitory receptor proteins on its surface that turn off important biochemical pathways, causing changes in the regulation of gene expression, altering metabolism to produce energy to fight infections and tumors, and promoting optimal immune function. Suppress development. Applying such assessments to exhausted T cells in immunotherapy clinical trials can identify patients who are likely to benefit from a particular combination of immunotherapies and respond to infection or cancer. We will be able to understand the underlying mechanisms of certain types of exhausted T cells.

New and highly effective immunotherapies targeting inhibitory receptors expressed by exhausted T cells (Tex), such as PD-1 and CTLA-1, represent a breakthrough therapeutic approach for the treatment of cancer. It should be possible.

In this regard, until recently cancer immunotherapy has focused on the adaptation-transfer of activated effector cells, immunization against relevant antigens, or the provision of non-specific immunostimulants such as cytokines to induce anti-tumor immune responses. Research has been focused on increasing methods. However, in the past decade, intensive efforts to develop specific immune checkpoint pathway inhibitors have begun to offer new immunotherapeutic approaches to treating cancer, which have not been shown to progress. inhibitory PD-1, such as ipilimumab, an antibody (Ab) that binds and inhibits cytotoxic T-lymphocyte antigen-4 (CTLA-4) to treat patients with melanoma Including the development of antibodies that block the pathway.

On the other hand, PD-1 (programmed death-1) is a major immune checkpoint receptor expressed by activated T and B cells and mediates immune inhibition. PD-1 is a 55 kDa type 1 membrane protein that forms part of the Ig superfamily of genes and is well known as a co-inhibitory molecule for T cells belonging to the immunoglobulin class of molecules. PD-1 is a member of the CD28 lineage receptor, which includes receptors expressed on activated B cells, T cells and myeloid cells, namely CD28, CTLA-4, ICOS, PD-1 and BTLA. . Two cell-surface glycoprotein ligands for PD-1, namely PD-L1 and PD-L2, have been identified, which are expressed on many human cancers as well as antigen-presenting cells and are associated with PD-1. It has been shown to downregulate cytokine secretion and T cell activation upon binding [Freeman et al. , 2000]; [Latchman et al. , 2001]. It is known that PD-1 is not expressed in general T cells under normal conditions, but its expression is increased upon activation. In the case of PD-L1, it has been reported that it is expressed to some extent even in T cells in a normal state, and that its expression also increases when activated. PD-L1 is abundant in many human cancers, and the interaction of PD-1 and PD-L1 transmits stimulatory or inhibitory signals to T cells. That is, the interaction between PD-1 and PD-L1 reduces tumor-infiltrating lymphocytes, reduces T-cell receptor-mediated proliferation, and causes immune evasion by cancer cells. Unlike CTLA-4, PD-1 binds to immunoinhibitory PD-L1 (B7-H1) and PD-L2 (B7-DC) ligands expressed by activated T cells by tumor and/or stromal cells It functions primarily in peripheral tissues [Flies et al. , 2011; Topalian et al. , 2012a]. When PD-L1 and PD-L2, which are proteins on the surface of cancer cells, bind to PD-1, which is a protein on the surface of T cells, T cells cannot attack cancer cells.

Intracellular signaling by PD-1 is essential for inducing immune exhaustion of T cells in chronic infection. As an example, PD-1 is known to play an important role in the chronicization process of viral diseases including chronic hepatitis B and C, thus inhibiting the PD-1/PD-L1 interaction. antitumor activity can be induced.

On the other hand, the specific antigen-antibody reaction system that exists in the body is generally understood as independent immunity and is regarded as unrelated to other diseases. I didn't. Rather, since immunity developed against bacteria or viruses that infect the same organ interferes with the formation of antibodies from other infectious agents, chimeric antigens containing antigens from both infectious agents at the same time have been proposed as a way to overcome this. There have been attempts at methods of administration, or attempts to administer a recombinant chimeric antigen of two antigens to improve the immune response to the other antigen. However, it is still extremely rare to find a successful case of effective pharmacological activity by a chimeric antigen.

Therefore, the present inventors have found that, as a method for effective treatment of cancer or infectious diseases, a specific external antigen, in which a specific antigen-antibody reaction system is formed in the body, binds to a target protein associated with the disease. When the peptide domain chimeric antigen is produced by fusion and the produced chimeric antigen is administered, the chimeric antigen specifically binds to cancer or infected cells whose target protein is labeled on the cell membrane, and the chimeric antigen Confirm that other domains of the antigen can effectively treat cancer or infectious diseases by inducing the antigen-antibody reaction system already formed in the body to effectively eliminate target cells. This completes the present invention.

The present invention uses in vitro transcribed and synthesized mRNA, i.e., chimeric antigen fused with IVT mRNA, to promote cellular immunity and humoral immunity by T cells and B cells. We have developed a new therapeutic agent that can maximize immunotherapeutic effects by simultaneously activating both humoral immunity and antibody-dependent cellular cytotoxicity.

Accordingly, an object of the present invention is to provide a chimeric antigen with enhanced multiple immune functions, in which an immune response-inducing region; and a target cell-specific binding-inducing region are fused.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising the chimeric antigen with enhanced multiple immune function of the present invention as an active ingredient.

It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating infectious diseases, comprising the chimeric antigen with enhanced multiple immune function of the present invention as an active ingredient.

It is still another object of the present invention to provide an immunopotentiator composition comprising, as an active ingredient, the chimeric antigen with enhanced multiple immune functions of the present invention.

Still another object of the present invention is to provide a vaccine composition against cancer or infectious disease, comprising the chimeric antigen with enhanced multiple immune function of the present invention as an active ingredient.

In order to achieve the above objects, the present invention provides a chimeric antigen with enhanced multiple immune functions, in which an immune response-inducing region; and a target cell-specific binding-inducing region are fused.

In one embodiment of the present invention, the immune response-inducing region may be a foreign viral antigen, and an antigen-antibody reaction may be formed with the antigen.

In one embodiment of the present invention, the immune response-inducing region is hepatitis B virus antigen, hepatitis C virus antigen, hepatitis A virus antigen, AIDS virus antigen, MERS virus antigen, influenza A virus antigen, human papilloma virus antigen, It may be selected from the group consisting of lymphocytic choriomeningitis virus (LCMV) antigens and herpes simplex virus (HSV) antigens.

In one embodiment of the present invention, the immune response-inducing region is a hepatitis B virus antigen, which is hepatitis B virus core protein (HBc), hepatitis B virus e antigen (HBe), or hepatitis B virus surface proteins (HBs) of

In one embodiment of the present invention, the target cell-specific binding-inducing region is a cell membrane protein capable of specifically binding to target cells, comprising PD-1, PD-L1, PD-L2, CTLA-4 , CD28, ICOS, OX40, OX40L, GITR, GITRL, CD40, CD40L, 4-1BB, 4-1BBL, B7-1, B7-2, B7-H1, B7-H2, B7-DC, CD80, CD160, BTLA , HVEM, DPP-4, NTCP, CD16 and Caveolin-1.

