Production of ultra-advanced humanized mouse model based on umbilical cord blood-derived hematopoietic stem cell transplantation and immune cell expansion technology, and method for producing same

Abstract

The present invention relates to the production of an ultra-advanced humanized mouse model based on umbilical cord blood-derived hematopoietic stem cell transplantation and immune cell expansion technology, and a method for producing same and, more specifically, to a humanized mouse based on human stem cell transplantation and immune boosting, which is produced by administering cephalosporin antibiotics, fluoroquinolone antibiotics, and vitamins to a mouse into which human stem cells have been transplanted, and to a method for producing same.

Description

Manufacture of the Advanced Humanized Mouse Model with Transplantation of Umbilical Cord Blood-Derived Hematopoietic Stem Cells and Immunoboosting, and Manufacturing Method Thereof

[0001] The present invention relates to the production of an ultra-advanced humanized mouse model based on umbilical cord blood-derived hematopoietic stem cell transplantation and immune cell expansion technology, and a method for producing the same.

[0002] Research on human diseases presents limitations in clinical trials due to technical and ethical issues. Therefore, alternative approaches utilizing animals, known to be anatomically and physiologically similar to humans, are being utilized effectively. To achieve this, human diseases are induced in laboratory animals and various treatment options are explored. In particular, with the recent growth in the development of cutting-edge biopharmaceuticals, preclinical research in various fields is becoming a key issue in linking clinical trials. This is because utilizing "more appropriate models" is not only enabling the application of cellular-level research results to the human body, but is also becoming an essential evaluation item for ensuring efficacy and safety.

[0003] The suitability and toxicity assessment of advanced biopharmaceuticals and the application of novel medical technologies through disease-reproducing models play a crucial role in evaluating various medical approaches. However, approaches utilizing conventional animal models have been criticized for failing to closely represent patients' diseases and conditions. Recently, as part of the effort to develop more appropriate research resources, technologies for improving laboratory animal models are actively being developed, and these are being directly utilized in the development of advanced biopharmaceuticals (new substances) and new drugs.

[0004] Advances in laboratory animals include genetically modified animals such as rodents, minipigs, and primates, as well as PDX (patient-derived xenograft) / CDX (cell-derived xenograft) mice (xenograft mice transplanted with human tissues or cells). These can be utilized more closely to humans and human diseases through the development of “more appropriate laboratory animal disease models.” In particular, humanized mice are laboratory animals that have had human immune systems, such as human T cells, B cells, and NK cells, introduced into the host, and are highly valuable for studying human immune responses to biopharmaceuticals.

[0005] However, xenograft mice have limitations in assessing human-specific drug responses, and human-immunized mice only have performance guarantees of less than 40% of human blood cells (human-specific CD45 positive cells). Furthermore, the relatively short survival period required for stability and efficacy verification (survival rates decline sharply after 25 weeks) presents a significant barrier to overcoming survival rates, and the high cost must also be considered.

[0006] Until now, mice used in new drug development have shown limitations in the validation phase of therapeutic development due to differences in the human immune system. While previously approved targeted anticancer drugs such as pembrolizumab (KEYTRUDA® from MSD) and nivolumab (Opdivo® from BMS) have shown low reactivity in existing preclinical results, many new drugs have been reported to have shown high reactivity in tumor models (CDX or PDX) using human-immunized mice. This is because the results of the preclinical testing phase, which essentially involves using experimental animals with immune systems different from humans, can lead to discrepancies between actual clinical results. In this respect, human-immunized mice can be applied as disease-specific avatar models as preclinical biomaterials, providing a high-end technology-based material that can verify and evaluate highly stable data in the preclinical phase.

[0007] Recently, the market for autologous, allogeneic, and xenogeneic cell therapies has been booming, aiming to overcome the limitations of current cancer treatments. In particular, with the strengthening of regulations on the approval and review of biological products, etc., the need for sophisticated validation in disease-reproducing environments that simulate human diseases is crucial. This means that T-cell therapy, NK cell therapy, hematopoietic stem cell therapy, and induced pluripotent stem cell therapies must demonstrate precisely validated mechanisms of action, including pharmacodynamics, pharmacokinetics, and clinical efficacy, as well as safety and efficacy.

[0008] Recent research papers in the field of anticancer drug development are being utilized in preclinical studies to elucidate the efficacy of targeted anticancer drugs alone or in combination with cell therapy after inducing various carcinogenesis models (breast cancer, colon cancer, lung cancer, etc.) (① Richard B Bankert et al. Humanized mouse model of ovarian cancer recapitulates patient solid tumor progression, ascites formation, and metastasis, PLoS One. 2011;6(9):e24420. ② J. Jason Morton et al. Humanized xenograft models: narrow the tumor-microenvironment gap. Cancer Res 2016, Nov 1; 76(21): 6153-6158. ③ Nina B. Horowitz et al. Humanized mouse model for advanced innate lymphoid cell-based cancer immunotherapies. Frontiers in immunology, ④ Ling Yin et al. Humanized mouse model: a review on preclinical applications for cancer immunotherapy. Am J Cancer Res. 2020; 10(12): 4568-4584. ⑤ Morgane M. Cogels et al. Humanized Mice as a Valuable Pre-Clinical Model for Cancer Immunotherapy Research. Front Oncol. 2021; 11: 784947.). In addition, as a prior art, Patent Publication No. 2005-0120863 discloses a method for testing the anticancer activity of an anticancer agent using an H-ras overexpressing mouse.

