JP2022513340A - Chimeric antigen with enhanced multiple immune function by specifically binding to target cells and its use - Google Patents

Abstract

The present invention relates to a chimeric antigen having enhanced multiple immune function by specifically binding to a target cell and its use. Specifically, the present invention has an immune response-inducing region and a target cell-specific binding-inducing region. A fused chimeric antigen with enhanced multiple immune function; a pharmaceutical composition for preventing or treating cancer containing the chimeric antigen with enhanced multiple immune function as an active ingredient; a pharmaceutical composition for preventing or treating an infectious disease. The present invention relates to an immunopotentiator composition; and a vaccine composition.

Description

The present invention relates to a chimeric antigen having enhanced multiple immune function by specifically binding to a target cell and its use.

The ability to prevent pathogens such as bacteria and viruses from invading the body from the outside, and to recognize and resist pathogens even if they invade, is called "immunity". Immunity includes innate immunity, which is an ability that everyone has, 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 is also called specific immunity, and lymphocytes and antibodies are important as protective actions that recognize the type of invading pathogen and respond accordingly. Play a role. That is, B lymphocytes and T lymphocytes that circulate in the body are in charge. They have receptors on the surface that recognize specific antigens, and when they recognize the antigen, 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 (non-self) such as pathogens, foreign substances, and organ tissues of other species and cause an acquired immune response. , Antibodies are substances produced by B lymphocytes to counter the invading specific antigen, and bind to the specific antigen to eliminate the pathogenicity. In addition, since it has a function of memorizing the type of pathogen that has invaded once, it is possible to effectively deal with the secondary invasion of the same pathogen. Acquired immunity is an action that occurs after the pathogen has completely invaded the body, and because it works after the innate immunity action, it is also called "secondary defense". Acquired immunity includes cell-mediated immunity and humoral immunity. The surfaces of T lymphocytes and B lymphocytes have receptors that recognize specific antigens, and when they recognize antigens, they have cell-mediated immunity and humoral immunity, respectively. Activates sexual immunity. In the former, antigen-recognizing toxic T lymphocytes (Cytotoxic T lymphocytes) directly attack and remove pathogen-infected cells and cancer cells, while the latter humoral immunity assists in recognizing antigens. T lymphocytes (Helper T lymphocytes) stimulate B lymphocytes to differentiate and proliferate into plasma cells that secrete antibodies and memory cells that store antigens. The resulting generation of memory cells allows for a faster and stronger immune response to later re-invading pathogenic antigens.

When plasma cells secrete an antibody into the blood, the antibody binds to the antigen and triggers an antigen-antibody reaction to remove the antigen. One antibody specifically binds to only one antigen, which is called the specificity of the antigen-antibody reaction. Antibodies impair the motility of invading microorganisms by adhering to them and prevent them from invading somatic cells. In addition, the antigen, which is surrounded by antibodies, undergoes a chemical chain reaction with complement, a series of proteins found in plasma. This complement reaction attracts macrophages that cause the degradation of invading microorganisms and phagocytose the invading microorganisms. The reaction and binding 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 period, lymphocytes differentiate to produce antibodies when an antigen first invades, and memory cells produce antibodies at this time. It is formed. The 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 re-enters, and induces a faster and stronger immune response.

The adaptive immune system, composed of T and B lymphocytes, has strong anticancer potential as well as the ability to respond to a variety of tumor antigens. In addition, the immune system has considerable plasticity and memory components, and the attributes of these adaptive immune systems make immunotherapy a more effective method of any cancer treatment. However, although endogenous immune responses to cancer have been observed in preclinical models and patients, such responses are ineffective and established cancers are considered "self" and accepted by the immune system. I also do. Due to such tolerated conditions, tumors may abuse other mechanisms to actively disrupt antitumor immunity, which are T cell signaling and inhibitory factors that have caused dysfunction. Utilize an endogenous "immune checkpoint" that protects normal tissues from the incidental damage caused by tumors and down-regulates the intensity of adaptive immune responses to avoid regulatory cells and immune destruction.

The long-term conflict between the human immune system and diseases such as chronic infections and cancer puts a lot of physiological burden on the host, resulting in a stalemate in the immune response. T cells will be exhausted and inactivated while giving dominance to viruses or bacteria and tumors. Based on a large number of molecular databases, researchers have investigated nine types of exhausted T cells (Tex) that are associated with chronic infection, autoimmunity, and cancer interactions. Exhaustion of normal T cells results in a deficiency in the ability to respond to bacteria and tumors. Tex expresses inhibitory receptor proteins on its surface that stop important biochemical pathways, altering the regulation of gene expression, altering metabolism that creates energy to fight infections and tumors, and optimal immune function. Suppress development. Applying such an assessment to exhausted T cells in immunotherapy clinical trials can identify patients who are likely to benefit from a particular combination of immunotherapy and respond to infection or cancer. You will be able to understand the underlying mechanism of certain types of exhausted T cells.

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

In this regard, until recently, cancer immunotherapy has an antitumor immune response by providing adaptation-metamorphosis of activated effector cells, immunization against related antigens, or non-specific immunostimulators such as cytokines. I have focused my research on increasing methods. However, over the last decade, intensive efforts to develop specific immune checkpoint pathway inhibitors have begun to offer new immunotherapeutic approaches to treat cancer, which is progressing. Suppressive PD-1 such as ipilimumab, an antibody (Ab) that binds to and suppresses cytotoxic T lymphocyte antigen-4 (CTLA-4) to treat patients with melanoma. Includes 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, which is a part of the Ig superfamily gene and is well known as a co-inhibitory molecule of T cells belonging to the epidemic globulin molecule group. PD-1 is a type of CD28 series receptor, including receptors expressed on activated B cells, T cells and bone marrow cells, ie CD28, CTLA-4, ICOS, PD-1 and BTLA. .. Two cell surface glycoprotein ligands for PD-1, namely PD-L1 and PD-L2, were identified, but they were expressed not only on antigen-presenting cells but also on many human cancers, 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 increases when activated. It has been reported that PD-L1 is expressed to some extent even in normal T cells, and its expression also increases when activated. PD-L1 is found in large numbers in many human cancers, and the interaction between 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 an antigenic escape phenomenon 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 in tumors and / or stromal cells. It mainly functions in peripheral tissues that can be used [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, the T cells cannot attack the cancer cells.