In one embodiment of the present invention, a linker peptide represented by SEQ ID NO: 19 or SEQ ID NO: 21 is used for structural stability of the chimeric antigen between the immune reaction-inducing region and the target cell-specific binding-inducing region. It can be the one that is

In one embodiment of the present invention, the chimeric antigen with enhanced multiple immune functions may consist of the peptide sequence of SEQ ID NO:9 or SEQ ID NO:11.

The present invention also provides a pharmaceutical composition for cancer prevention or treatment, comprising the chimeric antigen with enhanced multiple immune function of the present invention as an active ingredient.

In one embodiment of the present invention, the cancer is lung cancer, stomach cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous melanoma, uterine cancer, ovarian cancer, rectal cancer, colorectal cancer, colon cancer, breast cancer, uterine sarcoma, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, Laryngeal cancer, small bowel cancer, thyroid cancer, parathyroid cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, childhood solid tumor, differentiated lymphoma, bladder It may be selected from the group consisting of cancer, renal cancer, renal cell carcinoma, renal pelvic carcinoma, primary central nervous system lymphoma, spinal cord tumor, brain stem glioma and pituitary adenoma.

The present invention also provides a pharmaceutical composition for preventing or treating infectious diseases, comprising the chimeric antigen with enhanced multiple immune function of the present invention as an active ingredient.

In one embodiment of the present invention, the infectious diseases include hepatitis B virus, hepatitis C virus, hepatitis A virus, AIDS virus, MERS virus, influenza A virus, human papilloma virus, lymphocytic choriomeningitis virus ( LCMV) or herpes simplex virus (HSV) infectious disease.

The present invention also provides an immunopotentiator composition comprising the chimeric antigen with enhanced multiple immune functions of the present invention as an active ingredient.

In one embodiment of the present invention, the multiple immune-enhanced chimeric antigens (i) specifically bind to diseased target cells and (ii) promote recovery of exhausted CD8+ T cells to produce T cells. and (iii) induce antigen-antibody reactions to enhance humoral immune responses and activate cytotoxic effects by natural killer cells (NK cells).

In one embodiment of the present invention, the antigen-antibody reaction induction of (iii) above can enhance a specific antigen-antibody reaction formed in the body of an individual by the immune reaction-inducing region of the chimeric antigen.

The present invention also provides vaccine compositions against cancer or infectious diseases, comprising the chimeric antigen with enhanced multiple immune function of the present invention as an active ingredient.

The present invention relates to a chimeric antigen that binds specifically to target cells and has enhanced multiple immune functions, and uses thereof, wherein multiple immune functions are enhanced by fusing an immune reaction-inducing region and a target cell-specific binding-inducing region. In the case of the resulting chimeric antigen, it can specifically bind to diseased cells, promote the recovery of exhausted CD8+ T cells, induce T cell activity, and induce antigen-antibody reaction to enhance humoral immune response and natural It is possible to effectively prevent and treat cancer, infectious diseases and immune diseases by activating the cytotoxic effect of killer cells (NK cells), suppressing the immune evasion of diseased cells, and strengthening multiple immune functions. can.

It shows the process by which soluble chimeric antigen induces antibodies formed in the body and humoral immunity acts, and also recovers cell-specific CD8+ T cells in a state of immune exhaustion in target cancer or infected cells. Schematic diagram showing that CD8+ T cells specific to a specific external antigen of the chimeric antigen are simultaneously induced to the target cancer or infected cell site and reactivated T cells and antibody-dependent cytotoxicity act simultaneously. is. It shows the blueprints of fusion genes encoding single antigens of HBcAg, HBeAg, and HBsAg and chimeric antigens of PD-1/HBc and PD-1/HBe, which are expected to have various molecular biological structural changes. A recombinant DNA segment for production of IVT mRNA encoding a single antigen or a chimeric antigen consists of an open reading frame, a T7 promoter, a 5′ untranslated region (UTR), a 3′ It was designed to include untranslated regions, etc. 2 shows the results of production of DNA segments encoding HBcAg, HBeAg, HBsAg, PD-1 single antigens and PD-1/HBc, PD-1/HBe chimeric antigens. This figure shows the results of producing respective IVT mRNAs using DNA fragments encoding HBcAg, HBeAg, HBsAg and PD-1 single antigens and PD-1/HBc and PD-1/HBe chimeric antigens as templates. . After inserting IVT mRNA encoding the PD-1/HBc and PD-1/HBe chimeric antigens of the present invention into 293T animal cells, antigens actually produced in the cells were confirmed by an enzyme-linked immunosorbent test. be. After inserting IVT mRNAs encoding PD-1/HBc and PD-1/HBe chimeric antigens into 293T animal cells, the results of confirming the actual production of chimeric antigens in the cells by Western blotting are shown. It was confirmed by coimmunoprecipitation that the intracellularly expressed PD-1/HBc and PD-1/HBe chimeric antigens actually specifically bind to the PD-L1 ligand of cells. . 1 shows the results of measuring the size of particles using a nanoparticle analyzer (Zetasizer) after encapsulating single antigen and chimeric antigen IVT mRNA synthesized in an example of the present invention into LNP. 1 shows the results of measuring the actual encapsulation efficiency of mRNA in particles after encapsulating single antigen and chimeric antigen IVT mRNAs synthesized in an example of the present invention into LNPs. It was confirmed by an enzyme-linked immunosorbent assay that a chimeric antigen protein was actually produced in the cells after the LNP encapsulating IVT mRNA was inserted into 293T animal cells. After injecting LNP encapsulating the chimeric antigen PD-1/HBe IVT mRNA into C57BL/6 mice, it was confirmed by an enzyme-linked immunosorbent assay that the antigen protein was actually produced from the injected IVT mRNA in the body. It is. Prior to treating C57BL/6 mice with chimeric antigens, mice were pre-inoculated with single antigens HBcAg and HBeAg proteins, and enzyme-linked immunosorbent assays confirmed that mice actually produced antibodies against each antigen. It shows what you have done. HBcAg and HBeAg proteins were injected into mice in which antibodies were primarily formed against these proteins. After transplantation of B16F10 mouse melanoma cancer cells, PD-1/HBc and PD-1/ This shows that the growth of cancer tissue was inhibited by the chimeric antigen produced in vivo by injecting LNP encapsulating HBe-encoding IVT mRNA into mice. C57BL/6 mice in which antibodies were primarily formed against HBcAg and HBeAg were transplanted with B16F10, which is a melanoma cancer cell, to induce cancer tissue generation, and then PD-1/HBc and PD. This shows that the endogenously produced chimeric antigen increases the survival rate of mice when LNP encapsulating IVT mRNA encoding -1/HBe is injected into the mice. C57BL/6 mice in which antibodies were primarily formed against HBcAg and HBeAg were transplanted with B16F10, which is a melanoma cancer cell, to induce cancer tissue generation, and then PD-1/HBc and PD. Comparing survival rates before and after injection of LNPs encapsulating IVT mRNA encoding -1/HBe into mice, it was shown that the survival rate of mice was increased after chimeric antigen treatment. C57BL/6 mice in which antibodies were primarily formed against the HBcAg antigen were transplanted with melanoma cancer cells, B16F10, and induced to generate cancer tissue, which then encodes PD-1/HBc. Fig. 4 shows the correlation between the relative amount of antibody against HBcAg antigen and the size of cancer tissue before and after injection of LNP encapsulating IVT mRNA into mice.