[0009] In the case of human immunized mice developed so far, the proportion of human blood cells (human CD45 surface marker) in the host's blood is at the level of 40%, and it has been utilized to present in-depth research results in the field of oncology worldwide, but recently, with the growth of advanced biopharmaceuticals (cell therapy market), it is expected to be applied to general disease fields such as autoimmune diseases and infectious diseases (e.g., SARS-CoV2), and to reproduce human disease-specific diseases through partial humanization or integrated humanization models, and as a material for efficacy verification and evaluation, as a prevention and evaluation tool at the preclinical stage.

[0010] However, in the case of conventional human immunized mice, the ratio of human blood cells within the mouse and the ratio of human immune cells within the host are low and the survival period is not long, so there is a need for improvement, and there is a demand for the production of experimental subjects of human immunized mice with the same performance and the establishment of stability in the production stage of human immunized mice.

[0011] Meanwhile, cefazolin, a first-generation cephalosporin antibiotic, is only used to prevent infection when producing human-immunized mice, and there have been no reported cases of its application in research to increase the proportion of human-specific CD45 positive cells when producing human-immunized mice. In addition, a study has been published showing that when cefazolin is treated to lymphoid cells, their activity decreases by 44% (EA CHAPERON et al. suppression of lymphocyte response by cephaloporins, Infection and immunity, 1997).

[0012] Against this backdrop, the present inventors have continued their research to provide highly advanced humanized mice transplanted with human stem cells that have increased the proportion of human blood cells in the mouse, the proportion of human immune cells in the host, and the survival ability based on immune cell expansion (boosting) technology, and as a result, they have completed the present invention by elucidating the effect of significantly improving the immune cell expression rate of humanized mice based on triple combination boosting with cephalosporin antibiotics.

[0013]

[0014] The present invention is intended to solve the problems of the prior art, and the purpose of the present invention is to provide an advanced humanized mouse transplanted with human stem cells that have increased human blood cell ratios within the mouse, human immune cell ratios within the host, and survival ability.

[0015] Another object of the present invention is to provide an advanced humanized mouse by minimizing the host's graft rejection reaction by applying to the mouse a compound for suppressing metabolic disturbance and human cells donated to the host, and at the same time optimizing the appropriateness of the stem cell transplantation amount.

[0016] Another object of the present invention is to provide a method for producing an advanced humanized mouse having increased human blood cell ratio, various activated human immune cell ratios, and viability in the host by applying a boosting technique to donated human immune cells in the mouse.

[0017]

[0018] In order to achieve the above-described purpose, the present invention provides a humanized mouse based on human stem cell transplantation and immune boosting, which is produced by administering a cephalosporin antibiotic, a fluoroquinolone antibiotic, and vitamins to a mouse into which human stem cells have been transplanted.

[0019] Conventional approaches have involved suppressing host bone marrow cells through high-dose radiation exposure. However, radiation exposure throughout the body, not just the bone marrow, causes damage to tissues and all cells, leading to metabolic disruption and significantly lower survival rates. Furthermore, the host's immune rejection of xenotransplanted human cells limits the creation of high-performance humanized mice and the establishment of long-term models. Consequently, the blood analysis process, the final verification step, cannot replicate the sophisticated human immune system due to the relatively high host immunity. Even if it could be replicated, it could only measure the performance of representative blood cells (human CD45).

[0020] The present invention has the advantage of maintaining and preserving a stable, long-term human immune system by boosting immune cells by administering cephalosporin antibiotics, fluoroquinolone antibiotics, and vitamins to mice transplanted with human stem cells, thereby inducing cell-specific activity of the transplanted human cells and maximally suppressing the host's immunity. The vitamin is preferably vitamin D.

[0021] In one embodiment of the present invention, the mouse into which the human stem cells have been transplanted may be an immunodeficient mouse into which the human stem cells have been transplanted.

[0022] The above immunodeficient mouse refers to a non-obese diabetic (NOD) mouse that is double homozygous for a severe combined immunodeficiency (SCID) mutation and an interleukin-2Rγ (IL-2Rγ) allele mutation (γ null). The SCID mutation refers to a mutation that causes a defect in functional B cells and T cells. For example, the SCID mouse (CB-17-Prkdcscid) has a defect in the rearrangement of the B cell receptor (BCR) and the T cell receptor (TCR), causing a defect in functional B cells and T cells. The immunodeficient mouse is also called a NOD scid gamma (NSG), a NOD / SCID γnull mouse, or a NOD / SCID IL-2RγKO mouse. NSG mice were developed in the laboratory of Dr. Leonard Shultz at The Jackson Laboratory. NSG mice can be commercially obtained from Jackson Laboratory or prepared according to a known method (Shultz LD, et al., J. Immunol. 2005; 169: 204-209). For example, NSG mice can be obtained by backcrossing C57BL / 6J-γ null mice with NOD / SCID mice nine times. NSG mice are known to lack functional T cells and functional B cells, have reduced macrophage function, have abolished NK cells or NK activity, and have reduced dendritic cell function.

