Animal models for studying cancer immunotherapy

Abstract

This invention provides chimeric pheasant bird embryos, methods for obtaining them, and novel animal models that are particularly useful for screening immunotherapeutic agents and / or cells, as well as for classifying patients. [Solution] A chimeric pheasant avian embryo is provided, comprising both types of exogenous cells: a. a population of at least one type of cancer cells and b. a population of at least one type of immune cells, wherein the exogenous cancer cells are present in at least one tissue of the embryo and the exogenous immune cells are present in at least one tissue of the embryo and / or circulate in the blood vessels of the embryo.

Description

Technical Field

[0001] Field of the Invention The present invention relates to an animal model for studying cancer cells, particularly the relationship between cancer cells and immune cells. The animal model of the present invention is particularly relevant to the study of immunotherapy strategies.

[0002] Introduction Modeling of human cancers in experimental animals is a central issue in preclinical trials associated with the development of new anti-cancer therapies. The main evaluation criteria for animal models developed for this purpose are the reliability of the model and the speed and cost of implementation.

[0003] Animal models developed and currently used for tumor research are mainly mouse models. Preparing these models takes a relatively long execution time and high cost. In addition, certain cancer cells cannot be transplanted into mouse animal models.

[0004] Galliform bird embryos, particularly chicken or quail embryos, are attractive models for performing ex vivo experiments, particularly for studying embryonic development and xenograft experiments. It is actually inexpensive, very easy to obtain, and easy to handle. Furthermore, approval of the research by an ethics committee is not required. This is because the use of avian embryos during the first two-thirds of embryonic development is not considered "animal experimentation" (European Directive 2010 / 63 / EU). It is a model selected for studying cell proliferation, differentiation, and migration. This animal model can also be used to study tumors.

[0005] A classic research model using pheasant embryos involves the transplantation of exogenous cells into an extraembryonic structure, more precisely onto the chorioalangiomolecular membrane (CAM). Tumor cells are transplanted onto chicken embryonic membranes. After approximately two days of incubation, a tumor forms. This tumor utilizes the particularly well-developed embryonic membrane vascular structure for growth. This system has made it possible to reproduce in vivo human tumors, particularly glioblastoma, with cellular and molecular characteristics similar to those observed in tumors in vivo.

[0006] Furthermore, this chorioalangiocarcinoma (CHOLA) transplantation model is also widely used to screen for therapeutic molecules, particularly those that inhibit tumor angiogenesis. For example, WO 2015 / 074050 and US 2013 / 0171680 describe xenotransplantation of malignant human hematopoietic cells into extraembryonic structures, in which cancer cells are injected into the blood vessels of the amniotic sac, yolk sac, or CAM.

[0007] WO No. 2020 / 075168 describes grafts of cancer cells and immune cells dispersed in hydrogels on CAM.

[0008] Park et al. (2009) described the introduction of human hematopoietic endothelial progenitor cells into the yolk sac of chicken embryos, and these progenitor cells were able to be transplanted into the hematopoietic organs of chicken embryos.

[0009] Several cases have been reported of introducing cancer cells into the vascular or neural tube lumen of chicken embryos.

[0010] Carter et al. (2012) reported the injection of human neuroblastoma cells into the blood vessels of chicken embryos. Boulland et al. (2010) disclosed the injection of human stem cells into lesions formed in extraembryonic blood vessels or the neural tube.

[0011] Busch et al. (2012) describe the injection of melanoma cells into the lumen of rhombencephalic vesicles in chicken embryo brains.

[0012] Jayachandran et al. (2015) instructed the injection of melanoma cells into the lumen of the neural tube in chicken embryos. Meanwhile, Joel et al. (2013) instructed the injection of human glioblastoma cells into the same location.

[0013] None of these techniques enable the transplantation of tumor cells into embryonic tissue, and therefore tumor formation. On the contrary, cancer cells introduced into these locations tend to be reprogrammed into a more benign phenotype and quickly disappear.

[0014] WO2016 / 05398 and WO2017 / 103025 both describe a novel animal model in which cancer cells are transplanted into the tissue of a pheasant bird embryo, rather than into an extraembryonic structure, and are not injected into the lumens of any blood vessels or tubes of the embryo. Advantageously, the introduction of cancer cells into the embryonic tissue allows the cancer cells to follow migratory pathways induced by hormonal signals circulating within the embryo. In this animal model, the cancer cells migrate to specific tissues according to their properties, and then transplant into those tissues to form tumors.

[0015] This invention relates to the development of an alternative animal model for cancer research, specifically designed for studying the interaction between cancer cells and immune cells. This animal model is particularly useful for screening immunotherapeutic agents and / or cells.

[0016] Immunotherapy Treatment Strategies Immunotherapy can be defined as a type of treatment that helps / enhances the immune system of cancer patients to fight cancer. In fact, as part of its normal function, the immune system is capable of detecting and destroying abnormal cells, especially tumor cells, and is most likely to prevent the growth of many cancers that are destroyed early and therefore go undetected. In the case of a diagnosed tumor, immune cells are found within and around the tumor. The presence of these immune cells, called tumor-infiltrating lymphocytes or TILs, is a sign that the immune system is responding to the tumor. Generally, the presence of lymphocytes within or around a tumor is a marker of a good prognosis.

[0017] Unfortunately, some tumors have found ways to evade the immune system, and this has been established as a means of avoiding destruction.

[0018] For example, cancer cells, • In some cases, there may be genetic changes that make the immune system less detectable. • May have proteins on its surface that can stop immune cells and / or • It may alter the normal cells surrounding the tumor, interfering with how the immune system responds to cancer cells.