The present invention is characterized by providing a recombinant chimeric antigen with enhanced multiple immune functions. Specifically, the present invention provides a recombinant chimeric antigen with multiple immune functions, in which an immune response-inducing region and a target cell-specific binding-inducing region are fused. It is characterized by providing enhanced chimeric antigens.

In particular, the chimeric antigen with enhanced multiple immune function provided by the present invention specifically binds to a target protein associated with disease in a specific external antigen region (domain) in which a specific antigen-antibody reaction system is formed in the body. When the chimeric antigen of the present invention is administered to an individual, the chimeric antigen is a specific disease cell in which the target protein is labeled on the cell membrane, such as cancer cells or It specifically binds to infected cells, and the other domain of the chimeric antigen, that is, the immune response-inducing region (specific external antigen region), can induce the antigen-antibody reaction system already formed in the body to target the disease. Designed to efficiently remove cells.

Therefore, when the chimeric antigen of the present invention is used, the target disease can be treated very efficiently by reactivating the immune system (immune reaction) by the specific antigen-antibody system already formed in the individual. made it possible.

Considering the component of the chimeric antigen with enhanced multiple immune functions of the present invention, it is formed as a fusion protein in which an immune reaction-inducing region and a target cell-specific binding-inducing region are fused together.

The immune response-inducing region is a specific external antigen region (domain), which may be a foreign viral antigen, and the immune response-inducing region stably forms an antigen-antibody reaction in an individual to whom the chimeric antigen is administered. be able to.

The immune response-inducing region that can be included in the chimeric antigen of the present invention is a foreign antigen, which is a protein, glycoprotein, coat protein, core protein, or function of a virus, bacterium, or organism related to various infectious diseases. preferably, but not limited to, hepatitis B virus antigen, hepatitis C virus antigen, hepatitis A virus antigen, AIDS virus antigen, MERS virus antigen, influenza A virus antigen, It can be selected from the group consisting of human papillomavirus antigen, lymphocytic choriomeningitis virus (LCMV) antigen and herpes simplex virus (HSV) antigen, more preferably hepatitis B virus antigen, type B It may be hepatitis virus core protein (HBc) or hepatitis B virus e antigen (HBe) or hepatitis B virus surface proteins (HBs).

The hepatitis B virus-derived antigen core protein antigen (HBcAg) consists of the peptide sequence of SEQ ID NO: 1, and the hepatitis B virus e antigen (HBeAg) consists of the peptide sequence of SEQ ID NO: 3. and the hepatitis B virus surface protein antigen (HBsAg) may consist of the peptide sequence of SEQ ID NO:5.

Further, the polynucleotide encoding the peptide of SEQ ID NO: 1 is shown in SEQ ID NO: 2, the polynucleotide encoding the peptide of SEQ ID NO: 3 is shown in SEQ ID NO: 4, and the polynucleotide encoding the peptide of SEQ ID NO: 5 is SEQ ID NO: 6.

In addition, the target cell-specific binding-inducing region is a protein that is mainly involved in normal life activities in the body, and is a water-soluble protein or cell membrane protein that can physically bind to a receptor or ligand on the cell membrane. could be. The target cell-specific binding-inducing region of the present invention includes, but is not limited to, PD-1, PD-L1, PD-L2, CTLA-4, CD28, ICOS, OX40, OX40L, GITR, and GITRL. , CD40, CD40L, 4-1BB, 4-1BBL, B7-1, B7-2, B7-H1, B7-H2, B7-DC, CD80, CD160, BTLA, HVEM, DPP-4, NTCP, CD16 and Caveolin can be selected from the group consisting of -1;

In one embodiment of the present invention, PD-1, which is a cell membrane protein that can specifically bind to target cells, is used as the target cell-specific binding-inducing region, and the cell membrane protein is a core protein in the immune checkpoint system. PD-1 is attached to the cell membrane and is known as a form that is well secreted outside the cell (Cellular Immunology, 2005, 235: 109). Therefore, the present invention converts PD-1 into a protein that tightly binds to its receptor, PD-L1, and thus uses a recombinant PD-1 protein that is capable of extracellular secretion and has high binding avidity to its receptor. , the peptide sequence of the PD-1 protein used in the present invention is shown in SEQ ID NO:7, and the polynucleotide sequence encoding the peptide of SEQ ID NO:7 is shown in SEQ ID NO:8.

In addition, the chimeric antigen of the present invention has a linker peptide consisting of a specific sequence between the immune reaction-inducing region and the target cell-specific binding-inducing region to maximize the structural stability and immune reaction of the chimeric antigen. Designed to be articulated. It was confirmed that there is a difference in the structural stability of chimeric antigens depending on the sequence of the linker peptide, and based on this, effective linker peptides were selected for enhancing the stability and immune response of chimeric antigens. The linker peptide 1 usable in the present invention consists of 46 peptides represented by SEQ ID NO: 19, and the polynucleotide sequence encoding the linker peptide 1 is shown in SEQ ID NO: 20. The linker peptide 2 usable in the present invention consists of 24 peptides represented by SEQ ID NO:21, and the polynucleotide sequence encoding the linker peptide 2 is shown in SEQ ID NO:22.

In one embodiment of the present invention, soluble chimeric antigens PD-1/HBc and PD-1/HBe were prepared as chimeric antigens with enhanced multiple immune functions, respectively, which are used as immune reaction-inducing domains. Hepatitis B virus core protein (HBcAg) and hepatitis B virus e-antigen protein (HBeAg) are used, respectively, and secreted extracellularly as a target cell-specific binding-inducing region that has an affinity for PD-L1 It uses the water-soluble PD-1 that is commonly used. In one embodiment of the present invention, the PD-1 and HBc domains are linked by a linker peptide 1 of SEQ ID NO: 19, and the PD-1 and HBe domains are linked by a linker peptide of SEQ ID NO: 21. 2 were connected to each other.

In addition, HBcAg and HBeAg single antigens, PD-1/HBc and PD-1/HBe chimeric antigens are designed to contain mostly amino acid sequences that induce antibody formation, and each antigen protein is produced intracellularly. A signal peptide was added to the amino terminus of each protein to facilitate its extracellular secretion. Synthesis of the signal peptide was done by methods well known to those skilled in the art (PLoS One.2016.19:11; Mol Ther.2005, 11(3):435; Journal of Biotechnology, 2007, 128(4): 705; Trends in Cell and Molecular Biology, Improving mammalian cell factories: The selection of signal peptides has a major impact on recombinant protein synthes is and secretion in mammalian cells). Various types of signal peptide sequences were linked to the ends, and the produced antigens were tested for good extracellular secretion.

