Personalized humanized animal model transplanted with peripheral blood mononuclear cells (PBMCS) and production method therefor

Abstract

The present invention relates to a personalized humanized animal model transplanted with peripheral blood mononuclear cells (PBMCs) and a production method therefor, and more particularly, to a personalized humanized animal model generated by administering peripheral blood mononuclear cells (PBMCs) of a donor to an immunodeficient mouse, and a production method therefor, wherein the immunodeficient mouse is established as a personalized humanized subject by administering a compound containing busulfan or cyclophosphamide thereto.

Description

Personalized humanized animal model transplanted with peripheral blood mononuclear cells (PBMCS) and method for producing the same

[0001] The present invention relates to a personalized humanized animal model into which peripheral blood mononuclear cells (PBMCs) are transplanted, and a method for producing the same, and more particularly, to a personalized humanized animal model into which peripheral blood mononuclear cells (PBMCs) of a donor are administered to an immunodeficient mouse, wherein the immunodeficient mouse is administered a compound containing busulfan or cyclophosphamide to induce and maintain an extremely inactive host immune system, and is characterized in that the immunodeficient mouse is implemented as a non-clinical entity that completely replicates the immune system of an individual, and a method for producing the same.

[0002]

[0003] Establishing personalized diagnosis and treatment strategies is a new concept in the development of precision medicine (PM) technology. Precision medicine is strengthened by precisely diagnosing diverse diseases, identifying effective, specific drugs tailored to each patient, and developing diagnostic and treatment strategies. Especially in light of the diverse cancer types associated with genetic mutations and the high mortality rates of acute infectious diseases, the variability in patient responses and characteristics to drugs is significant. Therefore, effective approaches and advancements in related technologies for precisely diagnosing and utilizing diverse diseases are essential. In the field of oncology, humanized animal models have been highlighted as a key solution, even being selected as one of the "Top 10 Oncology Solution Providers in APAC 2024." The world is focusing on building a comprehensive approach and system across the entire cycle of "quarantine-prevention-diagnosis-treatment-dispersion prevention" ahead of long-term infectious diseases and large-scale pandemics like COVID-19. A representative example is CAR-T cell therapy, one of the innovative anticancer drugs, which has been reported to have problems such as CSS (cytokine storming syndrome) and CRS (cytokine release syndrome) in patients, so prior evidence of efficacy and safety in non-clinical trials is becoming increasingly important. In this regard, the U.S. FDA's industry guidelines for chimeric antigen receptor (CAR) T manufacturers (Guidance for Industry, the Office of Communication, Outreach and Development; OCOD, 2024) also require more appropriate prior validation data, such as disease-tailored humanized models, to present rigorous verification results on the precise efficacy, effectiveness, and safety of therapeutic agents corresponding to advanced biopharmaceuticals.

[0004] Ultimately, a highly advanced humanized mouse model with a fully equipped human immune system can be presented as a strategy for developing therapeutics and preventing technologies as an early preclinical approach not only in the field of oncology but also for infectious diseases or pandemic diseases that may occur in the future, and can be built as a technology to reproduce them in a complete medical setting and may become prevalent in the future.

[0005] Meanwhile, as a prior art, Patent Publication No. 2014-0133399 discloses a method for culturing primary cultured cancer cells derived from patients and a method for constructing a xenograft animal model identical to a cancer patient produced using primary cultured cancer cells. However, this is limited to a disease reproducibility model to represent the onset of a specific cancer type and clinical practice, and does not disclose a personalized humanized animal model in which peripheral blood mononuclear cells of a donor are transplanted into an immunodeficient mouse.

[0006] That is, the present invention relates to the development of an experimental animal that replicates and induces the same immune system as mine by transplanting peripheral blood mononuclear cells of an adult (human) into the experimental subject, and this means the development of optimal advanced biomaterials for the detailed verification of precise efficacy, effectiveness, safety, etc. prior to the commercialization of new therapeutic agents (new advanced biopharmaceuticals such as anticancer drugs and cell therapy agents) and the activation / deactivation of immune cells in the human body.

[0007]

[0008] Accordingly, the purpose of the present invention is to provide a personalized humanized animal model and a method for producing the same by administering (applying) peripheral blood mononuclear cells (PBMCs) of a donor to an immunodeficient mouse after inactivating the host's immune system.

[0009]

[0010] In order to achieve the above-described purpose, the present invention provides a personalized humanized animal model, characterized in that peripheral blood mononuclear cells (PBMCs) of a donor are administered to an immunodeficient mouse, and the immunodeficient mouse is implemented as a personalized humanized individual by administering a compound containing busulfan or cyclophosphamide.

[0011] The above immunodeficient mice are implemented as customized humanized individuals by administering compounds containing busulfan or cyclophosphamide to neutralize the host's immune system and then applying PBMCs from the donor's blood.

[0012] In this specification, the term peripheral blood mononuclear cells may be used interchangeably with PBMCs.

[0013] The term "personalized humanized animal model" in this specification refers to a personalized animal model (Personalized CDX / PDX-xenografted Animal Model) produced by orthotopically xenografting donor-derived cells or tissues into an immunodeficient animal, which can provide conditions that directly reflect the genetic, physiological, and environmental characteristics of the donor.

[0014] The term "immunodeficient animal" in this specification refers to an animal model manufactured through breeding and reproduction by artificially deleting or removing some components of the immune system at the genetic level so that a specific disease can develop. The immunodeficient animal is an individual in which the thymus is underdeveloped or defective and differentiation and developmental defects of T lymphocytes and B lymphocytes are induced due to a defect in a specific gene. Preferably, an immunodeficient mammal can be used, and more preferably, an immunodeficient rodent such as a mouse, rat, hamster, beaver, guinea pig, or nutria engineered to be immunodeficient, and most preferably, a nude mouse, a NSG (NOD scid gamma) mouse, a NOD (non-obese diabetic) mouse, a SCID (Severe combined immunodeficiency) mouse, a NOD-SCID mouse, or a NOG (NOD / SCID Il2rg) mouse. - / - ) can be, but is not limited to, a mouse, etc.