[0019] The main goal of immunotherapy is to restore the interaction between the immune system and cancer cells. Several types of immunotherapy are used to treat cancer and stimulate the detection and / or destruction of tumor cells by the cancer patient's own immune system. These are, • Immune checkpoint inhibitors (These are drugs that block immune "checkpoints." These checkpoints are normal parts of the immune system that maintain an immune response that is not too strong. By blocking them, these drugs allow immune cells to respond more strongly to cancer.) • Immune system modulator (which enhances the body's immune response to cancer), • Monoclonal antibodies (These are designed to bind to specific targets on cancer cells. Monoclonal antibodies can "mark" cancer cells so that they are more visible to the immune system and therefore more likely to be destroyed by the immune system. The use of monoclonal antibodies in cancer treatment was first introduced in 1997 with rituximab, an anti-CD20 antibody for the treatment of B-cell lymphoma.) • T-cell transfer therapy (This is a procedure that restores and / or enhances the T-cell's inherent ability to fight cancer. T-cell transfer therapy can also be called adoptive cell therapy, adoptive immunotherapy, or immunotherapy.) Includes.

[0020] In 1996, it was shown that blocking CTLA-4, a key component of T cell exhaustion, induced rejection of rectal cancer in mice (Leach et al., 1996).

[0021] T cell exhaustion T lymphocytes present in the tumor microenvironment (TME, i.e., cancer cells, inflammatory cells, and suppressive cytokines) are generally "exhausted." T cell exhaustion is characterized by loss of functionality and expression of inhibitory molecules. Exhausted T cells lack the ability to secrete effector molecules, such as IL-2, IFN-γ, TNF-α, and granzyme B, and express inhibitory receptors, such as PD1, TIM-3, LAG3, and CTLA-4 (McLane et al., 2019).

[0022] Therapeutic strategies aimed at reversing the exhausted phenotype of T cells have been a focus over the past decade. Significant progress has been made in the management of multiple types of cancer. In particular, clinical data for αPD1 (pembrolizumab, nivolumab) and / or αCTLA-4 (ipilimumab) have shown overwhelming success. Unsurprisingly, the expression of PDL1, a ligand for PD1, on tumor tissue is associated with a better response rate to these immunotherapies.

[0023] Targeting Specific Epitopes - CAR-T Another paradigm shift in immunotherapy is the emergence of chimeric antigen receptor-T cells, known as CAR-T cells. CAR-T cells are autologous T cells genetically modified to express a chimeric receptor that targets a specific epitope. The specific portion of the CAR is connected via a transmembrane domain to the signaling domains of CD28, 41BB, and CD3ε, enabling efficient activation and optimal survival of T cells upon engraftment (Feins et al., 2019).

[0024] The use of CD19-specific CAR-T cells has achieved remarkable success in B-cell leukemia. However, CAR-T cell strategies for solid tumors are likely to be much less efficient, probably due to multifactorial reasons such as lack of access to the tumor microenvironment (TME), the immunosuppressive capacity of the TME, and tumor-associated antigen immunogenicity / heterogeneity. Nevertheless, new constructs of CAR-T cells for solid tumors are still being investigated with several new targets, different addressing modes to the TME, and various strategies for CAR-T cell persistence / survival being tested (Martinez & Moon, 2019).

[0025] Furthermore, another focus is the development of donor-derived allogeneic CAR-T cells. In fact, this technology has many potential advantages over the autologous approach, such as the immediate availability of cryopreserved batches for patient treatment, the potential standardization of CAR-T cell products, time for multiple cell modifications, re-administration or combination of CAR-T cells against different targets, and finally, cost reduction using an industrialized process. Therefore, the development of allogeneic CAR-T cells is an active research area.

[0026] Models for Studying Immunotherapy The emergence of new treatment strategies and, to a greater extent, the evident heterogeneity in the response to immunomodulatory interventions have necessitated a practical model for classifying patients before treatment initiation.

[0027] From a clinical perspective, patient scoring can be based on the expression of biomarkers, such as the PD1 / PDL1 axis, genetic / transcriptome screening, the presence of appropriate TILs, and / or the ability of these TILs to achieve effector functions in relation to the tumor.

[0028] Furthermore, several mouse models of patient-derived xenografts (PDX) have been developed. A common strategy is to transplant patient-derived tumor tissue into immunodeficient mouse recipients and ultimately co-transplant patient-derived immune cells.

[0029] However, this approach is time-consuming, expensive, and may have a low transplantation success rate of 10%, making it unreliable. The effort and financial investment for generating such animal models are not compatible with the clinical situation (Jung et al., 2018).

[0030] Considering this situation, novel animal models for preferentially studying the in vivo interactions between cancer cells and immune cells on a patient-by-patient basis are being actively explored. There is also a need for preclinical models for testing CAR-T engineered cells (Siegler and Wang, 2018).

[0031] In this context, the present invention proposes a novel animal model consisting of chimeric quail embryos, which is particularly useful for screening immunotherapeutic agents and / or cells and for classifying patients.

[0032] Summary of the Invention The present invention relates to a chimeric quail embryo comprising both types of exogenous cells: a. a population of at least one type of cancer cell and b. a population of at least one type of immune cell The present invention relates to a chimeric pheasant avian embryo, wherein the exogenous cancer cells are present in at least one tissue of the embryo, and the exogenous immune cells are present in at least one tissue of the embryo and / or circulate in the blood vessels of the embryo.

[0033] Furthermore, the present invention relates to the following steps: The process involves introducing both a population of immune cells and a population of cancer cells into a pheasant bird embryo. The present invention relates to a method for obtaining the above-described chimeric pheasant bird embryo, comprising the step of incubating the chimeric embryo at a temperature within 35°C to 42°C for at least 6 hours.

[0034] At least two implementations of this method are described herein. 1) The following steps: The process involves co-transplanting both a population of immune cells and a population of cancer cells into at least one type of tissue of a pheasant bird embryo in the developmental stage between HH10 and HH30, A method comprising the step of incubating the chimeric embryo at a temperature within 35°C to 42°C for at least 6 hours. 2) The following steps: The process involves transplanting a population of at least one type of cancer cells into the tissue of at least one type of pheasant bird embryo in the developmental stage between HH10 and HH30, The step of injecting the population of at least one type of immune cells into the blood vessels of the embryo, The method is thought to include a step of incubating the transplanted embryo at a temperature within 35°C to 42°C for at least 6 hours.