In the present invention, 21 peptides represented by SEQ ID NO: 23 (signal peptide 1) or SEQ ID NO: 25 were used as the signal peptide in order to enhance the extracellular secretion of the chimeric antigen of the present invention by the above experiments. Using a signal peptide (signal peptide 2) consisting of 17 peptides represented by, wherein the polynucleotide sequence encoding the polypeptide of SEQ ID NO: 23 is shown in SEQ ID NO: 24, and the polypeptide of SEQ ID NO: 25 is The encoding polynucleotide sequence is shown in SEQ ID NO:26.

Thus, the chimeric antigen with enhanced multiple immune function according to the present invention has a form in which the immune reaction-inducing region; and the target cell-specific binding-inducing region; PD-1/HBcAg and PD-1/HBeAg chimeric antigens were prepared as chimeric antigens with enhanced immune function, respectively.

The peptide sequence for the PD-1/HBcAg chimeric antigen of the present invention produced in the present invention is shown in SEQ ID NO:9, and the polynucleotide sequence encoding it is shown in SEQ ID NO:10.

The polypeptide of SEQ ID NO: 9 representing the PD-1/HBcAg chimeric antigen of the present invention contains the sequence of signal peptide 1 for extracellular secretion, wherein the sequence of signal peptide 1 is from amino acids 1 to 21 of SEQ ID NO: 9. It consists of a total of 21 amino acid sequences up to the first.

In addition, the polypeptide showing the PD-1/HBcAg chimeric antigen of SEQ ID NO: 9 contains linker peptide 1, and the linker peptide 1 is a total of 46 amino acids from the 232nd amino acid to the 277th amino acid of SEQ ID NO: 9. is an amino acid.

Further, the polynucleotide sequence encoding the polypeptide against the PD-1/HBcAg chimeric antigen of SEQ ID NO: 9 is shown in SEQ ID NO: 10, and the polynucleotide sequence of SEQ ID NO: 10 encoding the signal peptide 1 is SEQ ID NO: 10. It consists of a total of 63 base sequences from the 1st base sequence to the 63rd base sequence, and the sequence of the polynucleotide encoding the linker peptide 1 in SEQ ID NO: 10 is from the 694th base sequence to the 831st base sequence of SEQ ID NO: 10. Become.

Also, the peptide sequence of the PD-1/HBeAg chimeric antigen of the present invention is shown in SEQ ID NO:11, and the polynucleotide sequence encoding it is shown in SEQ ID NO:12.

The polypeptide of SEQ ID NO: 11 representing the PD-1/HBeAg chimeric antigen of the present invention comprises the sequence of signal peptide 2 for extracellular secretion, said signal peptide 2 sequence being amino acids 1 to 17 of SEQ ID NO: 11. It consists of a total of 17 amino acid sequences up to the first.

In addition, the polypeptide showing the PD-1/HBeAg chimeric antigen of SEQ ID NO: 11 contains linker peptide 2, and the linker peptide 2 consists of a total of 24 amino acids from the 228th amino acid to the 251st amino acid of SEQ ID NO: 11. Consists of an amino acid sequence.

Further, the polynucleotide sequence encoding the polypeptide against the PD-1/HBeAg chimeric antigen of SEQ ID NO: 11 is shown in SEQ ID NO: 12, and the polynucleotide sequence of SEQ ID NO: 12 encoding the signal peptide 2 is SEQ ID NO: 12. Consists of a total of 51 base sequences from the 1st base sequence to the 51st base sequence, and the sequence of the polynucleotide encoding the linker peptide 2 in SEQ ID NO: 12 is a total of 72 base sequences from the 682nd base sequence to the 753rd base sequence of SEQ ID NO: 12 consists of a base sequence of

Meanwhile, the chimeric antigen with enhanced multiple immune functions according to the present invention can be used for the prevention or treatment of cancer or infectious diseases by activating immune responses.

Since the chimeric antigen of the present invention contains a target cell-specific binding-inducing region, it can specifically bind to diseased cells, which are target cells. Multiple immune functions by promoting the recovery of T cells and inducing the activity of T cells and inducing antigen-antibody reactions to promote humoral immune responses and simultaneously activate cytotoxic effects by natural killer cells (NK cells) It is characterized by being able to strengthen

In particular, the enhancement of multiple immune functions by the immune response-inducing region refers to the subject of the present invention containing the above-mentioned foreign antigen for individuals exposed to foreign antigens and having a specific and stable antigen-antibody reaction system against the above-mentioned foreign antigens. It refers to reactivation of the antigen-antibody reaction by treating the chimeric antigen, thus multiple activation of humoral immunity and cell-mediated immunity at the same time.

Therefore, the present invention can provide a pharmaceutical composition for cancer prevention or treatment, comprising the chimeric antigen with enhanced multiple immune function of the present invention as an active ingredient.

The above types of cancer include, but are not limited to, lung cancer, stomach cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous melanoma, uterine cancer, and ovarian cancer. Cancer, rectal cancer, colorectal cancer, colon cancer, breast cancer, uterine sarcoma, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, esophagus Cancer, laryngeal cancer, small bowel cancer, thyroid cancer, parathyroid cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, childhood solid tumor, differentiated lymphoma , bladder cancer, kidney cancer, renal cell carcinoma, renal pelvic carcinoma, primary central nervous system lymphoma, spinal cord tumor, brain stem glioma or pituitary adenoma.

In addition, the present invention can provide a pharmaceutical composition for preventing or treating infectious diseases, which contains the chimeric antigen with enhanced multiple immune function of the present invention as an active ingredient.

In the present invention, the term "infectious disease" means a disease in which various infectious agents or constituents, including foreign viruses, bacteria, protozoa, etc., cause, mediate, or otherwise contribute to pathological conditions in mammals. , The types of the above-mentioned infectious diseases include, but are not limited to, hepatitis B virus, hepatitis C virus, hepatitis A virus, AIDS virus, MERS virus, influenza A virus, human papilloma virus, lymphocytic choriomedullary It may be meningitis virus (LCMV) or herpes simplex virus (HSV) infectious disease.

In addition, the present invention can provide an immunoenhancer composition comprising, as an active ingredient, a chimeric antigen with enhanced multiple immune functions, and such an immunoenhancer composition can be used to treat immune disorders induced by immune dysfunction. It can be used as a pharmaceutical composition for prevention or treatment of diseases.

In the present invention, the term "immune disease" means a disease in which components of the mammalian immune system cause, mediate, or otherwise contribute to pathological conditions in mammals, and stimulation or disruption of the immune response is associated with the disease. can include all diseases that have a compensatory effect on the progression of

In one embodiment of the present invention, a PD-1/HBc chimeric antigen fused with HBcAg and PD-1 and a PD-1/HBe chimeric antigen fused with HBeAg and PD-1 were prepared by in vitro transcription of each mRNA ( After producing IVT mRNA; in vitro transcription mRNA), the produced PD-1/HBc mRNA and PD-1/HBe mRNA are introduced into diseased cells, respectively, and then the chimeric antigen selectively binds to the PD-L1 ligand. As a result of confirming the binding, it was confirmed that both the PD-1/HBc and PD-1/HBe chimeric antigens specifically bound to the PD-L1 ligand.