[0023] In addition, in one embodiment of the present invention, the human stem cell transplanted mouse may be a mouse in which the IL2 receptor common gamma chain (IL2 receptor common gamma chain) based on prkdc scid (DNA-PKcs Severe Combine Immuno Deficiency) is deficient or mutated, a mouse in which the RAG2 (Recombination activating gene2) gene is deficient or mutated, or a mouse in which the B2m (Beta-2-microglobulin) is deficient or mutated.

[0024] Here, it is preferable that the human stem cell is a CD34+ hematopoietic stem cell derived from human fetal liver or umbilical cord blood.

[0025] The present invention is characterized in that it boosts immune cells by administering cephalosporin antibiotics, fluoroquinolone antibiotics, and vitamins to mice transplanted with human stem cells, thereby maintaining immune activity for a long period of time.

[0026] That is, by boosting immune cells using the triple combination of the present invention's cephalosporin antibiotic, fluoroquinolone antibiotic, and vitamins, the host's immunity is maximally suppressed while simultaneously maintaining the activity of transplanted human cells, thereby maintaining the human immune system and immune activity for a long period of time. The immune cell boosting process is maintained for, but is not limited to, four weeks after transplantation.

[0027] In one embodiment of the present invention, the mouse into which the human stem cells have been transplanted is characterized in that the mouse is a mouse into which the human stem cells have been transplanted into a mouse administered a compound for suppressing metabolic disturbance between the host and donor cells, including busulfan and cyclophosphamide.

[0028] The present invention solves the problem of low survival rate due to metabolic disturbance caused by tissue and cell damage caused by conventional high-dose radiation exposure. It adopts a method that utilizes the side effects of anticancer drugs and minimizes immunosuppression of the host by using an anticancer drug and an immunosuppressant (busulfan + cyclophosphamide). In other words, while the conventional method performs blood analysis through radiation-based bone marrow suppression and stem cell transplantation, the present invention performs blood analysis through bone marrow suppression, stem cell transplantation, and immune boosting using a compound. The method using the compound of the present invention suppresses only the actual bone marrow cells of the host, making it a very easy step to obtain a long-term and stable model. As a result, the stem cell transplantation process also has the advantage of high efficiency with a small amount of cells by using 1 / 10 to 1 / 100 of the cells of the conventional method.

[0029] In this way, the present invention has the advantage of minimizing various factors that pose a risk to the host in order to transplant and maintain the human immune system into a xenobiotic animal, and the final blood analysis process can also be confirmed with various immune cell-specific markers.

[0030] Meanwhile, the present invention provides a method for producing a humanized mouse based on human stem cell transplantation and immune boosting, comprising the steps of: administering a compound for suppressing metabolic disturbance to a mouse (S10); transplanting human stem cells into the mouse to which the compound for suppressing metabolic disturbance has been administered (S20); and administering a cephalosporin antibiotic, a fluoroquinolone antibiotic, and vitamins to the mouse to which the human stem cells have been transplanted to perform immune boosting (S30).

[0031] In the above step S10, the compound for suppressing the metabolic disturbance may include busulfan and cyclophosphamide.

[0032] The above step S20 may include a step (S22) of controlling the human-specific immunization rate in the mouse by controlling the number of human stem cells and transplanting them into the mouse to which the compound for suppressing the metabolic disturbance has been administered.

[0033] At this time, the human stem cells are hematopoietic stem cells, and the human stem cells can be transplanted by administering them through a vein of the mouse.

[0034] The present invention produced humanized mice using immunodeficient mice, and as a result of comparing before and after production, the ratio of human-specific CD45 marker (human-specific blood cell surface marker) in the blood of the host was found to be 90% or higher. In addition, as a result of confirming the ratio of human-specific blood cell surface marker in the blood of the mouse of the present invention compared to conventional humanized mice, it was confirmed that it had a significantly higher ratio. In addition, as a result of confirming the distribution of lower-order precise immune cells by specifying human lymphoid immune cells in the host, adaptive immune T cells, helper T cells, cytotoxic T cells, NK cells, NKT cells, B cells, etc. were confirmed.

[0035] Therefore, the present invention has improved quantitative performance and established stability and processing methods during the manufacturing process. Furthermore, the present invention enables the production and manufacture of experimental specimens that achieve consistent performance across batches.

[0036]

[0037] According to the humanized mouse and the method for producing the same according to the present invention, an advanced humanized mouse can be provided in which human stem cells are transplanted, which have increased human blood cell ratios within the mouse, human immune cell ratios within the host, and survival ability.

[0038] Additionally, by administering human cells donated to the host and compounds for suppressing metabolic disturbances to mice, it is possible to minimize graft rejection and provide highly humanized mice with optimal stem cell transplantation (minimum and optimal transplantation amount).

[0039] The present invention can be applied to the production of humanized mice with a highly developed human immune system, enabling more appropriate pharmaceutical development strategies. The humanized mice, equipped with a highly developed human immune system, can be utilized as biomaterials that specifically represent various human diseases and enable close verification of the dynamics of human immune cells, including tracking the activation / inactivation of biopharmaceuticals, as well as evaluating their efficacy and safety.

[0040]

[0041] Figure 1a is a schematic diagram of a human immunization manufacturing method according to one embodiment of the present invention.

[0042] Figures 1b to 1c are diagrams illustrating the effects of immunodeficiency (failure) due to immunosuppression of the host.