[0015] In one specific example of the present invention, “immunodeficient mouse” refers to a nude mouse, a NSG (NOD scid gamma) mouse, a NOD (non-obese diabetic) mouse, a SCID (Severe combined immunodeficiency) mouse, a NOD-SCID mouse, a NOG (NOD / SCID Il2rg) mouse, - / - ) mouse, etc. exist.

[0016] In the present invention, the immunodeficient mouse is a nude mouse, a NSG (NOD scid gamma) mouse, a NOD (non-obese diabetic) mouse, a SCID (Severe combined immunodeficiency) mouse, a NOD-SCID mouse, or a NOG (NOD / SCID Il2rg) mouse. - / -) may be any one selected from the group consisting of mice. However, if it corresponds to a type of mouse with a deficient immune function, such as the mice listed in the restrictions above, it may include all of them without limitation.

[0017] In general, immunodeficient mice are known to lack functional T cells and functional B cells, have reduced macrophage function, abolished NK cells or NK activity, and reduced dendritic cell function.

[0018] In one specific embodiment of the present invention, “peripheral blood mononuclear cells (PBMCs)” refer to peripheral blood cells with round nuclei, which are composed of lymphocytes (T cells, B cells, NK cells) and monocytes among the white blood cells contained in human peripheral blood. PBMCs can be extracted and cultured in vitro, and are frequently used in research in biological and chemistry fields such as immunology, transplant immunology, hematology, vaccine development for malignant tumors and infectious diseases, and high-throughput screening used in drug discovery.

[0019] In the present invention, “administration” refers to parenteral administration excluding oral administration, and includes subcutaneous injection, dermal injection, intramuscular injection, intraperitoneal injection, intracerebral injection, injection into the sublingual vein, intravenous injection, injection into the penile vein, injection into the caudal vein, and injection into the dorsal metatarsal vein injection or infusion techniques.

[0020] In the present invention, "transplantation" means removing living tissue, organ, or cells from a living body and transplanting them to another part of the same individual or another individual. For the purpose of the present invention, it may be understood as a method of transplanting PBMCs separated from a donor to an immunodeficient animal, but is not particularly limited thereto.

[0021] In a personalized humanized animal model according to one embodiment of the present invention, the immunodeficient mouse is characterized in that a compound including busulfan and cyclophosphamide is administered to secondarily inactivate or suppress the immunity of the mouse in an immunodeficient state, and then peripheral blood mononuclear cells of the donor are administered to the immunodeficient mouse, thereby implementing the mouse as a personalized humanized individual.

[0022] The present invention, in order to implement a personalized humanized animal model, administers a compound containing busulfan and cyclophosphamide to immunodeficient mice prior to administering peripheral blood mononuclear cells from a donor to the mice. This allows for the implementation of the personalized humanized animal model of the present invention by maximally inactivating or suppressing the small amount of immunity remaining in the immunodeficient mice.

[0023] In addition, in the personalized humanized animal model according to one embodiment of the present invention, the peripheral blood mononuclear cells (PBMCs) are injected into the immunodeficient mouse at a concentration of 1×10 5 2×10 7 It is characterized by being injected at a concentration of Cell / mouse.

[0024] In addition, in a personalized humanized animal model according to one embodiment of the present invention, the animal model is characterized in that it can predict the prognosis of changes in the immune system by confirming the expression level of the donor's B cells or T cells and confirming the expression rate of the donor's immune cells.

[0025] In addition, in a personalized humanized animal model according to one embodiment of the present invention, the animal model is characterized in that the STR (short tandem repeat) locus or sex chromosome is identical to that of the donor.

[0026] The present invention provides a method for producing a personalized humanized animal model, comprising a step (S10) of administering a compound containing busulfan or cyclophosphamide to an immunodeficient mouse; and a step (S20) of administering peripheral blood mononuclear cells (PBMCs) of a donor to the immunodeficient mouse to which the compound has been administered.

[0027] In addition, in the method for producing a personalized humanized animal model according to one embodiment of the present invention, the step (S10) of administering the compound is characterized in that a compound including busulfan and cyclophosphamide is administered to the immunodeficient mouse.

[0028] In addition, in the method for producing a personalized humanized animal model according to one embodiment of the present invention, the peripheral blood mononuclear cells (PBMCs) are injected into the immunodeficient mouse at a concentration of 1×10 5 2×10 7 It is characterized by being administered at a concentration of cell / mouse.

[0029] In addition, in the method for producing a personalized humanized animal model according to one embodiment of the present invention, the step (S10) of administering the compound is characterized in that a compound containing busulfan or cyclophosphamide is administered to the immunodeficient mouse, thereby inactivating or suppressing the host's bone marrow stem cells.

[0030] In addition, in the method for producing a personalized humanized animal model according to one embodiment of the present invention, the step (S20) of administering peripheral blood mononuclear cells may include the step (S22) of isolating peripheral blood mononuclear cells from the blood of a donor; and the step (S24) of administering peripheral blood mononuclear cells of the donor to an immunodeficient mouse into which a compound has been administered.

[0031] In addition, in a method for producing a personalized humanized animal model according to one embodiment of the present invention, a step (S30) of distinguishing subtypes of B cells and T cells of the donor in the animal model and confirming the expression of the B cells and T cells may be further included.

[0032] In addition, in the method for producing a personalized humanized animal model according to one embodiment of the present invention, the animal model is characterized in that B cells and T cells are specifically expressed so as to be able to evaluate the prognosis or prediction of the immune system of the donor, and the STR (short tandem repeat) locus or sex chromosome is identical to that of the donor.