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

On the other hand, the specific antigen-antibody reaction system existing in the body is generally understood as an independent immunity and is considered to be unrelated to other diseases, and has been widely used for the treatment of other antigens or diseases. I didn't. Rather, immunity formed against bacteria or viruses that infect the same organ interferes with antibody formation from other sources of infection, and as a way to overcome this, a chimeric antigen containing antigens from both sources at the same time is used. There have been attempts at methods of administration or administration of recombinant chimeric antigens of two antigens to improve the immune response of the other antigen. However, successful cases showing effective pharmacological activity by chimeric antigens are still extremely rare.

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

The present invention uses cell-mediated immunity and humoral immunity by T cells and B cells using mRNA synthesized by transcribing in vitro, that is, a chimeric antigen fused with IVT mRNA. We have developed a new therapeutic agent that can maximize the immunotherapeutic effect by simultaneously activating (human immunity) or antibody-dependent cellular cytotoxicity.

Therefore, it is an object of the present invention to provide a chimeric antigen with enhanced multiple immune function, 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, which comprises, as an active ingredient, a chimeric antigen having an enhanced multiple immune function of the present invention.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating an infectious disease, which comprises a chimeric antigen having an enhanced multiple immune function of the present invention as an active ingredient.

Another object of the present invention is to provide an immunopotentiator composition comprising the chimeric antigen enhanced with the multiple immune function of the present invention as an active ingredient.

Another object of the present invention is to provide a vaccine composition for a cancer or an infectious disease, which comprises the chimeric antigen having an enhanced multiple immune function of the present invention as an active ingredient.

In order to achieve the above object, the present invention provides a chimeric antigen with enhanced multiple immune function, 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 in which an antigen-antibody reaction is formed by the antigen.

In one embodiment of the present invention, the immune response-inducing region is a hepatitis B virus antigen, a hepatitis C virus antigen, a hepatitis A virus antigen, an AIDS virus antigen, a MERS virus antigen, an influenza A virus antigen, a human papillomavirus antigen, and the like. It may be selected from the group consisting of lymphocytic choroiditis virus (LCMV) antigen and simple herpesvirus (HSV) antigen.

In one embodiment of the present invention, the immune response-inducing region is a hepatitis B virus antigen, which is a core protein of hepatitis B virus (HBc), an e-antigen of hepatitis B virus (HBe), or a hepatitis B virus. Can be 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 the target cell, and is 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, the immune response-inducing region and the target cell-specific binding-inducing region are linked by a linker peptide represented by SEQ ID NO: 19 or SEQ ID NO: 21 for structural stability of the chimeric antigen. It can be what has been done.

In one embodiment of the present invention, the chimeric antigen with enhanced multiple immune function 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 preventing or treating cancer, which comprises the chimeric antigen having the enhanced multiple immune function of the present invention as an active ingredient.

In one embodiment of the present invention, the cancers are lung cancer, stomach cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin melanoma, uterine cancer, ovarian cancer, Rectal cancer, colon cancer, colon cancer, breast cancer, uterine sarcoma, oviduct cancer, endometrial cancer, cervical cancer, vaginal cancer, genital cancer, esophageal cancer, Laryngeal cancer, small intestinal cancer, thyroid cancer, accessory thyroid cancer, soft tissue sarcoma, urinary tract cancer, penis cancer, prostate cancer, chronic or acute leukemia, solid tumor in childhood, differentiated lymphoma, bladder It may be selected from the group consisting of cancer, renal cancer, renal cell carcinoma, renal pelvis carcinoma, central nervous system primary lymphoma, spinal cord tumor, brain stem glioma and pituitary adenoma.

The present invention also provides a pharmaceutical composition for preventing or treating an infectious disease, which comprises the chimeric antigen having the enhanced multiple immune function of the present invention as an active ingredient.

In one embodiment of the present invention, the infectious disease is hepatitis B virus, hepatitis C virus, hepatitis A virus, AIDS virus, MERS virus, influenza A virus, human papillomavirus, lymphocytic choriomenelitis virus ( It can be an LCMV) or simple herpesvirus (HSV) infectious disease.

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

In one embodiment of the present invention, the chimeric antigen with enhanced multiple immune function (i) specifically binds to diseased cells, which are target cells, and (ii) promotes recovery of exhausted CD8 + T cells. It can induce cell activity and (iii) induce an antigen-antibody reaction to enhance the humoral immune response and activate the cytotoxic effects of natural killer cells (NK cells).

In one embodiment of the present invention, the antigen-antibody reaction induction of (iii) above may enhance the 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 a vaccine composition for cancer or infectious disease, which comprises the chimeric antigen having the enhanced multiple immune function of the present invention as an active ingredient.

The present invention relates to a chimeric antigen having enhanced multiple immune function by specifically binding to a target cell and its use, and has enhanced multiple immune function in which an immune reaction-inducing region and a target cell-specific binding-inducing region are fused. In the case of the chimeric antigen, it can bind specifically to diseased cells, promote recovery of exhausted CD8 + T cells, induce T cell activity, induce antigen-antibody reaction, and enhance humoral immune response and natural. It is possible to activate the cytotoxic effects of killer cells (NK cells), suppress immune evasion of diseased cells, enhance multiple immune functions, and more effectively prevent and treat cancer, infectious diseases and immune diseases. can.