[0043] FIGS. 2A to 2C are diagrams showing the infection history and purity information of human stem cells (CD34-positive HSCs) and the activity of the corresponding cells according to one embodiment of the present invention.

[0044] FIG. 2d is a diagram illustrating human stem cell (CD34-positive HSCs) transplantation according to one embodiment of the present invention.

[0045] FIG. 2e is a diagram illustrating controlling the human-specific immunization rate in a mouse by controlling the number of donated human stem cells and transplanting them according to one embodiment of the present invention.

[0046] FIGS. 3A to 3F are drawings illustrating the performance of an individual (activity of blood cells) according to a human immunization manufacturing technology according to one embodiment of the present invention.

[0047] FIGS. 4A to 8D are diagrams illustrating the verification of human lymphoid immune cells in a triple-combination immunoboosting-based humanization model according to one embodiment of the present invention.

[0048] Figure 9 is a diagram illustrating the effects of immunodeficiency (failure) according to the immune combination suppression technology.

[0049] Figures 10a to 11d are drawings comparing and explaining the performance of an object according to triple combination, double combination, and single boosting.

[0050] Figure 12 is a diagram comparing and explaining the survival rate according to triple combination, double combination, and single boosting concentrations.

[0051] Figures 13a and 13b are diagrams showing the results of immune boosting according to cefazolin, a first-generation cephalosporin antibiotic.

[0052]

[0053] The present invention is susceptible to various modifications and embodiments, and thus, a preferred embodiment will be described in detail. It will be apparent to those skilled in the art that the present invention can be embodied in other specific forms without departing from the spirit and essential characteristics of the present invention. Unless otherwise defined, all technical and chemical terms used herein have the same meaning as commonly understood by one skilled in the art to which the present invention pertains. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

[0054] <Example>

[0055] Example 1: Process of humanized model manufacturing technology and the effect of immunodeficiency (failure) due to immunosuppression of the host

[0056] Figure 1a is a schematic diagram of a humanized mouse manufacturing method according to one embodiment of the present invention. Referring to Figure 1a, the humanized mouse manufacturing technology of the present invention is reconstructed through a total of four steps. Namely, Step 1 is the host immunosuppression step, Step 2 is the stem cell transplantation step, Step 3 is the donor cell boosting step, and Step 4 is the performance verification step through blood analysis.

[0057] In order to maximize the efficiency of the host organism for the production of humanized mice, it is essential to maximize the suppression of the immune rejection response of human cells by inducing immunosuppression of the donor organism (host: laboratory animal). For this test, 4-week-old non-immunodeficient Balb / C mice (normal control group) and immunodeficient mice (NOD-scid IL2Rgammanull / mutation / KO / KD, NOD-scid IL2Rg null , NOD.Cg-Prkdc scid IL2rg tm1wijl / SzJ, NOD.Cg-Prkdc scid IL2rg tm1wijl / SzJ, NOD-scid IL2; immunodeficient mice) and experimental animals that had been introduced with human immunization technology.

[0058] Referring to Figure 1b, in order to track the change in blood leukocyte common antigen before / after immune control in relation to verification of the host's immunosuppression technology, 1) normal control group (non-immunodeficient mouse Balb / C), 2) immunodeficient mouse (NOD-scid IL2Rgammanull / mutation / KO / KD, NOD-scid IL2Rg null, NOD scid gamma, 3) We observed changes in the blood mouse-specific CD45 antigen in the additional immunosuppression group based on immunodeficient mice (administration of cephalosporin antibiotics, fluoroquinolone antibiotics, and vitamins to immunodeficient mice). CD45 is a leukocyte common antigen, an antigen that exists in all animals, including humans, and is an antigen with species specificity. The human leukocyte common antigen is called human-specific CD45, and in the case of mice, it is called mouse-specific CD45.

[0059] Referring to Figure 1b, after host immunosuppression, the mouse-leukocyte common antigen (mouse CD45, mDC45) was significantly reduced in response to immunosuppression. In contrast, the human-leukocyte common antigen (human CD45, hCD45) was not observed after host immunosuppression, as it was in the pre-human cell transplantation state.

[0060] Referring to Figure 1c, the verification results showed that the expression of mouse-specific leukocyte common antigen was predominant in the normal control group and the immunodeficient mouse group, whereas the expression of mouse-specific leukocyte common antigen was significantly reduced in the group that underwent additional immunosuppression. In the control group where immunosuppression was not induced, the blood mouse leukocyte common antigen (mCD45) remained high, whereas the group that induced immunosuppression showed a tendency for the expression of mouse leukocyte common antigen to significantly decrease.

[0061]

[0062] Example 2: Information on human stem cells (CD34-positive HSCs) and their activity

[0063] Before transplanting human cells into mice, the cell lines used for transplantation were checked for activity and contamination. Referring to Figure 2a, a total of three stem cell samples were used for this evaluation, human CD34-positive hematopoietic stem cells (umbilical cord blood-derived hematopoietic stem cells). The three cell lines were negative for HIV (acquired immunodeficiency syndrome; AIDS), HBV (hepatitis B), and HCV (hepatitis C) infection, and the activity of the corresponding cells was 93-95% or higher. Referring to Figure 2b, the actual purity and activity of the cell lines used in this study were confirmed by flow cytometry and live cell counting immediately before stem cell transplantation. As a result, the human CD34-positive hematopoietic stem cells were 99.6%, 99.3%, and 99.4%, respectively, confirming that they were highly pure stem cells. In addition, the activity of these cells was also verified to be over 95%, and it was confirmed that they were pure HSCs that were not contaminated with non-human xenogeneic cells (Fig. 2c).