[0033]

[0034] According to the personalized humanized animal model transplanted with peripheral blood mononuclear cells (PBMCs) according to the present invention and the method for producing the same, it is possible to provide a non-clinical subject that replicates the biological immune system of a donor in a short period of time (within 14 days) while possessing a highly developed human immune system.

[0035] In addition, the present invention can utilize the immune system of a cloned donor to conduct various evaluations that replace humans, and this can be utilized as a prognostic evaluation technology for drug efficacy, responsiveness, and safety of personalized therapeutic agents or administered substances.

[0036] In addition, the present invention can be utilized as a platform for evaluating the development of new drugs and the effectiveness of new drug candidates by producing a personalized animal model using donor PBMCs, as human leukocyte antigens (HLA) differ depending on the race or person, and personalized screening diagnosis for the efficacy and side effects of therapeutic agents with confirmed therapeutic effects is possible.

[0037]

[0038] Figure 1a is a schematic diagram of the process of isolating PBMCs through blood extraction from a donor and freezing the cells.

[0039] Figure 1b shows the results of verification of the amount of cells extracted from peripheral blood mononuclear cells (PBMCs) collected from blood donors and the history of infection within these cells.

[0040] Figure 1c shows the verification results for the major infection history in the collected blood of the donor.

[0041] Figure 1d is a process for evaluating the activity and viability of cells after thawing frozen cells for transplantation in the pre-stage of implementing a personalized humanized animal model.

[0042] Figure 2a is a schematic diagram of the production of a personalized humanized model based on peripheral blood mononuclear cell (PBMC) transplantation.

[0043] Figure 2b shows the results of flow cytometry analysis verifying the binding affinity to mouse-specific leukocyte common antigen (mCD45) antibodies in human or mouse blood and the expression level of target markers.

[0044] Figure 2c shows the results of verifying the bone marrow suppression effect of the host (mouse) following administration of a chemical immunosuppressant.

[0045] Figure 2d shows the results of verifying whether immunosuppression within the host was inactivated by visualizing the expression of leukocyte common antigen (mCD45) in the blood of mice after administration of a chemical immunosuppressant using a scatter plot.

[0046] Figure 2e shows the results of describing the expression level of mouse-specific leukocyte common antigen (mCD45) in the blood of mouse individuals as a percentage.

[0047] Figure 2f is a bar graph showing the average expression values ​​and standard deviations of mCD45 in the two groups.

[0048] Figure 2g is the result of analyzing the effect of Figure 2e using a histogram.

[0049] Figure 3a is a schematic diagram of a method for administering frozen PBMCs through the caudal vein of a laboratory animal after thawing.

[0050] Figure 3b shows that PBMCs from one donor were transplanted into five individuals in equal amounts, and includes information on the individuals of each donor-simulated model.

[0051] Figure 3c is a diagram showing the cell count information of PBMCs collected from donors A, B, and C, divided into equal amounts of 1 / 5, and the amount transplanted into the caudal vein of five individuals.

[0052] Figure 3d is a clinical scoring system that scores physiological observations and internal and external accompanying symptoms according to treatment / administration applied to experimental subjects after transplantation.

[0053] Figure 3e is a scoring sheet for clinical indicators of all groups (control group and donor groups A, B, C; total of 20 subjects) consisting of 5 subjects per group.

[0054] Figure 4a schematically illustrates information on treatment cell and sample collection prior to flow cytometry analysis.

[0055] Figure 4b is a schematic diagram of collecting blood from each donor after transplanting peripheral blood mononuclear cells (PBMCs) from each donor into five individuals.

[0056] Figure 4c shows the results of confirming the expression of human and mouse-specific leukocyte common antigens in a human-like model (personalized humanized model) through transplantation of peripheral blood mononuclear cells (PBMCs) from donors A, B, and C.

[0057] Figure 4d presents numerical values ​​for the expression levels of hCD45 / mCD45 collected from humanized whole organisms.

[0058] Figure 4e is the result of converting the figures presented in Figure 4d into a bar graph.

[0059] Figures 4f and 4g show the results of Kaplan-Meier survival function analysis between human-like models using blood from each donor versus all groups or the control group.

[0060] Figure 5a is a schematic diagram illustrating the process of precisely evaluating the rate of human immune system establishment and expression pattern and performance as a personalized identical model within a human simulant subject to which each donor blood was applied using flow cytometry and genetic analysis, and is a schematic diagram of information on flow cytometry.

[0061] Figure 5b is information on phenotypic markers of surface markers of B cells / T cells.

[0062] Figure 5c shows the results of distinguishing the subtypes of B cells / T cells in the donor-mimicking model using each donor as a control and confirming the presence or absence and pattern of their expression.

[0063] Figure 5d is a result of the percentage information obtained for the expression amount obtained from the expression pattern of human T / B cells.

[0064] Figure 6a is a schematic diagram illustrating the process of precisely verifying through genetic analysis the degree of system replication within the immune system between a donor and a human simulant (personalized humanized model).

[0065] Figure 6b shows information on the internationally used STR locus set and the currently used differential loci.

[0066] Figures 6c to 6e show the results of the matching rate of STR loci of recipients (simulated models; recipient A, B, C) compared to donors.

[0067] [Correction pursuant to Rule 91, March 11, 2025][Deleted]

[0068] Figure 7a is a schematic diagram of a test to find the optimal bone marrow suppression method using chemical-administrated method and high-dose irradiated method.

[0069] Figure 7b shows the results of a comparative test to determine which of the two bone marrow suppression methods is more effective, using chemical agents or radiation.

[0070] Figure 7c shows the results of a scatter plot showing the expression level of mCD45 among individuals within each group.