It shows the process of humoral immunity acting by inducing an antibody formed in the body by a lytic chimeric antigen, and recovers immunocompromised cell-specific CD8 + T cells in target cancer or infected cells. Schematic diagram showing that CD8 + T cells specific for the specific external antigen of the chimeric antigen are simultaneously induced to the target cancer or infected cell site and reactivated, and antibody-dependent cytotoxicity acts simultaneously. Is. It shows the design drawing of the fusion gene encoding the single antigen of HBcAg, HBeAg, HBsAg and the chimeric antigen that is expected to have various structural changes in molecular biology of PD-1 / HBc, PD-1 / HBe. Recombinant DNA sections for the production of IVT mRNAs that encode single or chimeric antigens are open reading frames, T7 promotors, 5'untranslated regions, 3'. Designed to include untranslated regions. The production results of DNA sections encoding HBcAg, HBeAg, HBsAg, PD-1 single antigen and PD-1 / HBc, PD-1 / HBe chimeric antigens are shown. The results showing the results of producing each VT mRNA using DNA sections encoding HBcAg, HBeAg, HBsAg, PD-1 single antigen and PD-1 / HBc, PD-1 / HBe chimeric antigens as templates. .. After inserting the VT mRNA encoding the PD-1 / HBc and PD-1 / HBe chimeric antigens of the present invention into animal cells 293T, the antigens actually produced in the cells were confirmed by an enzyme-bound immunoadsorption test. be. After inserting the VT mRNA encoding the PD-1 / HBc and PD-1 / HBe chimeric antigens into animal cells 293T, the results of confirming the actual production of the chimeric antigens in the cells by Western blotting are shown. It was confirmed by using the coimmunoprecipitation method that the PD-1 / HBc and PD-1 / HBe chimeric antigens expressed in the cells actually specifically bind to the PD-L1 ligand of the cells. .. The results of encapsulating the single antigen and chimeric antigen IVT mRNA synthesized in one example of the present invention in LNP and then measuring the particle size using a nanoparticle analyzer (Zetasizer) are shown. The results showing the results of measuring the efficiency of actually encapsulating the mRNA in the particles after encapsulating the single antigen and chimeric antigen IVT mRNA synthesized in one example of the present invention in the LNP. After inserting the LNP encapsulating IVT mRNA into animal cells 293T, it was confirmed by an enzyme-bound immunoadsorption test that the chimeric antigen protein was actually produced in the cells. After injecting LNP containing the chimeric antigen PD-1 / HBe IVT mRNA into C57BL / 6 mice, it was confirmed by an enzyme-bound immunoadsorption test that the antigen protein was actually produced from the IVT mRNA injected in the body. It is a thing. Prior to treating C57BL / 6 mice with the chimeric antigen, the single antigens HBcAg and HBeAg proteins were pre-inoculated into the mice, and it was confirmed by an enzyme-bound immunoadsorption test that the mice actually produced antibodies against each antigen. It shows that it was done. After transplanting B16F10, which is a mouse melanoma cancer cell, into mice in which antibodies were primarily formed against these proteins by injecting HBcAg and HBeAg proteins, then PD-1 / HBc and PD-1 / It is shown that the growth of cancer tissue was inhibited by the chimeric antigen produced in the body by injecting LNP encapsulating IVT mRNA encoding HBe into mice. After inducing C57BL / 6 mice, which had primary antibodies against HBcAg and HBeAg, to generate cancer tissue by transplanting B16F10, which is a melanoma cancer cell, PD-1 / HBc and PD. It is shown that when LNP encapsulating -1 / HBe-encoding IVT mRNA is injected into mice, the chimeric antigen produced in the body increases the survival rate of the mice. After inducing C57BL / 6 mice, which had primary antibodies against HBcAg and HBeAg, to generate cancer tissue by transplanting B16F10, which is a melanoma cancer cell, PD-1 / HBc and PD. Comparing the survival rates before and after injecting LNP encapsulated with -1 / HBe-encoding IVT mRNA into mice, it is shown that the survival rate of mice increased after treatment with chimeric antigen. After transplanting B16F10, which is a melanoma cancer cell, into C57BL / 6 mice in which an antibody was primarily formed against the HBcAg antigen and inducing it to generate cancer tissue, it encodes PD-1 / HBc. It shows the association between the relative amount of antibody against the HBcAg antigen before and after injecting LNP encapsulating IVT mRNA into mice and the size of cancer tissue.

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

In particular, the chimeric antigen with enhanced multiple immune function provided by the present invention specifically binds to a target protein associated with a disease in a specific external antigen region (domain) in which a specific antigen-antigen reaction system is formed in the body. When the chimeric antigen of the present invention is administered to an individual, the chimeric antigen is produced by fusing the regions (domains) that can be used, and the chimeric antigen is a specific diseased cell in which the target protein is labeled on the cell membrane, for example, cancer cells or. The other domain of the chimeric antigen, that is, the immune reaction-inducing region (specific external antigen region), can induce the antigen-antigen reaction system already formed in the body and is a target disease. Designed to efficiently remove cells.

Therefore, when the chimeric antigen of the present invention is used, it is possible to reactivate the immune system (immune response) by the specific antigen-antibody system already formed and existing in the individual to treat the target disease very efficiently. I made it possible.

Considering the components of the chimeric antigen with enhanced multiple immune function of the present invention, it is formed of a fusion protein in a form in which an immune response-inducing region and a target cell-specific binding-inducing region are largely fused.

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

The immune reaction-inducing region that can be contained in the chimeric antigen of the present invention is a foreign antigen, which is a protein, glycoprotein, coat protein, core protein or function of an organism involved in a virus, a bacterium or various infectious diseases. Sexual proteins can be included, 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 choriomyelitis virus (LCMV) antigen and simple herpesvirus (HSV) antigen, more preferably hepatitis B virus antigen, type B. It can be a hepatitis virus core protein (HBc) or a hepatitis B virus e-antigen (HBe) or a hepatitis B virus surface protein (HBs).

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

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: Shown in 6.

In addition, the target cell-specific binding-inducing region is mainly a protein related to normal life activity in the body, and is a water-soluble protein or a cell membrane protein that can physically bind to a receptor or a ligand on the cell membrane. possible. The target cell-specific binding-inducing region of the present invention is not limited to this, 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 It can be selected from the group consisting of -1.

In one embodiment of the present invention, PD-1, which is a cell membrane protein capable of specifically binding to a target cell, is used as the target cell-specific binding-inducing region, and the cell membrane protein is the core of the immune checkpoint system. PD-1 serves a role and is known as a form in which PD-1 is attached to the cell membrane and is often secreted extracellularly (Cellular Immunology, 2005, 235: 109). Therefore, the present invention uses a recombinant PD-1 protein that converts PD-1 into a protein that binds tightly to this receptor, PD-L1, and thus is capable of extracellular secretion and has high binding to the 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.

Further, the chimeric antigen of the present invention is a linker peptide consisting of a specific sequence in order to maximize the structural stability and immune response of the chimeric antigen between the immune reaction-inducing region and the target cell-specific binding-inducing region. Designed to be connected. It was confirmed that there was a difference in the structural stability of the chimeric antigen depending on the sequence of the linker peptide, and based on this, an effective linker peptide for enhancing the stability of the chimeric antigen and the immune response was selected. The linker peptide 1 that can be used 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. Further, the linker peptide 2 that can be used in the present invention comprises 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, the soluble chimeric antigens PD-1 / HBc and PD-1 / HBe were produced as chimeric antigens with enhanced multiple immune functions, respectively, but this is used as an immune reaction-inducing region. It uses the core protein of hepatitis B virus (HBcAg) and the e-antigen protein of hepatitis B virus (HBeAg), respectively, and is secreted extracellularly with PD-L1 as a target cell-specific binding induction region. The water-soluble PD-1 to be used is used. Further, in one embodiment of the present invention, the PD-1 and the HBc domain are linked between them by the linker peptide 1 of SEQ ID NO: 19, and the PD-1 and the HBe domain are linked between them by the linker peptide of SEQ ID NO: 21. They were connected in 2 respectively.