[0064] Referring to Figure 2d, HSC 1x10 for stem cell transplantation 3 ~ 1x10 5 (1,000 to 50,000 cells are injected into the caudal vein to induce systemic circulation (vein → ventricle → artery → capillary).

[0065] Referring to Figure 2e, the performance controllability of the humanized mouse manufacturing technology was verified by hematopoiesis of the amount of donated cells per mouse during the stem cell transplantation process. After transplanting 1,000, 10,000, and 50,000 cells into each host, the reproducibility of the human immune system was evaluated 8 weeks later (12 weeks after birth). For this purpose, human-specific CD45 was quantified and numerically expressed. When 1,000 human hematopoietic stem cells were transplanted, the proportion of human-specific CD45 in the host was 31.6%, and the proportion of mouse-specific CD45 was 65.2%. When 10,000 hematopoietic stem cells were transplanted, the expression rate of human-specific CD45 was 63.3%, and the expression rate of mouse-specific CD45 was 35.7%. Furthermore, when 50,000 hematopoietic stem cells were transplanted, the proportion of human-specific leukocyte common antigen was estimated to be 98%, and that of mouse-specific leukocyte common antigen was 1.3%. In other words, it was confirmed that the reproducibility of the human immune system within the host can be configured and manufactured to be approximately 30%, 60%, and 90% or more through transplantation of 1,000 to 50,000 cells using human immunization manufacturing technology.

[0066] Therefore, the present invention has an advantage in that it can control the human-specific immunization rate in mice by controlling the number of human stem cells and transplanting them into mice administered with a compound for suppressing metabolic disturbance.

[0067]

[0068] Example 3: Performance of an individual (blood cell activity) according to human immunization manufacturing technology

[0069] To verify the reproducibility of the human immune system according to the human immunization manufacturing technology manufactured by administering cephalosporin antibiotics, fluoroquinolone antibiotics, and vitamins (triple combination boosting) to mice transplanted with human stem cells, the non-human immunized group (normal control group) and the human immunized mouse group (at 12 weeks of age) were evaluated with human / mouse-specific leukocyte co-antigen (mouse / human-specific CD45).

[0070] Referring to Figure 3a, 24 hours after donor cell transplantation, a mixture of Cefaclor (cephalosporin) 0.01 ㎍ / ml to 10 mg / ml, Ciprofloxacin (fluoroquinolone) 0.01 ㎍ / ml to 10 mg / ml, and vitamin 0.1 ㎍ / ml to 100 mg / ml is administered. Preferably, a mixture of Cefaclor (cephalosporin) 0.3 ㎍ / ml, Ciprofloxacin (fluoroquinolone) 0.1 ㎍ / ml, and vitamin D3 1 ㎍ / ml is administered twice a week for 4 weeks to induce the activation of human immune cells (a total of 8 administrations: performed until the 8th week of life).

[0071] Referring to Figure 3b, a flowchart is provided for analyzing specific immune cells within the buffy coat of blood collected for human-specific lymphoid cell analysis in humanized mouse blood. Within the buffy coat, various cells, including leukocytes and platelets, exist, and among these, only lymphoid cells within singlet cells are specifically labeled and the expression level of human or host (mouse) leukocyte common antigens is measured.

[0072] Figure 3c shows the expression level of the common leukocyte antigen according to the presence / absence of immune boosting (performance / comparative evaluation: based on the 12th week). In the normal control group (non-human immunized group), only the expression of mouse-specific blood cells was observed, and no human cells were found. On the other hand, in the human immunized mouse group before immune boosting, human-specific CD45 was detected at an average of 41.8%, and the expression rate of mouse-specific CD45 was an average of 58.0%, with two types of cells observed in one individual. On the other hand, in the human immunized mouse group after immune boosting, human-specific CD45 was detected at an average of 87.9%, and the expression rate of mouse-specific CD45 was an average of 10.0%, with two types of cells observed in one individual. Compared to the human immunized mouse group before immune boosting, the expression rate of human-specific CD45 was significantly increased.

[0073] Referring to Fig. 3d, as verification data tracking the performance of the same individual before and after the human immunization manufacturing technology, the control group expressed human-specific CD45 at 0.08 to 0.03 from week 12 to week 40, whereas in the pre-boosting stage, the expression was maintained at an average of 38.8% at week 12, 39.8% at week 20, 40.7% at week 30, and 35.3% at week 40. However, in the case of the humanized model group to which triple combination boosting was applied, it was confirmed that the high human-specific CD45 expression was greatly increased and maintained at an average of 90.5% at week 12, 93.3% at week 20, 91.7% at week 30, and 82.8% at week 40.

[0074] Figure 3e is a numerical result of quantifying the specific expression rate of human leukocyte common antigen of all individuals used in this example, and Figure 3f is a bar graph of the humanization performance between groups according to the presence or absence of immune boosting.