[0071] Figure 7d shows the results of calculating the median and standard deviation among each of the 10 individuals in each group.

[0072] Figure 8 shows the results of verification of the effects of immunodeficiency according to the single and combined therapy of busulfan and cyclophosphamide, respectively, among the chemical suppression therapies for bone marrow suppression of the host.

[0073] Figures 9a and 9b show the results comparing the performance of human-like models according to low-dose / high-dose transplantation of peripheral blood mononuclear cells (PBMCs).

[0074] Figure 10a is a diagram that determines the capacity of transplanted cells.

[0075] Figure 10b shows the results of flow cytometry analysis on the expression of human CD45 in the blood following normal control, low-dose, and high-dose PBMC transplantation.

[0076] Figure 10c shows the results of flow cytometry analysis evaluated at 2 weeks (D+14), 6 weeks (D+42), and 10 weeks (D+70) after transplantation.

[0077] Figure 10d is the result of converting the figures in Figure 9c into a bar graph.

[0078] Figure 10e shows the results of external changes according to the application of control, low-dose, and high-dose PBMCs.

[0079] Figure 10f shows the results of Kaplan-Meier survival function analysis comparing all groups (control group, low-dose PBMCs, donors (Donor A, B, C), and high-dose PBMCs transplant group) including low-dose / high-dose.

[0080] Figure 10g is the result of separating and analyzing the results of Figure 10f into individual groups compared to the control group.

[0081]

[0082] 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.

[0083] <Example>

[0084] Example 1: Extraction of donor blood and peripheral blood mononuclear cells (PBMCs)

[0085] Figure 1a is a schematic diagram of the process for isolating PBMCs from donor blood and freezing the cells. Peripheral blood mononuclear cells (PBMCs) were isolated from 30 cc of blood collected from the donor using density gradient centrifugation using Ficoll-Hypaque. Ficoll-Hypaque solution is a polymerized compound of sucrose and epichlorohydrin, consisting of molecules ranging from low to high density. When centrifuged together with blood, pure PBMCs can be collected.

[0086] The collected PBMCs were separated into layers as shown in ④ of Fig. 1a, and only the corresponding layer was separated, and after cell counting, they were stored and preserved in a vial as shown in ⑤ of Fig. 1a by ultra-low temperature freezing at -180℃.

[0087] Figure 1b shows the results of verification of the amount of peripheral blood mononuclear cells (PBMCs) extracted from 30 cc of blood collected from donors and the history of intracellular infection. PBMCs isolated from donor A in each of the 30 cc of blood collected from three donors were 4,080,000,000 (4.08 x 10 7 Cell / blood), donor B is 3,150,000,000 (3.15 x 10 7 Cell / blood), donor C is 9,800,000,000 (9.8x10 7 Cells / blood) were counted.

[0088] Figure 1c shows the results of verifying the major infection history in the collected donor's blood. The PCR test for HIV, HBV, and HCV was performed and all results were negative.

[0089] Figure 1d illustrates the process of evaluating cell activity and viability after thawing cells frozen for transplantation prior to the implementation of a personalized humanized animal model. As a control, cells from donors A, B, and C mixed with phosphate-buffered saline (PBS) and buffers used for cell dilution and preservation were evaluated for cell viability. The results were 94% for donor A, 96% for donor B, and 93% for donor C. This indicates that the cells used for transplantation were stable and viable without reversible damage during the thawing process.

[0090]

[0091] Example 2: Host myelosuppressive effect for the creation of a humanized model

[0092] Figure 2a is a schematic diagram of the production of a personalized humanized model based on peripheral blood mononuclear cell (PBMC) transplantation, which consists of three steps.

[0093] The bone marrow suppression method belonging to the first stage is a chemical-based bone marrow suppression method that deviates from the conventional approach (high-dose irradiated method) using radiation. The human cells in the host blood were analyzed on day D+14 after transplantation of peripheral blood mononuclear cells (PBMCs) from each donor. The bone marrow suppression process is an essential process that reduces the production of blood cells in the host bone marrow for building a humanized model. This is to suppress the immune rejection reaction between the donor cells and the host immune cells and the side effects caused by it, and to ensure the stable engraftment of the donor cells.

[0094] Step 2 is the transplantation of PBMCs from the donor, and it is an important process to ensure an appropriate amount of cells and avoid graft-versus-host disease (GvHD) between the host and donor cells. In the present invention, 6x10 per mouse 6 ~1.9 x10 7Cell / mouse cell transplantation was performed.

[0095] Step 3 involved analyzing the phenotype and subtype of cells by comparing human-specific lymphoid immune cells within a customized humanized model through analysis of blood immune cells, and comparing their performance as a donor clone model.

[0096] In Figure 2b, flow cytometry was performed to confirm the accuracy of the binding affinity of the mouse-specific CD45 (mCD45), a species-specific surface marker, to human or mouse blood, and simultaneously to determine the positive expression rate of the target marker in the host (mouse) blood. CD45 is a leukocyte common 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.

[0097] Flow cytometry analysis results showed that mouse mCD45 was expressed in 0.7% of human peripheral blood mononuclear cells (PBMCs), and the same surface marker was positively expressed in 99.7% of mouse blood. This means that the surface marker used binds only to mouse blood mCD45 without non-specific binding to other protein surface receptors, and suggests that the antibody itself is a marker without false positive expression due to autoflourescence.

[0098] Figure 2c shows the results of verifying the bone marrow suppression effect of the host following administration of a chemical immunosuppressant, suggesting that the expression of the leukocyte common antigen (mCD45) in the bone marrow of mice administered with immunosuppressants is significantly reduced compared to individuals not administered immunosuppressants (control group).

[0099] Figure 2d is a scatter plot visualizing the expression of mouse leukocyte common antigen (mCD45) in 10 individuals in each group (total of 20 individuals), and is a diagram illustrating the effect of non-administration or administration of immunosuppressants.