In addition, according to the present invention, mice are pre-inoculated with HBcAg and HBeAg proteins for generation of HBcAg and HBeAg antibodies capable of binding to the chimeric antigens PD-1/HBc and PD-1/HBe, respectively. As a result of analyzing the presence or absence of antibody production, it was confirmed that specific antibodies against HBcAg and HBeAg were produced.

Based on these results, the present inventors believe that when the chimeric antigen of the present invention is administered to an individual, the chimeric antigen of the present invention can bind to disease cell-specific target sites, and antibodies present in the individual By binding to , it was confirmed that humoral immune activity can be promoted, and as a result, diseased cells can be removed by the immunopotentiating action to effectively prevent or treat the target disease.

Furthermore, in animal experiments, in order to confirm the anticancer activity using the chimeric antigen of the present invention, PD-L1 was labeled on the cell membrane of mice in which HBsAg and HBcAg were preinoculated and primary antibodies against them were formed. After the mouse cancer cell B16F10 cells were transplanted to generate cancer tissue, the chimeric antigen of the present invention was injected. On the other hand, in the group injected with PD-1/HBc mRNA and PD-1/HBe mRNA, which are the chimeric antigens of the present invention, the growth of cancer tissue was effectively inhibited. It was found that the survival rate of mice was greatly increased.

From the above results, it was found that the chimeric antigen with enhanced multiple immune functions of the present invention has the effect of preventing and treating cancer or infectious diseases. Inducing recovery from T cell exhaustion by cancer or infected cells by blocking the signaling pathways of PD-1 that mediate and the binding of PD-1 to PD-L1, and enhancing the ability of T cells to secrete cytokines. It has been found that it can be activated to enhance the immune response.

That is, it was found that the activity of disease cell target-specific CD8+ T cells that were already formed in the body but disabled by T cell exhaustion could be promoted. In addition, natural killer (NK) cells, macrophages, monocytes, which are humoral immune cells that are bound and activated by antibodies specific for foreign antigens, have Antibody-dependent cellular cytotoxicity activity prevents or treats diseases induced by the interaction of PD-1 and PD-L1 more rapidly and strongly than general single antibody therapeutic agents be able to. Diseases induced by the interaction of PD-1 and PD-L1 can be cancers or infectious diseases as described above.

The pharmaceutical composition of the present invention may contain a pharmaceutically effective amount of the chimeric antigen of the present invention, or may contain one or more pharmaceutically acceptable carriers, excipients or diluents. A pharmaceutically effective amount as described above refers to an amount sufficient to prevent, ameliorate and treat symptoms of cancer or infectious diseases.

The chimeric antigen according to the present invention can be contained in a mass of 1 g at 0.1 pg per kg of the patient's body weight, and the pharmaceutically effective amount can vary depending on the degree of disease symptoms, the patient's age, weight, health condition, sex, , route of administration, duration of treatment, and the like.

In addition, the above-mentioned "pharmaceutically acceptable" means a composition that is physiologically acceptable and does not usually cause gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions when administered to humans. say about Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose. , polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, fillers, anti-agglomeration agents, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.

The composition of the present invention can be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to mammals. . Dosage forms can be in the form of powders, granules, formulations, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.

In addition, the preventive or therapeutic composition for cancer or infectious diseases according to the present invention can be administered through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of the active ingredient is It can be appropriately selected according to various factors such as route of administration, patient age, sex, body weight and severity of patient. It can be administered in parallel with known compounds that are effective in preventing, ameliorating or treating symptoms of infectious diseases.

Furthermore, the present invention can provide a method for treating cancer or an infectious disease comprising administering the chimeric antigen of the present invention to a non-human individual induced with cancer or an infectious disease.

Suitable routes of administration also include, for example, oral, nasal, transmucosal, or intraseptal enteral administration, direct intraventricular, intraperitoneal, or intraocular injection, as well as intramuscular, subcutaneous, intravenous, bone marrow injection. Parenteral delivery can be included.

Furthermore, the present invention can provide a vaccine composition against cancer or infectious diseases, which contains as an active ingredient the chimeric antigen of the present invention with enhanced multiple immune functions.

Although the vaccine composition of the present invention can be administered alone, it may preferably contain an adjuvant. An adjuvant is a substance that non-specifically promotes an immune response to an antigen during the initial activation process of immune cells, and is not an immunogen to the host, but a preparation that enhances immunity by increasing the activity of cells of the immune system. , molecules, etc. (Annu. Rev. Immunol, 1986, 4:369). Adjuvants that can be administered with the vaccine composition of the present invention to enhance the immune response include any of a variety of adjuvants, typical adjuvants include Freund's adjuvant, aluminum compounds, Muramyl dipeptide, lipopolysaccharide (LPS), monophosphoryl lipid A, quill A, etc. are known. The adjuvants can be co-administered with the vaccine composition or administered sequentially at timed intervals.

In addition, the vaccine composition of the present invention may additionally contain solvents, excipients, and the like. The solvent includes physiological saline, distilled water, and the like, and the excipient includes, but is not limited to, aluminum phosphate, aluminum hydroxide, aluminum potassium sulfate, and the like. can further contain substances that are commonly used in vaccine production.

The vaccine composition of the present invention can be produced by a method commonly used in the technical field to which the present invention belongs. The vaccine composition of the present invention can be prepared as an oral or parenteral preparation, preferably as an injection solution that is a parenteral preparation, and administered intradermally, intramuscularly, intraperitoneally, intravenously, or subcutaneously. Administration can be by intranasal, intranasal or epidural routes.

A vaccine composition of the invention can be administered to an individual in an immunologically effective amount. The above-mentioned "immunologically effective amount" means an amount sufficient to exhibit a disease-preventing effect and an amount that does not cause side effects or serious or excessive immune reactions, and the exact dosage concentration is Varies depending on the specific immunogen to be administered, and is readily available to those skilled in the art, depending on factors well known in the medical arts, such as the age, weight, health, sex, individual drug sensitivity, route of administration, and method of administration of the subject to be vaccinated. and can be administered once or several times.

Hereinafter, the present invention will be described in more detail through examples. These examples are intended to describe the present invention more specifically, and the scope of the present invention is not limited to these examples.