In addition, HBcAg and HBeAg single antigens, PD-1 / HBc and PD-1 / HBe chimeric antigens are designed to contain most of the amino acid sequences that induce antibody formation well, and each antigen protein is produced intracellularly. After that, the amino end of each protein was made to contain a signal peptide so that it could be well secreted extracellularly. The synthesis of the signal peptide is carried out by a method 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 peptide has a major impact on recombinant protein synthesis and secretion in mammalian cells), amino targeting a number of the signal peptide produced antigenic proteins are in the present invention Various types of signal peptide sequences were ligated to the ends, and after undergoing a verification process to see if the produced antigen was well secreted extracellularly, the signal peptides to which each antigen was well secreted extracellularly were selected.

In the present invention, in order to ensure that the chimeric antigen of the present invention is well secreted extracellularly by the above experiment, the signal peptide is 21 peptides (signal peptide 1) represented by SEQ ID NO: 23 or SEQ ID NO: 25. A signal peptide (signal peptide 2) consisting of 17 peptides represented by is used, and the polypeptide 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 used. The encoding polypeptide sequence is shown in SEQ ID NO: 26.

As described above, the chimeric antigen having the enhanced multiple immune function according to the present invention has a form in which an immune response-inducing region; and a target cell-specific binding-inducing region; are fused, and in one embodiment of the present invention, the multiplex is performed. PD-1 / HBcAg and PD-1 / HBeAg chimeric antigens were produced 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 this is shown in SEQ ID NO: 10.

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

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

Further, the polynucleotide sequence encoding the polypeptide for the PD-1 / HBcAg chimeric antigen of SEQ ID NO: 9 is shown in SEQ ID NO: 10, and the polynucleotide sequence encoding the signal peptide 1 of SEQ ID NO: 10 is shown in 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 base sequence from the 694th base sequence to the 831st base sequence of SEQ ID NO: 10. Become.

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 this is shown in SEQ ID NO: 12.

The polypeptide of SEQ ID NO: 11 showing the PD-1 / HBeAg chimeric antigen of the present invention contains the sequence of the signal peptide 2 for extracellular secretion, and the sequence of the signal peptide 2 is 17 from the first amino acid of SEQ ID NO: 11. It consists of a total of 17 amino acid sequences up to the second.

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

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

On the other hand, the chimeric antigen with enhanced multiple immune function according to the present invention can be used in applications where cancer or infectious disease can be prevented or treated by activating an immune response.

Since the chimeric antigen of the present invention contains a target cell-specific binding-inducing region, it can specifically bind to a diseased cell which is a target cell, and since it contains an immune reaction-inducing region, it is exhausted CD8 + T cells. Multiple immune function by promoting the recovery of T cells and inducing antigen-antibody reaction to enhance the humoral immune reaction and simultaneously activate the cytotoxic effect of natural killer cells (NK cells). It has the feature that it can be strengthened.

In particular, the enhancement of the multiple immune function by the immune reaction-inducing region means that the present invention contains the foreign antigen for an individual who is exposed to the foreign antigen and has a specific and stable antigen-antibody reaction system against the foreign antigen. By treating the chimeric antigen, the antigen-antibody reaction is reactivated, and thus humoral immunity and cell-mediated immunity are simultaneously multi-activated.

Therefore, the present invention can provide a pharmaceutical composition for preventing or treating cancer, which comprises the chimeric antigen having the enhanced multiple immune function of the present invention as an active ingredient.

The types of the above cancers are not limited to these, but are lung cancer, stomach cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin melanoma, uterine cancer, and ovary. Cancer, rectal cancer, colon cancer, colon cancer, breast cancer, uterine sarcoma, oviduct cancer, endometrial cancer, cervical cancer, vaginal cancer, genital cancer, esophagus Cancer, laryngeal cancer, small bowel cancer, thyroid cancer, accessory thyroid cancer, soft tissue sarcoma, urinary tract cancer, penis cancer, prostate cancer, chronic or acute leukemia, solid tumor in childhood, differentiated lymphoma , Bladder cancer, kidney cancer, kidney cell cancer, renal pelvis cancer, central nervous system primary lymphoma, spinal cord tumor, brain stem glioma or pituitary adenoma.

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

In the present invention, the above-mentioned "infectious disease" means a disease in which various sources or components including foreign viruses, bacteria, protozoa, etc. cause, mediate, or contribute to the pathological condition of mammals. The types of the above-mentioned infectious diseases are not limited to this, but hepatitis B virus, hepatitis C virus, hepatitis A virus, AIDS virus, MERS virus, influenza A virus, human papillomavirus, lymphocytic choroidal spinal cord. It can be a membranous virus (LCMV) or simple herpesvirus (HSV) infectious disease.

Further, the present invention can provide an immunopotentiating agent composition containing a chimeric antigen having enhanced multiple immune functions as an active ingredient, and such an immunopotentiating agent composition is an immunity induced as an immune dysfunction. It can be used as a pharmaceutical composition for the prevention or treatment of diseases.

In the present invention, the above-mentioned "immune disease" means a disease in which components of the mammalian immune system cause, mediate, or otherwise contribute to the pathological state of the mammal, and the stimulation or interruption of the immune response is the disease. It can include all diseases that have a compensatory effect on the progression of.

In one embodiment of the present invention, the PD-1 / HBc chimeric antigen fused with HBcAg and PD-1 and the PD-1 / HBe chimera antigen fused with HBeAg and PD-1 are transferred in vitro to each mRNA ( After producing IVT mRNA (in vitro translation mRNA), the produced PD-1 / HBc mRNA and PD-1 / HBe mRNA were introduced into diseased cells, respectively, and then the above chimeric antigen was selectively used as a PD-L1 ligand. As a result of confirming whether or not they bind, it was confirmed that both PD-1 / HBc and PD-1 / HBe chimeric antigens specifically bind to the PD-L1 ligand.

In addition, after injecting HBcAg and HBeAg proteins into mice for the production of HBcAg and HBeAg antibodies capable of binding to the chimeric antigens PD-1 / HBc and PD-1 / HBe by pre-inoculation of mice in the present invention, 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, when the chimeric antigen of the present invention is administered to an individual, the present inventors can bind the chimeric antigen of the present invention to a target site specific to a diseased cell, and an antibody present in the individual. It was confirmed that by binding to, the humoral immune activity can be promoted, and as a result, the diseased cells can be removed by the immunopotentiating action to effectively prevent or treat the target disease.