[0075]

[0076] Example 4: Validation of a humanized model based on triple-combination immunoboosting.

[0077] To evaluate the composition or activity of human-specific major lymphoid immune cells in experimental animal subjects with a highly reproducible human immune system, we evaluated helper T cells, cytotoxic T cells, NK cells, NKT cells, and B cells using surface antigens of specific immune cells.

[0078] Referring to Fig. 4a, the expression of helper T cells (CD45+ / CD3+ / CD4+) specific cells among T lymphoid cells was confirmed through flow cytometry, and as a result, a very small amount existed in the body until 8 to 12 weeks and a strong increase was observed from the 20th week. T lymphoid cells are cells belonging to active immunity, and it takes time to mature and differentiate into specific helper T cells. Fig. 4b describes the expression rate and continuous flow of all individuals within the group, and according to Fig. 4c and Fig. 4d, the average expression amount of helper T cells slightly increased to 2.7% at the 12th week, 7.5% at the 20th week, 20.8% at the 30th week, and 23% at the 40th week.

[0079] Referring to Fig. 5a, the expression of cytotoxic T cells (CD45+ / CD3+ / CD8+) specific cells among T lymphoid cells was confirmed through flow cytometry, and the pattern of increase and maintenance from week 12 to week 40 was confirmed. Cytotoxic T cells are known as cells that destroy viruses, somatic cells, and tumor cells and are cells that stimulate the activity of helper T cells. Fig. 5b shows the results of the expression rate of all individuals, and according to Fig. 5c and Fig. 5d, the average expression amount of cytotoxic T cells was 5.3% at week 12, 7.5% at week 20, 6.0% at week 30, and 9.5% at week 40.

[0080] Referring to Figure 6a, the expression of natural killer cells (CD45+ / CD3- / CD56+) specific cells among T lymphoid cells was confirmed through flow cytometry to determine the degree of composition and activity within the host. NK cells are major cells responsible for innate immunity, have a similar role to cytotoxic T cells, and are activated 3 days after infection, rapidly influencing the response of infected cells to induce apoptosis. Figure 6b shows the quantitative numerical results of the T cell-specific expression levels of all individuals, which were tracked from the 12th to the 40th week after birth. According to Figures 6c and 6d, the average expression rate of blood natural killer cells was 2.5% at the 12th week, 0.6% at the 20th week, 4.1% at the 30th week, and 2.3% at the 40th week.

[0081] Referring to Figure 7a, the expression of natural killer T cells (CD45+ / CD3+ / CD56+) among T lymphoid cells was analyzed using flow cytometry to determine the degree of composition and activity within the host. NKT cells are a group of T cells that share characteristics with both T cells and natural killer cells. As a result, strong expression was observed at an average of 20.7% at week 12, but a rapid decline occurred from week 20, and a pattern similar to the control group was observed at week 40.

[0082] Referring to Figure 8a, the total B lymphocytes (CD45+ / CD19+) in the blood within the host were analyzed by flow cytometry, and no human-specific cell expression was detected in the control group. Figure 8b shows the quantitative numerical results of the B cell-specific expression levels of all individuals, which were tracked from the 12th week of life to the 40th week. According to Figures 8c and 8d, the human-immunized mouse group showed a strong increase and maintenance, with 56.2% at the 12th week, 52.8% at the 20th week, 43.9% at the 30th week, and 38.5% at the 40th week.

[0083]

[0084] Comparative Example 1: Effects of Immunodeficiency (Failure) According to Immune Combination Suppression Technology

[0085] The effects of immunodeficiency (failure) following immunosuppressive therapy alone and in combination with immunosuppressive agents were examined. Figure 9 shows the results of measuring the expression rate of mouse CD45 induced by immunosuppressive agents.

[0086] At this time, the immunosuppressive compound busulfan was administered at 20 mg / kg, and cyclophosphamide was administered at 20 mg / kg.

[0087] Referring to Figure 9, it can be confirmed that compared to the normal control group (Balb / c mice that have the host's original immunity) and the immunodeficient mice (NSG mice that have the host's original immunity suppressed and are often used as a control group for disease reproducibility experiments), the immunodeficient mice administered with the immunosuppressive compound Busulfan are more effectively immunosuppressed and thus immunodeficiency is induced.

[0088] In addition, the present invention confirmed that in the case of immunodeficient mice, when cyclophosphamide was administered alone instead of busulfan, immunodeficiency was induced to a degree equivalent to that in mice administered busulfan alone.

[0089] As shown in Fig. 9, immunodeficient mice administered with busulfan and cyclophosphamide in combination showed significantly more immunosuppression and induced immunodeficiency compared to mice administered with busulfan or cyclophosphamide alone. Therefore, the present invention confirmed that the immunosuppression of the host can be minimized by using the combination of an anticancer agent and an immunosuppressant (busulfan + cyclophosphamide).

[0090]

[0091] Comparative Example 2: Comparison of Individual Performance According to Triple Combination, Double Combination, and Single Boosting

[0092] Referring to Figure 10a, the activity of donor cells was boosted by triple combination (cephalosporin + fluoroquinolone + vitamins), double combination (cephalosporin + fluoroquinolone), and single combination (cephalosporin, fluoroquinolone, vitamins), and the performance of the human immune system according to each administration method was verified.