<Example 1>
[Production of fusion gene encoding HBc, HBe, HBs, PD-1/HBc or PD-1/HBe chimeric antigen]
Proteins of hepatitis B core antigen (HBcAg), hepatitis B e antigen (HBeAg), hepatitis B envelope antigen (HBsAg), PD-1, soluble chimeric antigens PD-1/HBc and PD-1/HBe FIG. 2 shows a schematic diagram of a DNA fragment for producing IVT mRNA (in vitro transcribed mRNA) synthesized by in vitro transcription of each encoding gene. As shown in FIG. 2, the DNA fragment for producing the IVT mRNA consists of an open reading frame, a T7 promoter nucleotide sequence, a 5′ untranslated nucleotide sequence (5′UTR), 3 The 'untranslated base sequence (3'UTR) was prepared by referring to the mRNA Express mRNA Synthesis kit (System Biosciences, USA). The DNA segment was designed so that the open reading frame, that is, the target gene was linked in the form of a cassette and inserted. At this time, the order of the open reading frames encoding the immune reaction-inducing region and the target cell-specific binding-inducing region of the chimeric antigen is not fixed, and the positions of the two induction regions may be changed. That is, the immune reaction-inducing region and the target cell-specific binding-inducing region of the chimeric antigen are located in the 'A gene' and the 'B gene' connected by a linker in FIG.

In addition, hepatitis B core antigen (HBcAg), hepatitis B e antigen (HBeAg), hepatitis B envelope antigen (HBsAg), PD-1, soluble chimeric antigen PD-1/HBcAg and PD-1/HBeAg SEQ ID NOs: 13 (HBcAg), 14 (HBeAg), 15 (HBsAg), 16 (PD-1), 17 (PD-1/HBcAg) and 18 for each IVT mRNA transcribed in vitro. (PD-1/HBeAg) respectively.

In addition, the nucleotide sequence of SEQ ID NO: 17 is the nucleotide sequence of a DNA template encoding PD-1/HBcAg IVT mRNA, and the nucleotide sequence includes not only the PD-1/HBcAg coding sequence but also the T7 promoter, 5'UTR and 3′UTR base sequences.

The base sequence of SEQ ID NO: 18 is also the base sequence of the DNA template encoding PD-1/HBeAg IVT mRNA, and the above base sequence includes not only the PD-1/HBeAg coding sequence but also the T7 promoter, 5'UTR and 3' It also contains the base sequence of UTR.

Also, after transcription is complete, a series of adjacent "polyadenylic acid (poly(A)) tails" are added to the 3' end of the RNA molecule by poly A polymerase, a process known to those skilled in the art. A well-known method was devised to insert a minimum of 50 adenine sequence poly(A) sequences at the time of design. The above-designed DNA fragment was synthesized by requesting IDT in the United States, and 10 ng of vector DNA containing each antigen gene produced by IDT was used as a template, and the polymerase chain reaction (PCR) of SEQ ID NOS: 27 and 28 below was performed. Gene DNA amplification reaction was performed using the primers for . The PCR reaction products were purified by elution methods well known to those skilled in the art and then used as templates for in vitro transcription reactions (Figure 3).

SEQ ID NO: 27 primer base sequence (primer 1):
5′-AGATCTTAATACGACTCACTATAGGGGAAATAAGA-3′

SEQ ID NO: 28 primer base sequence (primer 2):
5′-TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTCTTTTTTT TTTTTTTTTTTT TTTTTTTTTT TTTTTAATAT TCTTCCTACT CAGGCTTTAT TCAAAGACC-3′

<Example 2>
[IVT mRNA synthesis from a DNA segment encoding a single antigen or chimeric antigen]
In vitro transcription (IVT) was performed using the template DNA prepared in Example 1 to synthesize artificial mRNA. It was carried out using an mRNA kit with tailing. Specifically, 1 μg of each purified DNA fragment containing the nucleotide sequences of single or chimeric antigen genes of HBcAg, HBeAg, HBsAg, PD-1, PD-1/HBc and PD-1/HBe, 10 μl 2× ARCA /NTP Mix, 2 μl T7 RNA Polymerase Mix, and distilled water was added to the remainder to make a final 20 μl reaction mixture, which was reacted at 37° C. for 60 minutes. After the reaction was completed, 2 μl of DNase enzyme was added and treated at 37° C. for 15 minutes to remove any residual DNA. The IVT mRNA obtained from the reaction was purified using an RNA purification kit well known to those skilled in the art, and mRNA production was confirmed by 1.2% agarose electrophoresis using 0.5x TAE buffer solution ( Figure 4).

The polypeptide sequence of the single antigen HBcAg, which is a protein expressed from the mRNA of the single antigen produced by the above process, is shown in SEQ ID NO: 1, the polypeptide sequence of HBeAg is shown in SEQ ID NO: 3, and the polypeptide sequence of HBsAg is It is shown in SEQ ID NO:5 and the polypeptide sequence of PD-1 is shown in SEQ ID NO:7. Moreover, the polypeptide (amino acid) sequence of PD-1/HBc, which is a chimeric antigen protein expressed from the chimeric antigen mRNA (SEQ ID NO: 17), is shown in SEQ ID NO: 9, and another chimeric antigen mRNA (SEQ ID NO: 18). The polypeptide (amino acid) sequence of PD-1/HBe, which is a chimeric antigen protein expressed from , is shown in SEQ ID NO:11.

<Example 3>
[Confirmation of expression of IVT mRNA encoding chimeric antigen in human 293T cells]
The IVT mRNA purified in Example 2 above was transfected into human cell line HEK293T (ATCC, CRL-3216). The HEK293T cell line is human embryonic kidney-derived cancerous kidney cells, and the cells were cultured in a T75 flask with 10% FBS (Fetus Bovine Serum: GIBCO, #16000-028), 1 %ABAM (GIBCO BRL, #15240-O13) in MEM medium (GIBCO, #11095-080) for 3 days, followed by washing with 1x PBS buffer solution, followed by trypsinization to remove the bottom of the container. peeled off from Thereafter, the separated cells were centrifuged, the supernatant was discarded, and the cells were redispersed in 1x PBS buffer solution, and then redispersed at 5x10^5 cells/dish. After 24 hours, after confirming that the cells were well attached to the surface of the culture vessel, the chimeric antigen was transfected using Lipofectamine Messenger Max Transfection Reagent (Invitrogen, #LMRNA003), an mRNA-specific transfection reagent, according to the method provided by those skilled in the art. The cells were transfected with encoding IVT mRNAs (PD-1/Hbc and PD-1/Hbe). Transfected cells were dispersed in 60 mm culture vessels and cultured in a 5% _CO2 cell incubator for 2 days.

After culturing, a cell culture medium was obtained 24 hours later, and after 48 hours, the attached cells and the cell culture medium were collected in a 1.5 ml tube. Enzyme-linked immunosorbent assay (ELISA) was performed using PD-1 ELISA Kit (Arigo Biolaboratories) in order to confirm whether single or chimeric antigens were produced and secreted in the obtained cells and cell culture medium. , #ARG81342).

As a result, as shown in FIG. 5, after IVT-mRNA produced by the method of the present invention was transfected into HEK293T cell line, PD-1 protein was detected both in the culture medium and intracellularly. As a result, the present inventors were able to confirm that all the chimeric antigens of interest were sufficiently secreted extracellularly. In addition, protein was extracted from the obtained cells, and after SDS-PAGE electrophoresis, Western blotting was performed using a PD-1 antibody (R&D System, AF1021). As a result, as shown in FIG. It was confirmed that a chimeric antigen was produced.