[0100] Figure 2e is a graph depicting the percentage expression levels of mouse-specific leukocyte common antigen (mCD45) in all subjects according to immunosuppressant administration. In the control group (non-myelosuppressant group), mCD45 expression was confirmed to be 97.9% on average (standard deviation ±1.8) and 98.2% on median, while in the test group (immunosuppressant group), it was 21.7% on average (standard deviation ±4.0) and 21.3% on median.

[0101] Figure 2f is a bar graph showing the average expression value and standard deviation of mCD45 in the two groups, showing that the host's lymphoid cells were reduced by 77.8% following chemical inhibitor treatment compared to the control group.

[0102] Figure 2g is a result of analyzing the effect of Figure 2e in a histogram, showing the effect of reducing the leukocyte common antigen of 5 randomly selected mice within each group.

[0103]

[0104] Example 3: Peripheral blood mononuclear cell (PBMC) transplantation for the construction of a human-like platform and clinical measures following transplantation.

[0105] To build a human-like platform based on peripheral blood mononuclear cells (PBMCs), we tracked adverse events, including immune rejection, and physiological and clinical parameters following the infusion of xenogeneic cells after cell transplantation. Alternative testing methods using laboratory animals typically involve pain, distress, and stress due to the treatment and administration of medications, resulting in the observation of physiological characteristics and associated internal and external symptoms.

[0106] Figure 3a is a schematic diagram of a method for administering frozen PBMCs through the caudal vein of a laboratory animal after thawing.

[0107] Figure 3b shows that PBMCs from one donor are transplanted into five individuals in equal amounts, and includes information on the individuals of each donor simulant model (e.g., in the case of a simulant model of donor A, it is described as A(a) to A(e), etc.).

[0108] Figure 3c is a diagram showing the amount of PBMCs collected from donors A, B, and C divided into 5 parts and transplanted into the tail vein of 5 mice.

[0109] Figure 3d uses a clinical scoring system (CSC) to determine the humane endpoint and monitor physical activity, using a scoring chart that assesses physiological observations and internal and external accompanying symptoms following treatment / administration in experimental subjects after transplantation. The clinical endpoint was determined primarily by assessing the physical (weight and baseline information), skin and fur condition (external changes), and morbidity or mortality (behavioral changes). Risk and endpoint were determined based on the scores assigned to each clinical indicator.

[0110] Figure 3e shows the scoring sheet for clinical indicators of all groups (control group and donor groups A, B, and C; total of 20 individuals) consisting of 5 individuals per group. The control group is a group that has not received any surgical treatment such as immunosuppression or cell transplantation and represents a normal control group. Experimental groups A, B, and C were the groups that received cell donation and were observed for clinical scales once a week to track changes in indicators for a total of 12 weeks. No specific clinical symptoms were recorded or observed in the control group throughout the tracking period. The donor A simulant model was recorded with 1 point each in the texture fur item from the 8th week until the end of the experiment (12th week). Donor B's simulant model also showed symptoms of texture fur, weight loss, and diarrhea at week 8, and was scored as 2 points in total, 1 point at week 9, 2 points at week 10, 1 point at week 11, and 3 points at the end of week 12. Donor C also received additional points for texture fur, weight loss, and diarrhea from week 6 to week 10 after transplantation, and was scored as 2 points at week 11 and 3 points at week 12. The scoring sheet for the total observation results used a technique to identify the degree of pain and symptoms by category, and all experimental groups were in risk level 1 and generally maintained a stable condition (scoring sheet category: if the total score is 0 to 3, it belongs to risk level 0, risk level 1 represents 4 to 9 points, and risk level 2 represents 10 to 22 points). As the risk level increases, humane end points are implemented whenever possible to reduce the suffering of experimental animals (Naserian et al., Simple, Reproducible, and efficient clinical grading system for murine models of acute Graft-verse-Host Disease. Frontiers in Immunology (2018); HY Lai et al., Cytokine profiles in various Graft-verse-Host Disease Target Organs following Hematopoietic Stem Cell Transplantation.).

[0111]

[0112] Example 4: Performance of a human-like model based on PBMC transplantation: Activation of blood cells (human CD45-specific)

[0113] To verify the performance of a human-like animal model based on peripheral blood mononuclear cell (PBMC) transplantation, the activity of human-specific leukocyte common antigen (hCD45) in the blood was evaluated using flow cytometry.

[0114] Figure 4a schematically illustrates the information on treatment cell and sample collection prior to flow cytometry analysis.

[0115] Figure 4b is a schematic diagram of the blood collection process for each donor after transplanting peripheral blood mononuclear cells (PBMCs) from each donor into five individuals. The amount of blood extracted at this time is 200 ul / ml through orbital blood collection. PBMCs are isolated from the samples collected from each of the five individuals, and after preprocessing, they are analyzed for the expression of species-specific human / mouse common leukocyte antigens in the blood using flow cytometry. In the flow cytometry, single-cell clusters are targeted to identify only cells of a specific lymphoid lineage, and then the expression of human- or mouse-specific common leukocyte antigens (hCD45 & mCD45) is analyzed to classify the phenotype and subtype.

[0116] Figure 4c shows the results of confirming the expression of human and mouse-specific leukocyte common antigens in a human-simulated model through peripheral blood mononuclear cell (PBMC) transplantation from donors A, B, and C. The normal control group expressed only the mouse-specific leukocyte common antigen marker, while the transplanted individuals showed the expression of both human and mouse leukocyte common antigens.