Furthermore, in order to confirm the anticancer activity using the chimeric antigen of the present invention by animal experiments, HBsAg and HBcAg were pre-inoculated, and PD-L1 was labeled on the cell membrane of mice in which the primary antibody against the HBsAg was formed. As a result of injecting the chimeric antigen of the present invention after transplanting the mouse cancer cells B16F10 cells to generate cancer tissue, the cancer cell killing effect was obtained in the group in which the single antigens HBcAg and HBeAg were individually injected. In contrast to the fact that it was shown to be almost nonexistent, the growth of cancer tissue was effectively inhibited in the group injected with PD-1 / HBc mRNA and PD-1 / HBe mRNA, which are the chimeric antigens of the present invention. 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 function of the present invention has an effect of preventing and treating cancer or infectious disease, and in particular, the chimeric antigen of the present invention inhibits immunity. By blocking the mediating PD-1 signaling pathway and the binding of PD-1 to PD-L1, it induces recovery of T cell exhaustion by cancer or infected cells and enhances the ability of T cells to secrete cytokines. It was found that it can be activated to enhance the immune response.

That is, it was found that it can promote the activity of diseased cell target-specific CD8 + T cells already formed in the body but disabled by T cell exhaustion. Furthermore, natural killer (NK) cells, macrophages (macrophages, macrophages), and monosites (monospheres), which are humoral epidemic cells that are bound and activated by antibodies specific for foreign antigens, are antibodies. The activity of antibody-dependent cellular cytotoxicity prevents or treats diseases induced by the interaction of PD-1 and PD-L1 more rapidly and powerfully than general single antibody therapeutic agents. be able to. The disease induced by the interaction of PD-1 and PD-L1 can be the cancer or infectious disease described above.

In the present invention, the pharmaceutical composition 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. In the above, the pharmaceutically effective amount means an amount sufficient to prevent, ameliorate and treat the symptoms of cancer or infectious disease.

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

In addition, the above-mentioned "pharmaceutically acceptable" is a composition that is physiologically acceptable and does not usually cause an allergic reaction such as gastrointestinal disorder, dizziness or a similar reaction when administered to humans. Say that. Examples of the carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose. , Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like can be further contained.

After being administered to a mammal, the compositions of the invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient. .. Dosage forms can be in the form of powders, granules, formulations, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injection solutions, sterile powders.

In addition, the composition for the prevention or treatment of cancer or infectious disease according to the present invention can be administered through various routes including oral, transdermal, subcutaneous, venous or muscle, and the dose of the active ingredient is determined. The composition for the prevention or treatment of cancer or infectious disease according to the present invention can be appropriately selected depending on various factors such as the route of administration, the age, sex, weight of the patient and the severity of the patient. It can be administered in parallel with known compounds that have the effect of preventing, ameliorating or treating the symptoms of infectious diseases.

Furthermore, the present invention can provide a method for treating a cancer or an infectious disease, which comprises the step of administering the chimeric antigen of the present invention to an individual having a cancer or an infectious disease induced except human.

Also, suitable routes of administration include, for example, oral, nasal, permeabilized or intraseptal enteral administration, direct intraventricular, intraperitoneal, or intraocular injections, as well as intramuscular, subcutaneous, intravenous, and bone marrow injections. Parenteral transmission can be included.

Furthermore, the present invention can provide a vaccine composition for cancer or infectious disease, which comprises the chimeric antigen of the present invention having enhanced multiple immune function as an active ingredient.

The vaccine composition of the present invention can be administered alone, but preferably an adjuvant can be included together. An adjuvant is a substance that non-specifically promotes an immune response to an antigen during the initial activation process of epidemic cells, and is a preparation that enhances immunity by increasing the activity of cells of the immune system, although it is not an immunogen to the host. , Mole, etc. (Annu. Rev. Immunol, 1986, 4: 369). The adjuvants that can be administered with the vaccine compositions of the invention to enhance the immune response include any diverse adjuvants, typically adjuvants include Freund's adjuvant, aluminum compound, and the like. Muramyl dipeptide, lipopolysaccharide (LPS), monophosphoryl lipid A, quill A and the like are known. The adjuvant can be administered with the vaccine composition or sequentially at time intervals.

In addition, the vaccine composition of the present invention may further contain a solvent, an excipient and the like. The solvent includes physiological saline, distilled water and the like, and the excipient includes aluminum phosphate, aluminum hydroxide, potassium aluminum sulfate and the like, but is not limited thereto, and the field to which the present invention belongs. Can further contain substances commonly used in the production of vaccines in.

The vaccine composition of the present invention can be produced by a method usually used in the technical field to which the present invention belongs. The vaccine composition of the present invention can be produced as an oral or parenteral preparation, preferably as an injection solution which is a parenteral preparation, and is intradermal, intramuscular, intraperitoneal, intravenous, or subcutaneous. It can be administered by the internal, nasal or epidural route.

The vaccine composition of the present invention can be administered to an individual in an immunologically effective amount. The above-mentioned "immunologically effective amount" means an amount sufficient to exert a disease-preventing effect and an amount not to cause side effects or a serious or excessive immune reaction, and the exact administration concentration is It depends on the specific immunogen to be administered, and is easily performed by those skilled in the art due to factors well known in the medical field such as age, weight, health, sex, individual drug sensitivity, administration route, administration method, etc. It can be administered once or several times.

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

<Example 1>
[Production of a fusion gene encoding a chimeric antigen of HBc, HBe, HBs, PD-1 / HBc or PD-1 / HBe]
Hepatitis B core antigen (HBcAg), hepatitis B e-antigen (HBeAg), hepatitis B external antigen (HBsAg), PD-1, soluble chimeric antigen PD-1 / HBc and PD-1 / HBe proteins FIG. 2 shows a schematic diagram of a DNA section for producing an IVT mRNA (in vitro transcribed mRNA) synthesized by transcribing each encoding gene in vitro. As shown in FIG. 2, the DNA section for IVT mRNA production includes an open reading frame, a T7 promotor base sequence, a 5'untranslated base sequence (5'UTR), and 3 'The untranslated base sequence (3'UTR) was prepared with reference to the mRNA Express mRNA Synthesis kit of System Biosciences, USA. In the design of the DNA section, an open reading frame, that is, the gene of interest was ligated and inserted in a cassette form. At this time, the order of the open reading frames encoding the immune response-inducing region and the target cell-specific binding-inducing region of the chimeric antigen is not fixed, and the positions of both induction regions may change from each other. That is, the immune response-inducing region and the target cell-specific binding-inducing region of the chimeric antigen are located in the linker-linked "A gene" and "B gene" in FIG. 2, respectively, and the positions may change from each other.