[0035] Furthermore, the present invention relates to an animal model for the study of human cancer, comprising the chimeric pheasant bird embryo described above or a chimeric pheasant bird embryo obtained by the previously proposed method.

[0036] Furthermore, the present invention relates to the use of such animal models for screening at least one anti-cancer therapy selected from the following group: immunotherapy, chemotherapy, targeted therapy, hormone therapy, anticancer agents, and therapeutic enzymes. [Brief explanation of the drawing]

[0037] [Figure 1] Figure 1: Photographs of labeled human PBMCs in avian embryos 2 days after injection (1A) or transplantation (1B). (White) fluorescent cells are immune cells that have settled in the embryonic tissue. [Figure 2] Figure 2: FACS analysis of human PBMCs injected into bird embryos 2 days after injection. CFSE: Carboxyfluorescein succinimidyl ester; FSC: Forward scattering; SSC: Side scattering. [Figure 3] Figure 3: Survival of PBMCs after injection into chicken embryos. PBMCs were injected at various doses (horizontal axis); % cell survival is shown on the vertical axis. Each marker (triangle, square, rhombus, circle) represents an independent experiment for the number of injected PBMCs measured. [Figure 4] Figure 4: Analysis of PBMC subpopulations after transplantation by flow cytometry. Determination of the relative proportion of immune cell subpopulations after PBMC transplantation. PBMC: Cells before transplantation. AVI-PBMC: PBMC cells transplanted and analyzed after Percoll isolation from collagenase-treated embryos (after 48 hours of embryo incubation). (4A) Quantification of CD19+ and CD3+ cells. Values ​​are expressed as a ratio of total specific lymphocytes. CD3+ / CD4+ and CD3+ / CD8+ ratios are also shown. (4B) Comparison of immune cell subpopulations (across the living CD45+ population) in samples before transplantation (PBMC) and 2 days after transplantation (AVI-PBMC) into avian embryos by FACS analysis. (4C) Comparison of activation markers and exhaustion markers (across the living CD45+CD3+ population) of immune cells (across the living CD45+CD3+ population) before transplantation (PBMC) and 2 days after transplantation (AVI-PBMC) into avian embryos by FACS analysis. Markers of activation: human CD69 and CD25; markers of exhaustion: human TIM-3 and PD-1. [Figure 5]Figure 5: Micrograph of tumors formed after transplantation into avian embryos. PBMCs and tumor cells were co-transplanted into the target site of chicken embryos. Before transplantation, both cell pools were labeled with fluorescent vital tracers (red for PBMCs, green for tumor cells). Two days after transplantation, embryos were retrieved and imaged with a confocal microscope to visualize the transplanted cells. PBMC cell components were present within the formed tumors. (Merge) Both cell types are visualized; (PBMC) Only PBMCs are visualized; (Tumor cells) Only tumor cells are visualized. [Figure 6] Figure 6: Magnified view of immune cells (white) in a human cell tumor (brighter halo) formed in an avian embryo. PBMCs were injected into the vascular structure, and tumor cells were transplanted to the target site in the embryo. [Figure 7] Figure 7: Magnified view of individual tumor cells and immune cells (PBMCs) within a tumor (tumor) injected into the vascular structure of a chicken embryo. [Figure 8] Figure 8: Body-to-size area (BSA), an indicator of individual embryo size and weight, was measured for embryos transplanted with Pmela cells alone (Glo) and embryos co-transplanted with Pmela cells and PBMCs (GLO-PBMC). No statistical differences in BSA were observed. [Figure 9] Figure 9: Measurement of tumor volume comparing conditions when Pmela cells were transplanted alone or in combination with human PBMCs (Method 1). The graph shows no statistically significant difference between the two conditions. Data were normalized to embryonic body size area (BSA). [Figure 10]Figure 10: (10A) Display of the proportion of immune cell subpopulations after co-transplantation of PBMCs with two types of cancer cells (Pmela cells: GLO; MDAM cells: MDAM). Values ​​are expressed as the ratio of total specific lymphocytes (CD3+). (10B) Expression of surface cell markers (PD1, TIM-3, CD69, and CD25) is measured by FACS for CD4+ and CD8+ subpopulations of T lymphocytes: CD8+ on the left, CD4+ on the right (across living CD45+CD3+ populations). The selected cells are as follows: PBMC alone before transplantation (black), AVI-PBMC after single transplantation into embryo (dark gray), and AVI-PBMC+MDAMB231: after co-transplantation of tumor cells and PBMCs (light gray). Measurements are performed 48 hours after transplantation. [Figure 11]Figure 11: Effect of Keytruda. (11A) A subpopulation of cells was identified by FACS after Percoll isolation of PBMCs from collagenase-treated embryos. Detection of PD1 surface expression by FACS. Anti-PD1 (Keytruda) can bind to PD1 expressed by human T cells derived from transplanted PBMCs, and therefore competes with anti-PD1 antibodies used for PD1 detection in FACS analysis, resulting in a decrease in PD1 detection. Therefore, when Keytruda reaches its target, a decrease in detected PD-1 levels is observed. Analysis of PD1 expression in avian embryos transplanted with PBMCS after intravenous injection of Keytruda (diluted to 5 mg / ml) or, for negative control, NaCl. The negative control is arbitrarily considered to be 100% CD4+ and CD8+ cells (within the living CD45+CD3+ population). Quantification in CD8+ and CD4+ cells in Keytruda-treated embryos is expressed as normalized to the control. (11B) PDL1 expression in MDAMB436 breast cancer cells (upper figure) and MDAMB231 cells (lower figure) was measured by FACS after transplantation into embryos. (11C) PBMCs were co-transplanted with tumor cells: Pmela (PDL1-negative cell lineage) or MDAMB436 (PDL1-weakly positive cell lineage). Subsequently, transplanted and isolated PBMC cells were treated with or not treated with Keytruda(K), an immunostimulatory anti-human PD1: +K indicates the presence of Keytruda. Markers of activation (CD69, HLADR on the left) and markers of exhaustion (CTLA4, PD1 on the right) were determined in PBMCs after co-transplantation with human tumor cells (Pmela or MDAM) into avian embryos. Values ​​are expressed as the ratio of total specific lymphocytes (CD3+CD4+ and CD3+CD8+) and the frequency exceeding the CD4+ and CD8+ populations. Standard deviations show data from a pool of PBMCs derived from different healthy donors. Left graph: CD4+, Right graph: CD8+. Recombination of T CD8+ cells (left) and CD4+ cells (right) after 24-hour treatment with Keytruda (dark gray) or NaCl (light gray) in embryos co-transplanted with (11D) PBMCs and MDAMB231 tumor cells (across the living CD45+CD3+ population).Results are expressed as a percentage of the control dose; white arrows indicate a decrease in the expression of fatigue markers (PD1 and TIM-3) under the Keytruda (5 mg / ml) treatment condition. [Figure 12] Figure 12: Tumor size in transplanted chicken embryos. (12A) Analysis of the effect of anti-PD1 treatment (Keytruda) on tumor volume formed by co-transplantation of A375 melanoma cells with human PBMCs. The tumors are formed in an avian embryo model. (12B) Effect of anti-PD1 (Keytruda) administration on tumor volume in avian embryos co-transplanted with human PBMCs and MDAMB231 tumor cells. Left panel: Effect of Keytruda on body size area (BSA); Center panel: Histogram of original tumor volume; Right panel: Normalized tumor volume / BSA. All results show a significant reduction in tumor volume in the anti-PD1 treated state compared to NaCl-treated embryos. (12C) Effect of anti-PD1 (Keytruda) administration on avian embryos co-transplanted with human PBMCs and tumor samples from patients with metastatic melanoma. The number of metastases (left) and the volume of metastatic tumors (center) were evaluated. The photograph on the right shows tumor enlargement after NaCl treatment (top) and after Keytruda treatment (bottom). [Figure 13] Figure 13. Analysis of PBMCs at 48 and 72 hours post-transplant. Monitoring of PBMC subpopulation rearrangement after transplantation into avian embryos. Embryos were collected at 48 hours (light gray) or 72 hours (dark gray) post-transplant. Subpopulations were observed on a living CD45+ population and compared to pre-transplant PBMCs (PBMC D0).