[0117] Figure 4d presents the numerical values ​​for the expression levels of hCD45 / mCD45 collected from all humanized individuals, including the control group, at D+14, D+42, and D+84 after transplantation based on the flow cytometry results of the blood. Referring to Figure 4d, in the control group, the human leukocyte common antigen (hCD45) was confirmed to be less than 0.1% on average, and the mouse leukocyte common antigen (mCD45) was observed to be maintained at an average of more than 98%. This means that only mouse-specific leukocyte common antigens were present in the blood in the control group, making it appropriate as a normal control group. On the other hand, it was confirmed that both human and mouse-specific leukocyte common antigens were expressed in the blood in all individuals transplanted with PBMCs from donors A, B, and C. In the group transplanted with peripheral blood mononuclear cells (PBMCs) from donor A, the expression of human-mouse leukocyte common antigens was as follows: at week 2 (D+14): hCD45; 90.3% / mCD45; 7.4%, at week 6 (D+42): hCD45; 89.7% / mCD45; 7.9%, and at week 12 (D+84): hCD45; 85.8% / mCD45; 12.7%. In the group transplanted with PBMCs from donor B, the expression was as follows: at week 2: hCD45; 91.3% / mCD45; 7.2%, at week 6 (D+42): hCD45; 91.0% / mCD45; 7.9%, and at week 12 (D+84): hCD45; 87.4% / mCD45; It was confirmed to be 9.3%. In addition, donor C also showed that all experimental groups possessed a high level of human immune system, with hCD45; 91.1% / mCD45; 7.0% at week 2 (D+14), hCD45; 89.1% / mCD45; 9.0% at week 6 (D+42), hCD45; 88.0% / mCD45; 8.9% at week 12 (D+84).

[0118] Figure 4e is the result of converting the values ​​presented in Figure 4d into a bar graph, and visualizes the changes and expression patterns according to the analysis time by isolating the human / mouse leukocyte common antigens.

[0119] Figure 4f shows the Kaplan-Meier survival function analysis results among all human-simulated groups using blood from each donor, including the control group. The results show that 60,000,000 to 200,000,000 (6x10 6 ~2x10 7 The group that was transplanted with PBMCs (cells / mouse) showed a stable survival function for 12 weeks. Figure 4g shows the Kaplan-Meier survival function analysis result between each group compared to the control group, which means that there is no variable that threatens survival. As a result, 600,000 to 200,000,000 cells per individual (6x10 6 ~2x10 7 It was confirmed that the transplantation of cells / mouse was an appropriate transplant amount that did not threaten the life of the subject.

[0120]

[0121] Example 5: Performance of the human-like AVARTAR model: Activation of lymphoid immune cells (B cells & T cells)

[0122] Figure 5a illustrates the process of precisely evaluating the human immune system establishment rate, expression pattern, and performance as a personalized identical model within a human-like entity using each donor blood, using flow cytometry and genetic analysis. Specifically, Figure 5a is a table schematically illustrating the process of precisely analyzing human immune cells using flow cytometry, and includes the process of classifying the phenotype and subtype of human B cells / T cells using surface markers of specific immune cells (red box).

[0123] Figure 5b shows phenotypic information on surface markers of B / T cells. Human T cells are cells that simultaneously express human common leukocyte antigen (hCD45) and CD3 surface markers, while being negative for mouse common leukocyte antigen. Human B cells are cells that simultaneously express human common leukocyte antigen (hCD45) and hCD19 surface markers, while being negative for mouse common leukocyte antigen.

[0124] Figure 5c shows the results of distinguishing B cell / T cell subtypes within donor-mimicking models, using each donor as a control, and confirming their expression status and patterns. Comparison of human T cell expression patterns revealed that both B cells and T cells were present in the blood of donors and recipients (Donor & Recipient A, B, C), and their expression patterns were similar to those of each donor.

[0125] Figure 5d shows quantitative figures for the expression levels obtained from the expression patterns of human T / B cells. The differences in the measured values ​​for each surface marker can be explained by the difference in the analyzed blood volume relative to the total body weight of the individual, and the fact that during the transfusion process, B cells first react to external antigens to produce antigens, and then interact with other surrounding immune cells to promote T cell development. This leads to a temporal difference in the developmental processes of B and T cells, which can explain the pattern in Figure 5d where B cell expression was higher and T cell expression was lower in Recipients A, B, and C compared to the donor. (Reference 3, Reference 4)

[0126]

[0127] Example 6

[0128] Figure 6a precisely validates this through genetic analysis to assess the degree of system replication within the immune system between the donor and the human simulant. Specifically, Figure 6a is a schematic diagram illustrating the genetic analysis process (blue box).

[0129] Figure 6b shows the internationally used STR locus set and the information on the currently used differential loci. The internationally used loci include all 13 standard STR genes used in the CODIS test method of the US FBI and 9 genes used in the European Enforcement Nucleic Acids Research Institute (ENFSI), and are mainly used for crime solving, personal identification, paternity, and blood relationship confirmation. The 16 loci for identity determination in this experiment include all 13 standard STR loci in the US CODIS (Combined DNA Index System) and 9 loci in Europe, and are the final 16 STR genes that take into account markers with high discrimination or heterozygosity and the size of the amplification product (Jin et al., 2007; Forensic Genetic Analysis for the PowerPlex-16 system in the Korean Population; Korean J Genetics 29:498-496). The above 16 STR genes were composed of allele A and allele B to evaluate the matching rate of blood genes of each donor (A, B, C) and recipient (recipient A, B, C). The information of the genes used is D821179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, S16S539, D2S1338, S19S433, vWA, TPOX, D18S51, D5S818, FGA, Amelogenin.

[0130] [Revised on 11.03.2025 under Rule 91] Figures 6c to E show the results of the match rate of STR loci of recipients (simulated models; recipient A, B, C) compared to the donors. Among them, Amelogenin is an allele indicating a sex chromosome, and was detected as XX (female chromosome) in Figure 6c and XY (male chromosome) in Figures 6d and 6e, which presents results that are actually consistent with the sex of each donor. This suggests that this is a technology that can fully represent the human immune system in a simulated model and simultaneously implement an individually customized model that is not limited by gender (all mice used in this study were female).