In addition, hepatitis B core antigen (HBcAg), hepatitis B e-antigen (HBeAg), hepatitis B external antigen (HBsAg), PD-1, soluble chimeric antigens PD-1 / HBcAg and PD-1 / HBeAg. On the other hand, the DNA base sequences for each VT mRNA transcribed in vitro are sequenced in SEQ ID NOs: 13 (HBcAg), 14 (HBeAg), 15 (HBsAg), 16 (PD-1), 17 (PD-1 / HBcAg) and 18. It is shown in (PD-1 / HBeAg) respectively.

The base sequence of SEQ ID NO: 17 is the base sequence of the DNA template encoding PD-1 / HBcAg IVT mRNA, and the base sequence is not only the coding sequence of PD-1 / HBcAg but also the T7 prober motor, 5'UTR. And 3'UTR base sequence are also included.

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 is not only the coding sequence of PD-1 / HBeAg but also the T7 prober motor, 5'UTR and 3'. It also includes the base sequence of UTR.

Also, after transcription is complete, a series of adjacent "polyadenylate (poly (A)) tails" are added to the 3'end of the RNA molecule by poly A polymerase, a process to those skilled in the art. It was devised by a well-known method to insert a polyA base sequence of a minimum of 50 adenine sequences at the time of design. The above-designed DNA section was synthesized by requesting IDT of the United States, and 10 ng of vector DNA containing each antigen gene produced by IDT was used as a template for the polymerase chain reaction (PCR) of SEQ ID NOs: 27 and 28 below. A gene DNA amplification reaction was performed using the prima for. The PCR reaction product was purified by an elution method well known to those skilled in the art and then used as a template for an in vitro transcription reaction (FIG. 3).

SEQ ID NO: 27 Primer Nucleotide Sequence (Primer 1):
5'-AGATTTAATACGACTCACTATAGGGAAAATAAGA-3'

SEQ ID NO: 28 Primer Nucleotide Sequence (Primer 2):
5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTACTGCTTTATT

<Example 2>
[IVT mRNA synthesis from DNA sections encoding single or chimeric antigens]
In vitro transcription (IVT) was performed using the template DNA produced in Example 1 to synthesize artificial mRNA, which is a kit for producing mRNA, HiScribe T7 ARCA of New England Biolabs, USA. It was performed using mRNA kit with tailing. Specifically, each purified DNA section containing the nucleotide sequences of 1 μg of HBcAg, HBeAg, HBsAg, PD-1, PD-1 / HBc and PD-1 / HBe single or chimeric antigen genes, 10 μl 2x ARCA. / NTP Mix, 2 μl T7 RNA Polymerase Mix was mixed, the rest was added with distilled water, and finally 20 μl of the reaction product was composed and then reacted at 37 ° C. for 60 minutes. When the reaction was completed, 2 μl DNase enzyme was added and then treated at 37 ° C. for 15 minutes to remove all 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 then mRNA production was confirmed by 1.2% agarose gel electrophoresis using 0.5 x TAE buffer solution (2). FIG. 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 shown in SEQ ID NO: 3. The polypeptide sequence of PD-1 is shown in SEQ ID NO: 5. The polypeptide (amino acid) sequence of PD-1 / HBc, which is a chimeric antigen protein expressed from the mRNA of the chimeric antigen (SEQ ID NO: 17), is shown in SEQ ID NO: 9, and the mRNA of another chimeric antigen (SEQ ID NO: 18) is shown. 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 the human cell line HEK293T (ATCC, CRL-3216). The HEK293T cell line is a cancer cell-derived kidney cell derived from human embryonic kidney, and the cells are cultured in a T75 flask at 10% FBS (Fetus Bovine Serum: GIBCO, # 16000-028), 1 Incubate in MEM medium (GIBCO, # 11095-080) supplemented with% ABAM (GIBCO BRL, # 15240-O13) for 3 days, and after culturing, wash with 1x PBS buffer solution, and then perform tripsin treatment to the bottom of the container. I peeled it off from. Then, the separated cells were centrifuged, the supernatant was discarded, and the cells were redispersed in 1x PBS buffer solution and then redispersed to 5X10 ^ 5cells / dish. After 24 hours, after confirming that the cells adhered well to the surface of the culture vessel, the chimeric antigen was prepared by a method provided by those skilled in the art using Lipofectamine Messenger Max Transfection Reagent (Invitrogen, # LMRNA003), a transfection reagent for mRNA. The encoding VT mRNAs (PD-1 / Hbc and PD-1 / Hbe) were transfected into the cells. Transfected cells were dispersed in 60 mm culture vessels and cultured in a 5% CO ₂ cell incubator for 2 days.

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

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

The cell extract obtained at this time was also used as a sample for an immunoprecipitation analysis method (Immuno-precipitation) capable of showing PD-L1 protein binding present on the cell membrane.

<Example 4>
[Confirmation of binding between PD-L1 protein present on the cell membrane and chimeric antigen expressed in 293T using immunoprecipitation method]
Using immunoprecipitation (IP), PD-L1 present on the cell membrane and the single antigen (PD-1) and chimeric antigen (PD-1 / HBc or HBsAg / PD-1) produced by the present invention. It was confirmed whether or not there was a bond with. For this purpose, PD-L1 / PD-1 / anti-PD-L1 antibody, PD-L1 / PD-1 / HBc / anti-PD-L1 antibody, PD-L1 / HBsAg are added by adding an antibody against PD-L1. A complex precipitate of / PD-1 / anti-PD-L1 antibody was obtained, and after electrophoresis of this precipitate, PD-L1 and PD-1, PD-1 / HBc or HBsAg / PD-1 were subjected to western blotting. Interactions were analyzed by coimmunoprecation (co-IP).

Specifically, electroporation or Lipofectamine Messenger Max Transfection Reaction (Invitrogen, Invitrogen, # LMRNA003) Transfection was performed using a transfection reagent dedicated to mRNA, cells were obtained 2 days later, and then the cells were lysed in a RIPA buffer solution. After removing the 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. After mixing the re-immobilized Protein A or Protein G agarose gel with this complex, the cells were rotated for 2 hours to allow them to adhere to the gel, and then the number of proteins not bound from the precipitated complex was increased. After washing and removing the precipitate complex by SDS-PAGE electrophoresis, Western blotting was performed using each antigen-specific antibody.