[0093] Referring to Figure 10b, the analysis was performed using flow cytometry, and among these, a higher expression of lymphatic cells was confirmed in the group that induced double combination boosting compared to single boosting, and the highest expression of lymphatic cells was confirmed in the group that induced triple combination boosting.

[0094] Figures 11a to 11d show the verification results for the performance as a humanized model and the stable state of the immunized subject for the minimum and maximum doses of the drug for immune boosting, respectively.

[0095] Figure 11a shows the results of confirming the expression rate of human leukocyte common antigen according to triple combination, double combination, and single boosting, and verified the performance of the humanized model according to the minimum / maximum treatment dose. The minimum treatment of the triple combination of 0.01 μg / ml ciprofloxacin, 0.01 μg / ml cefaclor, and 0.1 μg / ml vitamin D3 confirmed an average of 40.2% expression of human leukocyte common antigen. On the other hand, when the maximum drug dose was 10 mg / ml ciprofloxacin, 10 mg / ml cefaclor, and 100 mg / ml vitamin D3, the expression of the marker was confirmed to be 38.9%.

[0096] In the single-dose group treated with the lowest dose of the triple-combination booster drug, ciprofloxacin 0.01 μg / ml, cefaclor 0.01 μg / ml, and vitamin D3 0.1 μg / ml, human leukocyte common antigen expression was observed in 35.6%, 33.4%, and 42.6%, respectively. Even in the single-dose group treated with the highest dose of ciprofloxacin 10 mg / ml, cefaclor 10 mg / ml, and vitamin D3 100 mg / ml, expression rates of 32.2%, 41.3%, and 35.7% were recorded.

[0097] As a result of confirming the effect of boosting by a double combination or boosting alone by a double combination compared to the performance of a triple combination, an average of 32.0% of human leukocyte common antigen was confirmed at a mixed dose of 0.01 ㎍ / ml of ciprofloxacin and 0.01 ㎍ / ml of cefaclor, and an average of 41.6% of the marker of the antigen was expressed at the maximum dose of 10 mg / ml of ciprofloxacin and 10 mg / ml of cefaclor.

[0098] Among the dual combination boosters, the expression rate of leukocyte common antigen was also measured at the minimum and maximum doses in each single treatment group, and it was confirmed to be 33.4% at the 0.01 μg / ml dose of ciprofloxacin and 35.5% at the maximum dose of 10 mg / ml of ciprofloxacin. In addition, the expression of leukocyte common antigen was confirmed to be relatively similar to that of the single group of the triple combination at the minimum dose of 0.01 μg / ml of cefaclor and the maximum dose of 10 mg / ml of cefaclor. The corresponding values ​​were confirmed to be 32.6% at the minimum dose and 37.6% at the maximum dose.

[0099] Figure 11b is the analysis result for the quantitative analysis of each of the human / host (mouse) leukocyte common antigens, and is dot plot data for the quantitative values ​​of Figure 11a.

[0100] Figure 11c is a bar graph of the values ​​calculated in Figure 11a, showing the mean values ​​and standard errors of the mice in the triple combination boosting, triple combination boosting with single boosting (A, B, C), double combination boosting, and double combination boosting with single boosting (A, B) groups. Each group consisted of 10 individuals, and the expression rate of human leukocyte common antigen due to the minimum / maximum dose treatment was measured for a total of 7 groups.

[0101] Figure 11d shows the results of collecting the expression rates of human and mouse leukocyte common antigens of the humanized model entity confirmed in Figure 11c.

[0102]

[0103] Comparative Example 3: Comparison of survival rates according to triple combination, double combination, and single boosting concentrations.

[0104] Figure 12a shows the survival rate according to the triple combination, double combination, and single boosting concentrations. The concentration range for optimal survival safety among the humanized model groups is 0.01 ㎍ / ml to 10 mg / ml for Cefaclor (cephalosporin), 0.01 ㎍ / ml to 10 mg / ml for Ciprofloxacin (fluoroquinolone), and 0.1 ㎍ / ml to 100 mg / ml for Vitamin.

[0105] When the concentration range was exceeded, the number of animals continuously decreased by 3 at 12 weeks, 1 at 13 weeks, 2 at 14 weeks, 1 at 15 weeks, 2 at 20 weeks, and 1 at 25 weeks at 0.009 ㎍ / ml, which is within the appropriate standard of Cefaclor (cephalosporin). At 10.5 mg / ml, which is above the appropriate standard of the treatment, 1 at 12 weeks, 1 at 15 weeks, 1 at 12 weeks, 2 at 25 weeks, and 1 at each of 30 and 33 weeks died.

[0106] In the case of ciprofloxacin (fluoroquinolone), when treated within the appropriate standard, all animals died: 1 at 12 weeks, 1 at 13 weeks, 2 at 14 weeks, 2 at 15 weeks, 2 at 16 weeks, 1 at 27 weeks, and 1 at 30 weeks. When treated with a high dose (10.5 mg / ml) exceeding the appropriate standard, all animals died: 1 at 15 weeks, 20 weeks, 22 weeks, 25 weeks, 30 weeks, 37 weeks, and 38 weeks.

[0107] Vitamin D3 has been shown to have no toxicity that inhibits survival, regardless of whether it is administered in low or high doses, and as a booster of donated immune cells, survival rates have been maintained. However, it has been shown to have no effect on boosting human cells.