The cell extract obtained at this time was also used as a sample for immuno-precipitation, which can show PD-L1 protein binding present in the cell membrane.

<Example 4>
[Confirmation of presence or absence of binding between PD-L1 protein present in cell membrane and chimeric antigen expressed in 293T using immunoprecipitation method]
Using immunoprecipitation (Immunoprecipitation: IP), single antigen (PD-1) and chimeric antigen (PD-1/HBc or HBsAg/PD-1) produced by PD-L1 present in the cell membrane and the present invention The presence or absence of bonding with For this purpose, PD-L1/PD-1/anti-PD-L1 antibody, PD-L1/PD-1/HBc/anti-PD-L1 antibody, PD-L1/HBsAg by adding an antibody against PD-L1 A complex precipitate of /PD-1/anti-PD-L1 antibody is obtained, and after electrophoresis of this precipitate, PD-L1 and PD-1, PD-1/HBc or HBsAg/PD-1 are analyzed by Western blotting. interactions were analyzed by coimmunoprecipitation (co-IP).

Specifically, IVT mRNA encoding the PD-1, PD-1/HBc or PD-1/HBe gene is applied to cancer cells labeled with PD-L1 on the cell membrane by electroporation or Lipofectamine Messenger Max Transfection Reagent (Invitrogen, #LMRNA003) mRNA was transfected using a transfection reagent dedicated to mRNA, and after 2 days the cells were harvested and then lysed in RIPA buffer solution. After removing cell debris by centrifugation, transfer the supernatant to another 1.5 ml test tube, add an appropriate amount of anti-PD-L1 antibody or specific labeled protein antibody, mix, and incubate for 1 hour. An antigen-antibody complex (immune complex) was formed. The complexes were mixed again with immobilized Protein A or Protein G agarose gel and incubated with rotation for 2 hours to adhere to the gel. After removing by washing twice, the precipitated complex was subjected to SDS-PAGE electrophoresis, followed by Western blotting using each antigen-specific antibody.

As a result, as shown in FIG. 7, an antibody against PD-L1 was added to cell extracts of HEK293T cell lines transfected with IVT-mRNA of a single or chimeric antigen according to the present invention, and analyzed. It could be confirmed that PD-L1 interacts with PD-1, PD-1/HBc or PD-1/HBe.

<Example 5>
[Fabrication of Lipid Nanoparticles (LNPs) to Increase the Transduction Efficiency of IVT mRNA Encoding Chimeric Antigens]
Lipid nanoparticles (LNPs) composed of various lipids were fabricated to increase the stability of mRNA and the efficiency of delivery to cells (Cell, 2017, 168: 1). First, four kinds of lipids, SS-OP, DOPC, Cholesterol, and PEG-lipid, were dissolved in 100% ethanol and mixed at a molar ratio of 50:10:38.5:1.5, respectively. IVT mRNA encoding the above-mentioned single antigen and chimeric antigen of the present invention contained in a 50 mM citrate buffer solution (pH 3.0) and a lipid mixture were each prepared into LNP using a Microfluidic device of Precision Nanosystems of Canada. bottom. The fabricated LNP was transferred to a dialysis membrane cassette, dialyzed against 1x PBS buffer solution for 18 hours, and adjusted to an appropriate concentration using a centrifugal filter (Merk Millipore, Germany). After concentration matching, it was filtered through a 0.22 μm filter and stored refrigerated at 4° C. until used for intracellular loading or mouse injection.

<Example 6>
[Measurement of size and mRNA encapsulation efficiency of LNP encapsulating IVT mRNA encoding chimeric antigen]
The particle size and polydispersity index (PDI) of each LNP prepared in Example 5 were measured using a Zetasizer nanoparticle analyzer manufactured by Malvern, UK.

As a result, as shown in FIG. 8, the IVT mRNA-encapsulated LNP exhibited a particle size of 90-150 nm, which was slightly larger than that of the naked LNP without IVT mRNA. In addition, the mRNA encapsulation efficiency of IVT mRNA encapsulated in each LNP production process was measured by Ribogreen assay using the Quanti-iT Ribogreen kit of Thermo Fisher Scientific, USA. The encapsulation efficiencies of each IVT mRNA were all high in the range of about 86-94% during the production of LNPs encapsulating the single and chimeric antigens of the present invention.

<Example 7>
[Confirmation of antigen expression efficiency at the cellular level of LNP encapsulating IVT mRNA encoding chimeric antigen]
In order to know whether the LNPs produced in Example 5 above can actually produce proteins in cells, LNPs encapsulating the single antigens HBcAg and HBeAg IVT mRNA produced in the present invention were transfected into human cell lines. and HeLa (ATCC, CCL-2) were transfected, respectively. HeLa is a cancer cell derived from cervical cancer, and in order to culture it, 10% FBS (Fetus Bovine Serum: GIBCO, #11095-080), 1% ABAM (GIBCO BRL, #15240-013) was added to MEM medium (GIBCO, #11095-080) for 3 days, washed with 1x PBS buffer solution, trypsinized and detached from the bottom of the vessel, and the detached cells were separated. After seeding in a 24-well culture vessel at a concentration of 5×10 5 cells/well, the cells were cultured in a cell culture vessel with a 5% CO ₂ concentration for 24 hours. The next day, the LNP of the present invention and apolipoprotein (Apolipoprotein E4, ApoE4) were mixed, cultured at 37° C. for 10 minutes, and then transfected into cells. After obtaining the culture solution of the transfected cells, the amount of antigen secreted by the cells was confirmed by Enzyme Linked Immunosorbent Assay (ELISA).

As a result, as shown in FIG. 10, LNPs encapsulating PD-1/HBc and PD-1/HBeIVT mRNA, respectively, were introduced into HeLa cells, and each antigen was efficiently produced. We were able to.

<Example 8>
[Confirmation of antigen expression in mice of LNP encapsulating IVT mRNA encoding chimeric antigen]
In addition, the present inventors decided to confirm the results of antigen expression confirmed at the cell level in Example 7 also through experimental animals. For this purpose, the LNP produced in Example 5 above was injected into the left tibialis anterior muscle of 6-week-old C56BL/6 female mice to confirm the antigen expression efficiency. was analyzed for its efficiency as a carrier of IVT mRNA to For this purpose, mice were inoculated with a total of 80 μl of LNP encapsulating 2 μg of IVT mRNA of the present invention with 1× PBS buffer solution. Whole blood was collected from the heart on days 1, 3, and 6 after inoculation, and the presence or absence of production of each antigen protein was confirmed by enzyme-linked immunosorbent assay.

As a result, as shown in FIG. 11, it was found that the PD-1/HBe chimeric antigen was produced from day 1 and continued until day 6. It was found that sufficient monoantibodies and chimeric antibodies were indeed produced in mice when injected into mice.