[0038] Detailed description of embodiments of the present invention This invention relates to a chimeric pheasant bird embryo, comprising both types of exogenous cells: a. A population of at least one type of cancer cell and b. A population of at least one type of immune cell The present invention relates to a chimeric pheasant avian embryo, wherein the exogenous cancer cells are present in at least one tissue of the embryo, and the exogenous immune cells are present in at least one tissue of the embryo and / or circulate in the blood vessels of the embryo.

[0039] Unless otherwise specified, the following terms and expressions used herein are intended to have the meanings set forth below in the context of this invention.

[0040] The term "Phasianidae birds" refers to birds of the order Galliformes (or Galliformes), including chickens, quail, turkeys, pheasants, peacocks, guinea fowl, and other domesticated birds. Preferably, the embryos would be derived from chickens (Gallus gallus) or quail (Coturnix japonica). These two species are commonly used in laboratories.

[0041] The term "pheasant bird embryo" refers to a fertilized pheasant bird egg that develops normally under appropriate conditions by being placed in an incubator heated to a temperature of 35°C to 42°C.

[0042] In a specific embodiment of the present invention, the pheasant bird embryo is a chicken embryo.

[0043] The term "chimeric embryo" refers to an embryo having cells derived from the aforementioned embryo, referred to as endogenous cells, and further having exogenous cells derived from at least one other organism, the exogenous cells becoming an integral part of the embryo after acceptance as a graft and continuing their development within the tissues of the recipient embryo. This chimeric embryo is not intended to develop sufficiently to produce an adult chimeric organism and is used only for a short period to support the exogenous cells. This pheasant bird embryo will not produce a chimeric organism and will be euthanized in accordance with valid ethical rules as soon as research on the population of exogenous cells is completed.

[0044] The term "exogenous cells" refers to cells of another organism introduced into the recipient organism, in this example, a pheasant bird embryo.

[0045] The term "population of cells" refers to a group of similar cells that all share the same properties and functions. For example, a "population of immune cells" might be T lymphocytes, another population might be B lymphocytes, and yet another population might be macrophages.

[0046] The term "cancer" refers to a pathology characterized by the presence of malignant cells in an organism that are formed by the transformation, mutation, or genetic instability of initially normal cells.

[0047] The term "cancer cells" refers to malignant cells originating from either liquid or solid tumors. A "population of cancer cells" could be, for example, melanoma cells, another population could be glioblastoma cells, and yet another population could be breast cancer cells.

[0048] The term "immune cells" all refer to cells of the immune system, specifically those called "white blood cells." These cells can be classified into two main families: neutrophils and peripheral blood mononuclear cells (PBMCs), i.e., blood cells with round nuclei. Although both originate from the same hematopoietic progenitor cells, the term "immune cells" does not include erythrocytes, also known as "red blood cells."

[0049] The aforementioned PBMC is - Lymphocytes including T cells, B cells, and NK (natural killer) cells - Monocytes, macrophages, and dendritic cells Includes.

[0050] The term "tissue" refers to an assembly of similar cells and their extracellular matrices from the same origin that perform a specific function in an organism. Tissues can be organs, muscles, connective tissue, epithelium, glands, nerve tissue (central or peripheral), or embryonic tissue whose specific function is still unknown. Thus, "embryonic tissue" refers to any embryonic tissue that is homologous to typical or adult tissue but has not yet been fully differentiated.