[0108] The results in Figure 12b are the results of evaluating the expression rate of human and mouse leukocyte common antigens in the blood at the time of individual death, and it was confirmed that the proportion of host (mouse) immune cells was significantly higher at week 23, the time of individual death, compared to week 12 (Cefaclor alone: ​​mCD45 expression; increased from 61.6% at week 12 to 92.5% at week 23. Ciprofloxacin alone: ​​mCD45 expression; increased from 57.9% at week 12 to 82.9% at week 23). In addition, it was shown that the expression of the host (mouse) leukocyte common antigen increased by about 20-25% at the 30th week, when the individual died, even at a high concentration exceeding the appropriate level (Cefaclor alone: ​​mCD45 expression; increased from 58.7% at 12 weeks to 77.3% at 30 weeks. Ciprofloxacin alone: ​​mCD45 expression; increased from 55.6% at 12 weeks to 71.0% at 30 weeks). This confirmed that the individual's death was induced by an immune rejection reaction due to a disturbance in the ratio of heterologous immune cells within an individual, and the maintenance of performance was significantly reduced.

[0109]

[0110] Comparative Example 4: Comparison of immune boosting effects according to first-generation antibiotics.

[0111] Figures 13a and 13b show the results of immune boosting according to cefazolin, a first-generation cephalosporin antibiotic.

[0112] Table 1 shows the results of measuring the expression ratio of blood leukocyte co-antigens, B cells, T cells, NK cells, and NKT cells in humanized mice based on single boosting with cefazolin, a first-generation cephalosporin antibiotic.

[0113] Expression and proportion of human-specific cells in the host (%) Human leukocyte common antigen (hCD45) 20.6 Total B lymphocytes (hCD45+ / hCD19+) 25.1 Total T lymphocytes (hCD45+ / hCD3+) 12.3 Helper T lymphocytes (hCD45+ / hCD3+ / hCD4+) 3.1 Cytotoxic T lymphocytes (hCD45+ / hCD3+ / hCD8+) 5.2 Natural killer cells (hCD45+ / hCD56+) 7.1 Natural killer T cells (hCD45+ / hCD56+ / CD3+) 11.1

[0114] According to Table 1, the human leukocyte common antigen (hCD45) expression rate in humanized mice based on single boosting with cefazolin, a first-generation cephalosporin antibiotic, was only 20.6%, whereas the human leukocyte common antigen (hCD45) expression rate in humanized mice based on triple combination boosting with Cefaclor, a third-generation cephalosporin antibiotic of the present invention, was 92.4%, indicating a significantly improved effect.

[0115]

[0116] Meanwhile, the detailed description above should not be construed as limiting in any respect and should be considered illustrative. The scope of the present invention should be determined by a reasonable interpretation of the appended claims, and all modifications within the equivalent scope of the present invention are intended to be included within the scope of the present invention.

Claims

1. A humanized mouse based on human stem cell transplantation and immune boosting, produced by administering cephalosporin antibiotics, fluoroquinolone antibiotics, and vitamins to a mouse transplanted with human stem cells.

2. In paragraph 1, The above human stem cell transplanted mouse is a humanized mouse based on human stem cell transplantation and immune boosting, characterized in that human stem cells are transplanted into an immunodeficient mouse.

3. In paragraph 1, The human stem cell transplanted mouse is a humanized mouse based on human stem cell transplantation and immune boosting, characterized in that the human stem cell is transplanted into a mouse with a defective or mutated IL2 receptor common gamma chain based on prkdc scid (DNA-PKcs Severe Combine Immuno Deficiency), a mouse with a defective or mutated RAG2 (Recombination activating gene2) gene, or a mouse with a defective or mutated B2m (Beta-2-microglobulin).

4. In paragraph 1, A humanized mouse based on human stem cell transplantation and immune boosting, characterized in that the human stem cell transplanted mouse is a mouse in which human stem cells are transplanted into a mouse administered a compound for suppressing metabolic disturbance between host and donor cells, including busulfan and cyclophosphamide.

5. In paragraph 1, A humanized mouse based on human stem cell transplantation and immune boosting, characterized in that the human stem cells are CD34+ hematopoietic stem cells derived from human fetal liver or umbilical cord blood.

6. Step of administering a compound for suppressing metabolic disturbance to a mouse (S10); A step (S20) of transplanting human stem cells into a mouse to which a compound for suppressing the above metabolic disturbance has been administered; and A step of boosting immunity by administering cephalosporin antibiotics, fluoroquinolone antibiotics, and vitamins to the mouse into which the human stem cells have been transplanted (S30); A method for producing a humanized mouse based on human stem cell transplantation and immune boosting, comprising:

7. In paragraph 6, The above S20 step is, A step (S22) of controlling the human-specific immunization rate in the mouse by controlling the number of human stem cells and transplanting them into the mouse to which the compound for suppressing the above metabolic disturbance has been administered; A method for producing a humanized mouse based on human stem cell transplantation and immune boosting, characterized in that it comprises:

8. In paragraph 6, The above human stem cells are hematopoietic stem cells, A method for producing a humanized mouse based on human stem cell transplantation and immune boosting, characterized in that the human stem cells are transplanted by administering them through a vein of the mouse.

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