As a result, as shown in FIG. 7, as a result of adding and analyzing an antibody against PD-L1 to a cell extract of a HEK293T cell line transfected with the IVT-mRNA of a single or chimeric antigen according to the present invention, the results were analyzed. It was confirmed that PD-L1 interacts with PD-1, PD-1 / HBc or PD-1 / HBe.

<Example 5>
[Production of Lipid Nanoparticles (LNP) to Increase Transmission Efficiency of IVT mRNA Encoding Chimeric Antigen]
Lipid nanoparticles (LNP) consisting of various lipids were produced to increase the stability of mRNA and the efficiency of transmission to cells (Cell, 2017, 168: 1). First, four kinds of lipids, SS-OP, DOPC, Cholesterol, and PEG-lipid, were dissolved in 100% ethanol, and then these were mixed at a molar ratio of 50:10: 38.5: 1.5, respectively. LNP was prepared by using the Microfluidic device of Precision Nanosystems of Canada for each of the VT mRNA encoding the above-mentioned single antigen and chimeric antigen of the present invention and the lipid mixture contained in 50 mM citric acid buffer solution (pH 3.0). did. The prepared LNP was transferred to a dialysis membrane cassette and then dialyzed against 1x PBS buffer solution for 18 hours, and the appropriate concentration was adjusted using a centrifugal filter manufactured by Merck Millipore of Germany. After adjusting the concentration, the mixture was filtered through a 0.22 μm filter and refrigerated at 4 ° C. until it was used for intracellular injection or mouse injection.

<Example 6>
[Measurement of LNP size and mRNA encapsulation efficiency in which IVT mRNA encoding a chimeric antigen is encapsulated]
Each LNP produced in Example 5 was measured for particle size and polydispersity index (PDI) using a Zetasizer nanoparticle analyzer manufactured by Malvern in the United Kingdom.

As a result, as shown in FIG. 8, the LNP encapsulated with IVT mRNA showed a particle size of 90 to 150 nm, which was slightly larger than that of Naked LNP containing no IVT mRNA. In addition, the efficiency of mRNA encapsulation of VT mRNA encapsulated in each LNP production process into the LNP was measured by Ribogreen assy using the Quanti-iT Ribogreen kit manufactured by Thermo Fisher Scientific in the United States, as shown in FIG. In the process of producing LNP in which the single antigen and the chimeric antigen of the present invention were encapsulated, the encapsulation efficiency of each VT mRNA showed a high efficiency in the range of about 86 to 94%.

<Example 7>
[Confirmation of antigen expression efficiency at the cellular level of LNP encapsulating IVT mRNA encoding chimeric antigen]
In order to know whether or not the LNP produced in Example 5 can actually produce a protein in cells, a human cell line containing the single antigens HBcAg and HBeAg IVT mRNA produced in the present invention. HeLa (ATCC, CCL-2) was transfected into each of these. HeLa is a cancer cell derived from cervical cancer (Cerical cancer), and 10% FBS (Fetus Buffer Serum: GIBCO, # 11095-080), 1% ABAM (GIBCO BRL,) in a T75 flask for culturing the cells. After culturing in MEM medium (GIBCO, # 11095-080) supplemented with # 15240-013) for 3 days, the cells were washed with 1x PBS buffer solution, treated with trypsin, and peeled from the bottom of the container. After seeding in a 24-well culture vessel at a concentration of 5X10 ^ 5cells / well, the cells were cultured in a cell incubator at a 5% CO ₂ concentration for 24 hours. The next day, the LNP of the present invention and an apolipoprotein (Apolipoproteinin E4, ApoE4) were mixed, cultured at 37 ° C. for 10 minutes, and then transfected into cells. After obtaining a 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, it was confirmed that the LNP encapsulated with PD-1 / HBc and PD-1 / HBeIVT mRNA, respectively, was introduced into HeLa cells and then 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 antigen expression results confirmed at the cellular level in Example 7 through experimental animals. For this purpose, the LNP produced in Example 5 was injected into the left tibialis anterior of a 6-week-old C56BL / 6 female mouse to confirm the expression efficiency of the antigen in the animal body of LNP. Efficacy as an IVT mRNA transmitter to. To this end, mice were inoculated with a total of 80 μl of LNP encapsulating 2 μg of the IVT mRNA of the present invention together with 1 x PBS buffer solution. Whole blood was collected from the heart on the 1st, 3rd, and 6th days after inoculation, and the presence or absence of production of each antigen protein was confirmed by an enzyme-bound immunoadsorption analysis method.

As a result, as shown in FIG. 11, it was found that the PD-1 / HBe chimeric antigen was produced from the 1st day to the 6th day, and the PD-1 / HBe chimeric antigen was produced by producing LNP. It was found that when injected into mice using, sufficient single and chimeric antibodies were actually produced in the mouse body.

<Example 9>
[Confirmation of primary antibody formation against these antigens in mice pre-inoculated with HBcAg and HBeAg proteins]
HBcAg (US, MyBioSource, # MBS355629) and HBeAg protein (US, MyBioSource, # MBS355623) were adjuvanted to 6-week-old C57BL / 6 female mice on the first day (US, Sigma, # F5881). After mixing with, 2 μg per mouse was injected into the left tibialis anterior in 3 divided doses of 30 μl. On the 11th day after the first inoculation, the same amount of antigen protein was injected into each mouse by the same method. Serum was collected from the tail artery of mice infected on the 7th, 21st, 35th, and 44th days after the first inoculation, and the presence or absence of antibody production was searched by the enzyme-bound immunoadsorption analysis method.

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

<Example 10>
[Confirmation of 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]
On the 21st day of pre-inoculation of the antigen, about 1X10 ⁶ melanoma cancer cells B16F10 were injected subcutaneously into the lateral surface of C57BL / 6 mice in which antibodies against each antigen were formed by HBcAg and HBeAg antigen protein inoculation in Example 9. Ten days after the administration of cancer cells, VT mRNA-LNP encoding the chimeric antigens PD-1 / HBc and PD-1 / HBe of the present invention were injected into the left tibialis anterior muscle of the mouse (main inoculation), respectively. After the cancer cells were introduced, the changes in body weight of the mice and the formation of cancer tissues were observed every 3 days, and the size of the cancer tissues was measured. At this time, the number of mice used in the above experiment was 6 to 7 for each test group.