[0051] Furthermore, in the sense of the present invention, "embryonic tissue" is particularly, -Extraembryonic appendages of the embryo, such as chorioalinus (CAM) and - Luminous structure of a tube (e.g., nerve tube) and blood vessels (e.g., blood vessels) This refers to any tissue of the embryo, excluding the cerebral cortex.

[0052] The phrase "present in at least one tissue of the embryo" refers to the presence of exogenous cells in the tissue of the avian embryo in the sense of the present invention. These cells are preferably aggregated into a tumor morphology.

[0053] The phrase "circulating in the blood vessels of the embryo" refers to the presence of exogenous immune cells circulating in the blood circulation of the avian embryo, that is, in the blood vessels of the embryo.

[0054] A group of immune cells present in bird embryos Chimeric pheasant bird embryos contain a population of at least one species of immune cells.

[0055] Preferably, this population of at least one type of immune cell is selected from lymphocytes, including peripheral blood mononuclear cells (PBMCs), T cells, B cells, and NK (natural killer) cells.

[0056] In specific embodiments, all immune cell populations originate from a single organism, specifically the same human being. Human-derived immune cells can be easily collected by taking a blood sample and subsequently separating different types of blood cells.

[0057] In a specific embodiment of the present invention, the embryo of a chimeric pheasant bird contains only a population of one type of immune cell.

[0058] In a more specific embodiment of the present invention, the population of immune cells consists of peripheral blood mononuclear cells (PBMCs).

[0059] In another specific embodiment of the present invention, the population of immune cells consists of lymphocytes.

[0060] In another specific embodiment of the present invention, the population of immune cells consists of B lymphocytes (B cells).

[0061] In another specific embodiment of the present invention, the population of immune cells consists of T lymphocytes (T cells).

[0062] In another specific embodiment of the present invention, the population of immune cells consists of genetically modified CAR-T cells.

[0063] Genetically modified CAR-T cells refer to CAR-T cells that have been genetically modified according to any method well known to those skilled in the art in order to express specific receptors that target specific tumor epitopes. These cells are capable of targeting, recognizing, and destroying tumor cells. In the sense of the present invention, genetically modified CAR-T cells can be autologous (derived from a patient and subsequently reinjected into the same patient) or allogeneic (derived from a donor and modified to be injectable into any human).

[0064] Advantageously, these genetically engineered CAR-T cells express chimeric receptors that can recognize tumor-specific antigens, induce T cell activation, and sustain simultaneous stimulation signals.

[0065] A cluster of cancer cells present in bird embryos Chimeric pheasant bird embryos contain a population of at least one type of cancer cell.

[0066] In specific embodiments, the entire population of cancer cells originates from a single organism, in particular, from the same human being. Human-derived cancer cells can be obtained by any technique known to those skilled in the art.

[0067] In a specific embodiment of the present invention, the embryo of a chimeric pheasant bird contains only a population of one type of cancer cell.

[0068] Exogenous cancer cells present in at least one tissue of the embryo may be of any origin. In particular, these cancer cells may be derived from immortalized cancer cell lines or tumor samples.

[0069] In a specific embodiment of the present invention, exogenous cancer cells originate from the patient's tumor.

[0070] According to embodiments of the present invention, the exogenous cancer cells are selected from the group consisting of cells derived from primary or secondary brain tumors, such as glioblastoma cells or glioma cells, lung cancer cells, particularly "EGFR mutant" lung cancer cells, breast cancer cells, particularly HER2+ / ER+ breast cancer cells, prostate cancer cells, sarcoma cells, melanoma cells, germ cell tumor cells, lymphoma cells, particularly follicular lymphoma cells, liver cancer cells, gastrointestinal cancer cells, and ovarian cancer cells.

[0071] Characteristics of both cell populations The chimeric pheasant bird embryo of the present invention contains both types of exogenous cells. In specific embodiments of the present invention, both populations of exogenous cells are human cells.

[0072] In another specific embodiment of the present invention, both populations of exogenous cells originate from the same organism, in particular from the same cancer patient.

[0073] In another embodiment of the present invention, both exogenous cell populations arise from at least two different organisms. For example, -A population of at least one type of cancer cells originates from the patient's tumor. - A population of at least one type of immune cell is a population of genetically engineered allogeneic CAR-T cells derived from a donor.

[0074] In this configuration, chimeric embryos are used to test the ability of allogeneic CAR-T cells to recognize and / or destroy cancer cells derived from a specific patient.

[0075] Another example of this embodiment is as follows: - A population of at least one type of cancer cell originates from an immortalized cell lineage, - A population of at least one type of immune cell originates from the patient or a healthy donor.

[0076] In this configuration, chimeric embryos are used to test the ability of patient-derived immune cells to recognize and / or destroy specific cancer cells.

[0077] Another example of the embodiment is as follows: -A population of at least one type of cancer cell originates from patient-derived xenograft (PDX) mice. - A population of at least one type of immune cell originates from the patient or a healthy donor.

[0078] In this configuration, chimeric embryos are used to test the ability of immune cells derived from the patient / healthy donor to recognize and / or destroy specific cancer cells.

[0079] In a different configuration, -A population of at least one type of cancer cell originates from a patient or human cell lineage or mouse PDX mouse. - A population of at least one type of immune cell arises from a humanized mouse model in which the human immune system has been reconstituted.

[0080] In this configuration, chimeric embryos are used to test the ability of immune cells to recognize and / or destroy specific cancer cells.

[0081] In another preferred embodiment of the present invention, a population of at least one type of exogenous cells is labeled. Preferably, both a population of immune cells and a population of cancer cells are labeled, in particular, with two different colors or markers to distinguish them.

[0082] Localization of exogenous cells in avian embryos In the present invention, in the embryo of a chimeric pheasant bird, - Exogenous cancer cells are present in at least one type of tissue of the embryo. - Exogenous immune cells are present in at least one tissue of the embryo and / or circulate in the blood vessels of the embryo.