[0131]

[0132] <Comparative Example>

[0133] Comparative Example 1: Exploring Effective Methods for Myelosuppression (Chemical vs. Irradiated)

[0134] Figure 7a of Comparative Example 1 is a schematic diagram of a test to find the optimal bone marrow suppression method using chemical-administered method or high-dose irradiated method compared to a control group. The control group is a normal control group that was not administered a bone marrow suppressant, and the chemical suppression therapy of test group A was combined with busulfan 20 mg / kg and cyclophosphamide 100 mg / kg. Test group B was a bone marrow suppression method using radiation, exposing the whole body to 150 cGy of radiation (X-ray or gamma ray).

[0135] Figure 7b shows the results of a comparative study to determine the most effective method among two bone marrow suppression methods, applying chemotherapy or radiation. The untreated control group expressed mouse common leukocyte antigen (mCD45); 99.4%, the chemotherapy group 7.17%, and the radiation group 39.8%. These results suggest that chemotherapy suppresses the activation and production of host immune cells compared to radiation, while simultaneously offsetting immune rejection of donor cells, thereby reducing the side effects and risks of xenogeneic cell transplantation.

[0136] Figure 7c shows the results of a scatter plot showing the expression level of mCD45 among individuals in each group, and Figure 7d shows the results of calculating the median and standard deviation among 10 individuals in each group. The median of the control group was 98.2%, the median of the chemotherapy group was 21.3%, and the median of the radiotherapy group was 36.3%, and the standard deviations were ±1.79%, ±3.99%, and ±15.51%, respectively.

[0137]

[0138] Comparative Example 2: Analysis of the Impact of Monotherapy vs. Concurrent Chemotherapy for Myelosuppression on Immunodeficiency

[0139] Figure 8 of Comparative Example 2 shows the results of verifying the effects of immunosuppression according to the single and combined therapies of busulfan and cyclophosphamide, respectively, among the chemosuppressive therapies for bone marrow suppression in the host. When evaluating the expression level of mouse-specific leukocyte common antigen (mCD45) present in the host after applying the single and combined therapies of busulfan or cyclophosphamide, it was confirmed to be 99.7% in the control group, 33.6% in the single-administration of busulfan (Single A), and 37.1% in the single-administration of cyclophosphamide (Single B). On the other hand, it was confirmed to be 11.9% in the combined suppressive agent busulfan / cyclophosphamide. This suggests that the immunosuppressive effect of the combined therapy is a more effective method than the immunosuppressive effect through single application of the chemosuppressive agent.

[0140]

[0141] Comparative Example 3: Clinical Outcomes Following High-Dose / Low-Dose Peripheral Blood Mononuclear Cell (PBMC) Transplantation

[0142] Figures 9a and 9b of Comparative Example 3 are the results of a test to compare the performance of a human-like model according to low-dose / high-dose transplantation of peripheral blood mononuclear cells (PBMCs) and to secure a reference point for the transplant amount and lethal dose. The low-dose PBMCs were 100,000 (1x10) per individual. 5 The group transplanted with 100,000,000 (1x10 cells / mouse) cells was designated as PBMCs Low-dose, and the group transplanted with 100,000,000 (1x10 8The group that received 100 cells / mouse was designated as PBMC High-dose, and the internal and external changes were evaluated using the Clinical Scoring System for both groups. The control group was a normal control group, and only phosphate buffered saline (PBS) used for cell dilution and preservation was administered 100ul / mouse via the caudal vein. The experimental group, the low-dose PBMC transplant group, received 100,000 cells per animal, and the high-dose PBMC transplant group received 100,000,000 cells per animal diluted in phosphate buffered saline and administered via the caudal vein.

[0143] Referring to Fig. 9b, in the group transplanted with low-dose PBMCs in the clinical scoring system, all items that increased the risk were recorded as 0 points, but in the case of the high-dose group, physical changes (weight loss, diarrhea, eye), external changes in skin and fur (texture fur, dry skin, and depilation), and morbidity (movement) were observed from the first week after transplantation. 1 point was added for each indicator item, for a total of 7 points. In addition, at the second week, the score for symptoms in all items (physical changes, external changes in skin / fur, morbidity) was increased by 1 or 2 points, for a total of 17 points. The total of 17 points corresponds to the risk level 3 and was determined and determined as the humane end point, but 2 animals died on D+15 and 3 animals on D+16, so the subsequent records could not be confirmed.

[0144]

[0145] Comparative Example 4: Individual performance according to the amount of PBMC transplantation applied

[0146] We compared and evaluated the performance of humanized models following low-dose and high-dose peripheral blood mononuclear cell (PBMC) transplantation.

[0147] Figure 10a of Comparative Example 4 is a diagram that determines the dose of transplanted cells. The control group was treated with phosphate buffered saline (PBS), and the low-dose group was treated with 1x10 5 Cells / mouse cells, high dose group is 1x10 8 Cells / mouse were transplanted. At this time, phosphate-buffered saline was used for cell dilution, and 100ul volume / mouse was administered equally per individual.

[0148] Figure 10b shows the results of flow cytometry analysis of the expression of human CD45 in blood following normal control, low-dose, and high-dose PBMC transplantation.