As a result, as shown in FIG. 13, the growth of cancer tissue in the mice pre-inoculated with the HBcAg antigen was higher when the chimeric antigen PD-1 / HBc IVT mRNA-LNP of the present invention was inoculated than in the control group. Similarly, the growth of cancer tissue in mice pre-inoculated with the HBeAg antigen was found to be significantly suppressed when the chimeric antigen PD-1 / HBe IVT mRNA-LNP of the present invention was inoculated effectively. It turned out 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 44-mouse mortality rate as compared with the control group. It was found that the survival rate increased by about 2 times with a significant decrease during the test period of the day. It can be seen that such a decrease in mouse mortality is due to the injection of the chimeric antigen of the present invention. As a result of comparing the survival rates of mice before and after inoculation with the chimeric antigen of the present invention, it was confirmed that the survival rates were significantly increased only in the experimental group inoculated with the chimeric antigen of the present invention (FIG. 15).

In addition, FIG. 16 shows PD- after transplanting B16F10, which is a melanoma cancer cell, into C57BL / 6 mice in which an antibody was primarily formed against the HBcAg antigen to induce cancer tissue generation. The results show the association between the relative amount of antibody against the HBcAg antigen before and after injecting LNP encapsulating 1 / HBc-encapsulating VT mRNA into mice and the size of cancer tissue. It was found that the amount of antibody against the HBcAg antigen was relatively slightly reduced in the individuals whose size was significantly reduced. This is thought to be because the antibody was depleted by the natural killer cell attachment process in the process of inhibiting the growth of cancer tissue by the activity of antibody-dependent cellular cytotoxicity (ADCC) of the antibody against the HBcAg antigen in the mouse body. It can be said that the present inventor initially proved the prediction that the chimeric antigen can inhibit the growth of cancer tissue by antibody-dependent humoral immunity.

In conclusion, the present inventors have 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 an antigen-antibody reaction system specific to the body has already been formed. When administered, the chimeric antigen is capable of specifically binding the target protein to cancer or infected cells labeled on the cell membrane, while at the same time providing an antigen-antibody reaction system already formed in the body by other domains of the chimeric antigen. To be able to treat cancer or infectious disease by inducing and effectively removing target disease cells, and to provide a new treatment method for cancer or viral infectious disease that can greatly increase the survival of patients. I understood.

So far, the present invention has been considered mainly on its desirable embodiments. Those who have ordinary knowledge in the technical field to which the present invention belongs should understand that the present invention can be embodied in a modified form without departing from the essential properties of the present invention. Therefore, the disclosed examples must be considered from a descriptive point of view rather than a limiting point of view. The scope of the invention is set forth in the claims rather than the description above, and all differences within the equivalent scope should be construed to be included in the invention.

Claims (15)

A chimeric antigen with enhanced multiple immune function, in which an immune response-inducing region; and a target cell-specific binding-inducing region; are fused. The chimeric antigen according to claim 1, wherein the immune reaction-inducing region is a foreign viral antigen, wherein an antigen-antibody reaction is formed by the antigen. The immune reaction-inducing region includes hepatitis B virus antigen, hepatitis C virus antigen, hepatitis A virus antigen, AIDS virus antigen, MERS virus antigen, influenza A virus antigen, human papillomavirus antigen, and lymphocytic choriomenelitis virus. The chimeric antigen with enhanced multiple immune function according to claim 2, characterized in that it is selected from the group consisting of (LCMV) antigen and simple herpesvirus (HSV) antigen. The immune reaction-inducing region is a hepatitis B virus antigen, which is a core protein (HBc) of hepatitis B virus, an e-antigen (HBe) of hepatitis B virus, or a surface protein (HBs) of hepatitis B virus. The chimeric antigen according to claim 3, wherein the multiple immune function is enhanced. The target cell-specific binding-inducing region is a cell membrane protein capable of specifically binding to a target cell, and is 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, the chimeric antigen with enhanced multiple immune function according to claim 1. The immune response-inducing region and the target cell-specific binding-inducing region are characterized by being linked by a linker peptide represented by SEQ ID NO: 19 or SEQ ID NO: 21 for structural stability of the chimeric antigen. The chimeric antigen according to claim 1, wherein the multiple immune function is enhanced. The chimeric antigen with enhanced multiple immune function according to claim 1, wherein the chimeric antigen with enhanced multiple immune function comprises the peptide sequence of SEQ ID NO: 9 or SEQ ID NO: 11. A pharmaceutical composition for preventing or treating cancer, which comprises the chimeric antigen having enhanced multiple immune function according to claim 1 as an active ingredient. The above cancers are lung cancer, stomach cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin melanoma, uterine cancer, ovarian cancer, rectal cancer, colon cancer, Colon cancer, breast cancer, uterine sarcoma, oviduct cancer, endometrial cancer, cervical cancer, vaginal cancer, genital cancer, esophageal cancer, laryngeal cancer, small bowel cancer, Thyroid cancer, accessory thyroid cancer, soft tissue sarcoma, urinary tract cancer, penis cancer, prostate cancer, chronic or acute leukemia, solid tumor in childhood, differentiated lymphoma, bladder cancer, renal cancer, kidney From cell cancer, renal pelvic carcinoma, primary central neurosis system lymphoma, spinal cord tumor, brain stem glioma and pituitary gland tumor 8. The pharmaceutical composition for preventing or treating cancer according to claim 8. A pharmaceutical composition for preventing or treating an infectious disease, which comprises the chimeric antigen having enhanced multiple immune function according to claim 1 as an active ingredient. The above-mentioned infectious diseases include hepatitis B virus, hepatitis C virus, hepatitis A virus, AIDS virus, MERS virus, influenza A virus, human papillomavirus, lymphocytic choriomyelitis virus (LCMV) or simple herpes virus (HSV). ) The pharmaceutical composition for preventing or treating an infectious disease according to claim 10, which is characterized by being an infectious disease. An immunopotentiating agent composition comprising the chimeric antigen having enhanced multiple immune functions according to claim 1 as an active ingredient. The chimeric antigen with enhanced multiple immune function is
(I) It specifically binds to diseased cells, which are target cells, and
(Ii) Promotes recovery of exhausted CD8 + T cells to induce T cell activity, and (iii) Induces antigen-antibody reaction to enhance humoral immune response and cytotoxic effects by natural killer cells (NK cells). The immunopotentiator composition according to claim 12, which is characterized by activation. The immunopotentiator according to claim 13, wherein the antigen-antibody reaction induction according to (iii) is enhanced by a specific antigen-antibody reaction formed in an individual body by an immune reaction-inducing region of the chimeric antigen. Composition. A vaccine composition for cancer or infectious disease, which comprises the chimeric antigen having enhanced multiple immune function according to claim 1 as an active ingredient.

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