[0083] Therefore, the present invention relates to three distinct configurations. (1) Chimeric pheasant bird embryo, in which cancer cells and immune cells are present in at least one tissue of the embryo; (2) Chimeric pheasant bird embryo, in which cancer cells are present in at least one tissue of the embryo and all immune cells circulate in the blood vessels of the embryo; and (3) Chimeric pheasant bird embryo, in which cancer cells and immune cells are present in at least one tissue of the embryo, and some immune cells circulate in the blood vessels of the embryo.

[0084] In the three configurations, cancer cells are present in at least one type of tissue of the embryo. All cancer cells may be reorganized into only one type of tissue of the embryo, or they may be present in two, three, four, five or more different tissues.

[0085] In a specific embodiment, cancer cells form at least one tumor, i.e., a mass of cancer cells inserted into tissue.

[0086] In preferred embodiments of configurations (1) and (3), both types of exogenous cells are localized in the same at least one type of embryonic tissue, and in particular, immune cells are present in tumors formed by cancer cells. Among other things, immune cells infiltrate the tumor. Cancer cells can form one or more tumors.

[0087] This configuration is particularly advantageous because it reflects the in vivo situation in which tumors are infiltrated by the body's immune cells. This physical association of exogenous cancer cells and immune cells in the recipient embryo reflects the structure of tumors in the body. Therefore, the chimeric pheasant bird embryo of the present invention is a relevant animal model for studying the interaction between tumor cells and immune cells.

[0088] In preferred embodiments of the present invention, both types of exogenous cells are physically associated. In particular, immune cells and cancer cells form a solid tumor infiltrated by immune cells in at least one type of tissue of the embryo.

[0089] Advantageously, the tumors are infiltrated by various subpopulations of immune cells, particularly CD4+ T cells and CD8+ T cells, and examples illustrate the diversity of immune cell populations present in tumors formed in transplanted embryos. As shown in Figure 4B, all subpopulations of transplanted PBMCs are maintained in the post-transplant embryo.

[0090] Advantageously, tumors are infiltrated by immune cells expressing markers of activation and / or exhaustion. In particular, these immune cells express PD1 and / or TIM-3 exhaustion markers. Interestingly, immune cells expressing PD1 respond to treatment with anti-PD1 compounds.

[0091] In specific embodiments of the present invention, the tumor formed in the embryo comprises exogenous immune cells expressing markers of activation (CD69, CD25) and / or exhaustion (PD1, TIM-3).

[0092] Advantageously, in tumors formed in transplanted pheasant embryos, there is a "dialogue" between immune cells and tumor cells, which is a typical physiological process for tumors within the patient's body.

[0093] In preferred embodiments of the three configurations, the exogenous cancer cells are present in at least one embryonic tissue that is representative of the tissues / organs in which primary or secondary tumors are formed in cancer patients, i.e., equivalent tissues or organs, relating to the nature of the cancer cell population.

[0094] Both terms, “representative tissue” and “equivalent tissue,” are used interchangeably and refer to pheasant embryonic tissue that is equivalent to non-embryonic mammalian tissue but possesses its embryonic state and morphology as a pheasant bird organism. These terms refer to tissues that mimic the tissue, cellular, and / or molecular aspects of a particular non-embryonic mammalian tissue.

[0095] For example, the embryonic colon, which arises from the endoderm, is a typical colon organ in mammals, and if extrinsic cancer cells are cancerous colon cells, they are located in the aforementioned endoderm (endodermic germ layer).

[0096] Another example is breast cancer cells that tend to metastasize to bone tissue, in which case the exogenous cancer cells may be present in embryonic bone, which originates from the mesoderm germ layer.

[0097] Method for obtaining chimeric pheasant bird embryos according to the present invention Furthermore, the present invention comprises the following sequential steps: The process involves introducing both a population of exogenous immune cells and a population of cancer cells into a pheasant bird embryo. The present invention relates to a method for obtaining the aforementioned chimeric pheasant bird embryo, comprising the step of incubating the chimeric embryo for at least 6 hours at a temperature preferably in the range of 35°C to 42°C.

[0098] The term "introduction" refers to any technique useful for creating a chimeric embryo that contains exogenous cells in addition to its own cells. In particular, cell introduction is the transplantation of exogenous cells into specific tissues of the embryo.

[0099] The incubation process shall be carried out in accordance with standard techniques, in particular, in a humidity-saturated incubator.

[0100] The incubation temperature will be adapted to the properties of pheasant bird embryos according to the knowledge of those skilled in the art. In specific embodiments, the incubator temperature is in the range of 35°C to 42°C, where a range boundary value is included. Preferably, the incubation temperature is in the range of 37°C to 40°C, and preferably about 38.5°C for chicken embryos.

[0101] The incubation time is at least 6 hours, which is the time required to obtain the initial aggregation of cancer cells into at least one type of tissue in the embryo. Preferably, the incubation period is at least 8 hours, at least 10 hours, at least 12 hours, at least 16 hours, at least 24 hours, at least 36 hours, or at least 48 hours. The incubation period is generally about 24 hours or about 48 hours.

[0102] Ideally, embryo incubation and analysis should be stopped before the embryo is fully developed (i.e., 21 days post-fertilization for chicken embryos), and even more ideally, the incubation and analysis process should be completed before the first two-thirds of the embryonic development period, i.e., before 14 days post-fertilization.

[0103] Both types of methods are the objectives of the present invention and are described in detail below.

[0104] In the first embodiment, the method of the present invention comprises the following sequential steps: The process involves co-transplanting both a population of immune cells and a population of cancer cells into at least one type of tissue of a pheasant bird embryo in the developmental stage between HH10 and HH30, • This chimeric embryo is incubated at a temperature within 35°C to 42°C for at least 6 hours. Includes.

[0105] In the sense of the present invention, the term "co-transplantation" refers to introducing both types of exogenous cells into at least one type of tissue in the recipient embryo during a single treatment.

[0106] Exogenous cells are as follows: - Suspended cells injected into target tissue; - A solid piece (block) of tumor tissue containing both immune cells and cancer cells; - Aggregates / homogenates of isolated cells It can be transplanted in its morphological form.