[0149] In Fig. 10c, flow cytometry analysis was performed at 2 weeks (D+14), 6 weeks (D+42), and 10 weeks (D+70) after transplantation. The control group was the group treated with phosphate-buffered saline, and within that group, the expression of human leukocyte common antigen was confirmed to be 0.1%, and the average expression of mouse leukocyte common antigen was 98.4%. In contrast, the groups treated with low and high doses of PBMCs showed the presence of heterologous leukocyte common antigens, and the proportions are described in Fig. 10c. In the group transplanted with low doses of PBMCs, the average expression rates of human / mouse-specific leukocyte common antigens were evaluated as follows: 2 weeks (D+14): hCD45;22.4% / mCD45;71.3%, 6 weeks (D+42): hCD45;20.2% / mCD45;64.7%, 10 weeks (D+70): hCD45;13.0% / mCD45;85.4%. In addition, in the group transplanted with high doses of PBMCs, 2 weeks (D+14): hCD45;91.1% / mCD45;7.3%, indicating that the proportion of host immune cells was very low and the donated human immune system was highly replaced. However, all individuals in the group died after 2 weeks (2 individuals died on day 15, 3 individuals died on day 17) and follow-up was impossible. This suggests that while this method can be utilized as a humanized replacement for mice, it cannot guarantee stable performance and its ability to preserve humanized functions is reduced. Furthermore, in Comparative Example 4, the average expression levels of human / mouse-specific leukocyte common antigens for all individuals were recorded, along with the standard deviation between individuals.

[0150] Figure 10d shows the results of analyzing the changes between groups for human- and mouse-specific surface markers by converting the values ​​in Figure 9c into a bar graph. The results in Figures 10c and 10d include the maintenance period for humanization performance and stable performance of the humanized entity. When low-dose PBMCs were transplanted, the expression value of human leukocyte common antigen showed a tendency to gradually decrease while maintaining a level of human lymphoid cells below 25% in the body, and no internal or external abnormalities were observed. On the other hand, when high-dose PBMCs were transplanted, human lymphoid cells accounted for a high proportion of more than 90%, but an immune rejection reaction was observed due to the influx of xenogeneic cells into the host, and death occurred quickly after 2 weeks.

[0151] Figure 10e shows data on external changes following application of control, low-dose, and high-dose PBMCs. Facial beard / body hair loss and severe pale skin due to lack of circulating blood were observed due to rejection of xenogeneic immune cells.

[0152] Figure 10f compares the Kaplan-Meier survival function analysis for all groups of donors (Donor A, B, C) including low-dose and high-dose PBMCs. The results are as follows: low-dose PBMCs and 60,000,000 to 200,000,000 (6x10 6 ~2x10 7 A stable survival function was observed within the transplantation application of 100,000,000 cells / mouse. On the other hand, a high-dose PBMCs group of 100,000,000 cells (1x10 8 Cell / mouse) confirmed that the survival of all individuals was difficult after two weeks. This means that the survival is threatened by a series of changes in the body due to the high-dose cell transplantation technology. Therefore, when constructing a humanized model using PBMCs as in Fig. 4, including Fig. 10f, 100,000 to 200,000,000 (1x10) per individual is required. 5 ~2x10 7Transplantation of cells / mouse is suggested to be an appropriate treatment dose that does not threaten the life of the subject. Figure 10g shows the results of Figure 10f, which are separated and analyzed into individual groups compared to the control group.

[0153]

[0154] 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 personalized humanized animal model in which peripheral blood mononuclear cells (PBMCs) from a donor are administered to immunodeficient mice. The above immunodeficient mouse is a personalized humanized animal model characterized in that it is implemented as a personalized humanized individual by administering a compound containing busulfan or cyclophosphamide.

2. In paragraph 1, A personalized humanized animal model characterized in that the immunodeficient mouse is implemented as a personalized humanized individual by administering a compound containing busulfan and cyclophosphamide to suppress the immunity of the mouse and then administering peripheral blood mononuclear cells of the donor to the immunodeficient mouse.

3. In paragraph 1, The above peripheral blood mononuclear cells (PBMCs) were injected into the immunodeficient mice at a density of 1×10 5 2×10 7 A personalized humanized animal model characterized by administration at a concentration of cell / mouse.

4. In paragraph 1, The above animal model is a personalized humanized animal model characterized in that it can predict the prognosis of the immune system of the donor by confirming the expression level of the B cells or T cells of the donor.

5. In paragraph 1, The above animal model is a personalized humanized animal model characterized by having the same STR (short tandem repeat) locus or sex chromosome as the donor.

6. Step (S10) of administering a compound containing busulfan or cyclophosphamide to an immunodeficient mouse; and A step (S20) in which peripheral blood mononuclear cells (PBMCs) of a donor are administered to an immunodeficient mouse to which the compound has been injected; A method for producing a personalized humanized animal model, comprising:

7. In paragraph 6, A method for producing a personalized humanized animal model, characterized in that the step (S10) in which the compound is administered is a compound containing busulfan and cyclophosphamide, to the immunodeficient mouse.

8. In paragraph 6, The above peripheral blood mononuclear cells (PBMCs) were injected into the immunodeficient mice at a density of 1×10 5 2×10 7 A method for producing a personalized humanized animal model characterized by being administered at a concentration of cell / mouse.

9. In paragraph 6, The step (S10) of administering the compound is a step in which a compound containing busulfan or cyclophosphamide is administered to the immunodeficient mouse, thereby inactivating or suppressing the host's bone marrow stem cells. The step (S20) in which the peripheral blood mononuclear cells are administered is: Step of isolating peripheral blood mononuclear cells from the donor's blood (S22); and A method for producing a personalized humanized animal model, characterized by comprising a step (S24) of administering peripheral blood mononuclear cells of the donor to an immunodeficient mouse into which a compound has been injected.

10. In paragraph 6, Further comprising a step (S30) of distinguishing the subtypes of B cells and T cells of the donor in the animal model and confirming the expression of the B cells and T cells; A method for producing a personalized humanized animal model characterized in that the animal model specifically expresses B cells and T cells so as to enable prognosis or prediction of the immune system of the donor, and has the same STR (short tandem repeat) locus or sex chromosome as the donor.

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