<Example 9>
[Confirmation of primary antibody formation against these antigens in mice pre-inoculated with HBcAg and HBeAg proteins]
HBcAg (MyBioSource, USA, #MBS355629), HBeAg protein (MyBioSource, USA, #MBS355623) was adjuvanted (Sigma, USA, #F5881) to 6-week old C57BL/6 female mice on day 1. 2 μg per mouse was injected into the left tibialis anterior muscle in 3 aliquots of 30 μl. Eleven days after the first inoculation, each mouse was injected with the same amount of antigen protein in the same manner. Sera were collected from tail arteries of mice immunized 7 days, 21 days, 35 days and 44 days after the first inoculation, and the presence or absence of antibody production was examined by enzyme-linked immunosorbent assay.

As a result, as shown in FIG. 12, it was found that each mouse inoculated with the antigen formed respective antibodies against each HBcAg and HBeAg.

<Example 10>
[Confirmation of the anticancer activity of the chimeric antigen (PD-1/HBc or PD-1/HBe) of the present invention in cancer cells using a mouse Xenograft model]
About 1×10 ⁶ melanoma cancer cells B16F10 were injected subcutaneously into the lateral surface of C57BL/6 mice in which antibodies against each antigen had been formed by inoculation with HBcAg and HBeAg antigen proteins in Example 9 on day 21 after antigen pre-inoculation. Ten days after administration of the cancer cells, IVT mRNA-LNP encoding the chimeric antigens PD-1/HBc and PD-1/HBe of the present invention were injected (main inoculation) into the left tibialis anterior muscle of the mice. After injection of cancer cells, changes in body weight of the mice and generation of cancer tissue were observed every 3 days, and the size of the cancer tissue was measured. At this time, the number of mice used in the above experiments is 6-7 for each test group.

As a result, as shown in FIG. 13, the growth of cancer tissue in mice pre-inoculated with the HBcAg antigen was higher than that in the control group when the chimeric antigen PD-1/HBc IVT mRNA-LNP of the present invention was inoculated. Similarly, the growth of cancer tissue in mice pre-inoculated with the HBeAg antigen was effectively inhibited when the chimeric antigen PD-1/HBe IVT mRNA-LNP of the present invention was inoculated. found to be suppressed.

In addition, as shown in FIG. 14, the experimental group inoculated with the chimeric antigens PD-1/HBc and PD-1/HBe IVT mRNA-LNP of the present invention had a mortality rate of 44% compared to the control group. It was found that the survival rate increased by about 2 times with a significant decrease during the 1-day test period. It can be seen that such a reduction in mouse mortality is due to the injection of the chimeric antigen of the invention. As a result of comparing the survival rate of mice before and after inoculation with the chimeric antigen of the present invention, it was confirmed that the survival rate was greatly increased only in the experimental group inoculated with the chimeric antigen of the present invention (Fig. 15).

In addition, FIG. 16 shows that C57BL/6 mice in which antibodies were primarily formed against the HBcAg antigen were transplanted with melanoma cancer cells, B16F10, and induced to generate cancer tissues. 1/HBc-encoding IVT mRNA-encapsulated LNPs before and after injection into mice, showing the relationship between the relative amounts of antibodies to the HBcAg antigen and the size of cancer tissue. It was found that the amount of antibody against the HBcAg antigen was relatively slightly reduced in individuals with a large decrease in the size of . This is thought to be due to the antibody being exhausted in the natural killer cell attachment process during the process in which cancer tissue growth is inhibited by the antibody-dependent cellular cytotoxicity (ADCC) activity of the antibody against the HBcAg antigen in the mouse body. , it can be said that this result proved the present inventor's initial prediction that the chimeric antigen can inhibit the growth of cancer tissue through antibody-dependent humoral immunity.

In conclusion, the present inventors proposed a chimeric antigen in which a peptide domain capable of binding to a target protein associated with a disease is bound to a specific external antigen for which a specific antigen-antibody reaction system has already been formed in the body. When administered, the chimeric antigen can specifically bind to cancer or infected cells whose target protein is labeled on the cell membrane, and at the same time, the antigen-antibody reaction system already formed in the body by other domains of the chimeric antigen. To be able to treat cancer or infectious diseases by inducing and effectively eliminating target disease cells, and to provide new methods of treating cancer or viral infections that can greatly increase patient survival. I found out.

So far, the discussion of the present invention has centered on its preferred embodiment. Those skilled in the art to which the present invention pertains should understand that the present invention can be embodied in various forms without departing from the essential characteristics of the present invention. Accordingly, the disclosed embodiments should be considered in an illustrative rather than a restrictive perspective. The scope of the invention is indicated in the appended claims rather than in the foregoing description, and all differences within the scope of equivalents thereof are to be construed as encompassed in the present invention.

Claims (9)

The hepatitis B virus core protein (HBc) immune response- inducing region; and the target cell-specific binding-inducing region PD-1; 1 chimeric antigen; and
Hepatitis B virus e-antigen (HBe) immune response-inducing region; and PD-1 target-cell-specific binding-inducing region; a chimeric antigen of 2,
A chimeric antigen with multiple immune enhancements selected from Chimeric antigen according to claim 1 , characterized in that said first chimeric antigen consists of the peptide sequence of SEQ ID NO:9 and said second chimeric antigen consists of the peptide sequence of SEQ ID NO:11. A pharmaceutical composition for preventing or treating cancer, comprising the chimeric antigen according to claim 1 as an active ingredient. The above cancers include lung cancer, stomach cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous melanoma, uterine cancer, ovarian cancer, rectal cancer, colon cancer, colon cancer, breast cancer, uterine sarcoma, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, laryngeal cancer, small intestine cancer, Thyroid cancer, parathyroid cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, childhood solid tumor, differentiated lymphoma, bladder cancer, renal cancer, kidney selected from the group consisting of cell carcinoma, renal pelvic carcinoma, primary central nervous system lymphoma, spinal cord tumor, brain stem glioma and pituitary adenoma The pharmaceutical composition according to claim 3, characterized in that: A pharmaceutical composition for preventing or treating hepatitis B virus infection , comprising the chimeric antigen according to claim 1 as an active ingredient. An immune enhancer composition comprising the chimeric antigen according to claim 1 as an active ingredient. The chimeric antigen is
(i) specifically binds to target cells, diseased cells;
(ii) promoting recovery of exhausted CD8+ T cells to induce T cell activity, and (iii) inducing antigen-antibody responses to enhance humoral immune responses and cytotoxic effects by natural killer cells (NK cells). 7. Composition according to claim 6 , characterized in that it is activating. 8. The composition according to claim 7 , wherein the induction of the antigen-antibody reaction in (iii) enhances the specific antigen-antibody reaction formed in the body of the individual by the immune reaction-inducing region of the chimeric antigen. . A vaccine composition against cancer or hepatitis B virus infection , comprising the chimeric antigen according to claim 1 as an active ingredient.

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