[0107] The transplantation of exogenous cells into recipient pheasant bird embryos is carried out according to methods well known to those skilled in the art. In fact, pheasant bird embryos can be easily accessed after making a small opening in the eggshell. In particular, the transplantation of cancer cells is carried out using an air-powered microinjector (Picopump PV830, World Precision Instruments). This technique is presented in particular in WO 2016 / 05398 and WO 2017 / 103025.

[0108] Several developmental stages of pheasant bird embryos have been previously defined as a function of the incubation time after fertilization and are determined according to the criteria defined by Hamburger and Hamilton (1951, J Morphol.).

[0109] According to the present invention, at the time of transplantation of exogenous cells, the pheasant bird embryo is in a developmental stage between HH10 and HH30.

[0110] Preferably, at the time of exogenous cell introduction, the pheasant bird embryo is in the developmental stage between HH12 and HH25. This developmental stage of the embryo, occurring 40 hours to 4.5 days after fertilization, is characterized by many important events of embryogenesis, including the appearance of somites, the subdivision of cerebral regions, the curvature of various regions of the embryo, and the formation of numerous organs.

[0111] In another preferred embodiment, upon introduction of exogenous cells, the pheasant bird embryo is in a developmental stage between HH10 and HH18, between HH10 and HH15, or between HH12 and HH16.

[0112] Preferably, the cancer cells are transplanted into recipient pheasant bird embryos that are in the developmental stage between H13 and HH15, that is, 48 ​​to 55 hours after fertilization, preferably 50 to 53 hours after fertilization (HH14).

[0113] In a second embodiment, the method of the present invention comprises the following steps: The process involves transplanting a population of at least one type of cancer cells into at least one type of tissue of a pheasant bird embryo in the developmental stage between HH10 and HH30, The step of injecting the population of at least one type of immune cells into the blood vessels of the embryo, • The transplanted embryo is incubated at a temperature between 35°C and 42°C for at least 6 hours. Includes.

[0114] The transplantation and incubation processes are described and will be carried out as previously indicated.

[0115] In the implementation of this method, the step of "injecting cells" corresponds to the step of introducing immune cells into the blood circulation of the embryo by any technique known to those skilled in the art.

[0116] This injection step can be performed simultaneously with, before, or after the cancer cell transplantation step. In particular, the immune cell injection step can be performed in the later stages of embryonic development, i.e., after the HH30 stage. However, in a preferred embodiment, both types of exogenous cells are introduced into the embryo simultaneously.

[0117] In some cases, as shown in the Examples section, immune cells will bind to cancer cells transplanted into at least one type of tissue and invade the tumor. In some other cases, immune cells will circulate within the embryo's blood vessels, and their ability to act on the development of cancer cells through blood circulation will be tested. In some other cases, immune cells will both bind to cancer cells and circulate within the embryo's blood vessels.

[0118] In specific embodiments, the transplantation of a population of cancer cells is performed in at least one embryonic tissue that is representative of the tissues / organs in which primary and / or secondary tumors are formed in cancer patients, in relation to the nature of the population of cancer cells.

[0119] In the sense of the present invention, the “related” tissue is either a tissue that is the origin of the cancer cells or a tissue that is typically colonized by the cancer cells, i.e., a tissue in which secondary tumors (metastases) are embedded.

[0120] Exogenous cancer cells can be transplanted into a recipient embryo in different tissues, such as the tissue constituting the neural tube (not the lumen), brain tissue, digestive structures, such as embryonic tissue that preforms the liver, intestines, or pancreas, or embryonic tissue or somite regions that preform the skin, bone, and bone marrow.

[0121] Animal models and their use Furthermore, the present invention relates to an animal model for the study of human cancer that is easily obtained by either the above-described chimeric pheasant bird embryo or by either of the above-described methods.

[0122] Furthermore, the present invention relates to an animal model for the study of human cancer obtained by any one of the methods described above.

[0123] This animal model of the present invention has many applications.

[0124] In particular, this animal model can be used to monitor patients with cancer. Indeed, such an animal model makes it possible to monitor the development of a patient's tumor ex vivo, especially at time T0 (e.g., before the start of the treatment) and at times T1, T2, and T3 (e.g., after the start of treatment for the cancer patient) after the treatment has been initiated.

[0125] Furthermore, animal models can also be used to screen therapeutic molecules intended for treating cancer.

[0126] In particular, the present invention relates to the following group: - Immunotherapy: A therapeutic strategy consisting of promoting the antitumor activity of the patient's immune cells. - Chemotherapy: Drugs that affect the survival, metabolism, motility and / or proliferation of cancer cells. - Targeted therapy: The use of compounds such as antibodies that target specific molecules expressed by cancer cells. - Hormone therapy: Use of compounds that target hormone molecular signaling, - Anticancer drugs and - Therapeutic enzymes The present invention relates to the use of an animal model for screening at least one anti-cancer therapy selected from among the following.

[0127] In particular, the animal model of the present invention can be used to evaluate the activity of genetically modified CAR-T cells. In specific embodiments, these CAR-T cells are allogeneic CAR-T cells intended for use in any patient. The efficacy and safety of these allogeneic cells can be tested in the animal model of the present invention.

[0128] Examples In this specification, the present invention has been described with reference to specific embodiments, but it should be understood that these embodiments are merely illustrative of the principles and applications of the present invention. Therefore, it should be understood that many modifications can be made to the exemplified embodiments, and other configurations can be devised without departing from the spirit and scope of the invention as defined by the appended claims.

[0129] Materials and methods Gallus gallus fertilized eggs were obtained from a specialized farm, e.g., Earl Morizeau, Dangers, France, and maintained at 14°C until use. For running experiments, the eggs were incubated at 38.5°C for 52 hours to obtain embryos at developmental stages HH13-HH16, according to Hamiltonian nomenclature.

[0130] To perform the operation, a window was opened in the shell.

[0131] Human PBMCs and tumor cells were transplanted using a microinjector (Pipopump PV830, World Precision Instruments) for insertion.

[0132] (1) In some experiments, human PBMCs were injected into the embryonic blood vessels through blood vessels present on the chorioalangiate. Cancer cells were transplanted according to the methods described in WO 2016 / 05398 and 2017 / 103025.

[0133] (2) In other experiments, both types of cells were co-transplanted into selected sites of embryos within tissues where human tumor cells form tumors, establishing tumors with a human immune cell stroma.

[0134] Embryos were kept in incubation for 24 to 48 hours after transplantation of cancer cells. They were collected from the eggs and processed for various experiments. In some experiments, embryos were digested with collagenase and human cells were isolated for further analysis. In other experiments, embryos were fixed overnight at 4°C with 4% paraformaldehyde. Embryos were clarified for imaging using an optical sheet microscope (LaVision Biotec).

[0135] I. Demonstrating the feasibility of transplanting PBMCs into avian embryo models. Example 1 Condition (1): The shell of an embryo-forming chicken egg was opened. Human PBMCs from a healthy donor were injected into the vascular structure (blood circulation) of the embryo.

[0136] Condition (2): PBMCs were prepared, labeled with vital fluorescent tracers, and implanted into target tissue of avian embryos by injection using microcapillaries.

[0137] Two days after PBMC injection (1) or transplantation (2), the embryos were retrieved, fixed in paraformaldehyde, imaged using a stereomicroscope, then clarified, and imaged again using an optical sheet microscope.

[0138] In other experiments, embryos were digested with collagenase, and PBMCs were purified using a Percoll gradient.

[0139] Under both experimental conditions, living PBMCs were found in avian embryonic tissue, as shown in Figures 1A and 1B.

[0140] Example 2 In another experiment, PBMCs from healthy donors were labeled with a vital fluorescent tracer (CFSE: carboxyfluorescein succinimimidyl ester) and then injected into the vascular structure of chicken embryos (HH13-HH15) 56 hours after fertilization.

[0141] Two days later, the embryos were retrieved, heads removed, and digested with collagenase. PBMCs were isolated using FICOLL and PERCOLL gradient techniques, and fluorescent PBMC cells were analyzed by FACS.

[0142] The results are shown in Figure 2. The results demonstrate that the injected PBMCs survived 48 hours after introduction into the embryo.

[0143] Example 3 PBMCs were injected into the vascular structure of embryos at different doses. Two days (48 hours) after injection, isolated PBMCs were counted to determine their % survival rate.

[0144] The results are shown in Figure 3. Cell survival was observed at high doses, e.g., 6 × 10⁶ 5 Cells / μl (black triangle) showed impaired survival compared to injections of fewer PBMCs (2-fold dilution) where good survival was observed. It appears that the concentration of injected cells, rather than the absolute number of cells, is a crucial parameter for cell survival.

[0145] Example 4 PBMCs were prepared from human blood samples and transplanted into avian embryo tissue 56 hours after egg-laying (HH14).

[0146] Two days after transplantation, embryos were retrieved and digested using collagenase. Immune cells were purified, and subpopulations of immune cells were analyzed by molecular markers in FACS to determine the proportion of B lymphocytes (CD19+) and T (CD3+CD4+ and CD3+CD8+) populations to the total immune cell population. Subsequently, all major PBMC subpopulations across the entire PBMC population (CD45+ cells) were monitored: lymphocytes (B and T), natural killers, and bone marrow cells (hereinafter referred to as monocytes).

[0147] The results are shown in Figures 4A, 4B, and 4C. "PBMC" refers to immune cells before transplantation, and "AVI-PBMC" refers to PBMC cells that were transplanted and analyzed after Percoll isolation from collagenase-treated embryos.

[0148] In Figure 4A, the values ​​are expressed as the ratio of total specific lymphocytes (CD19+ and CD3+).

[0149] Under these conditions, the proportion of the B lymphocyte population to the total immune cell population was lower in chicken embryos compared to the population of fresh PBMCs. This difference in proportion was also observed in a mouse model of xenograft (PDX).

[0150] Figure 4B shows the subpopulation redistribution of immune cells before (PBMC) and 48 hours after (AVI-PBMC) transplantation into chicken embryos. Interestingly, all PBMC subpopulations were maintained after transplantation into chicken embryos.

[0151] Figure 4C shows the expression of immune cell activation markers (CD69, CD25) and exhaustion markers (PD1, TIM-3) before (PBMC) and 48 hours after (AVI-PBMC) transplantation of immune cells into chicken embryos. Two populations of T lymphocytes, CD8+ and CD4+, are analyzed.

[0152] Except for a significant increase in CD69 after transplantation of both CD4+ and CD8+ lymphocyte T cells, the expression of other markers was maintained or slightly increased after transplantation. PBMC activation was fairly weak, indicating a weak allogeneic response. PBMC transplantation did not interfere with embryonic development, indicating that the host and transplanted PBMCs can coexist.

[0153] II. Demonstrating the feasibility of co-transplanting PBMCs and tumor cell lines into avian embryo models. Example 5. Introduction of both an exogenous population of immune cells and an exogenous population of cancer cells. Human PBMCs were labeled with a vitals fluorescence tracer.

[0154] Two fluorescent human tumor cell lines were used. - Primary melanoma cell lineage derived from patient samples (Pmela, metastatic BRAF V600 melanoma) (also known as GLO) and - MDAMB436 cells (commercially available triple-negative breast cancer cell lineage) (also known as MDAM)

[0155] Both types of cells are introduced into the embryo as presented in the Materials and Methods section.

[0156] Condition (1): PBMCs were co-transplanted with tumor cells into selected tissue of the embryo.

[0157] Condition (2): PBMCs were injected into the vascular structure. Tumor cells were transplanted into selected tissue of the embryo.

[0158] Two days after transplantation, the embryos were imaged using a stereoscopic microscope. The retrieved embryos were measured, and their weights were determined. The embryos were clarified and imaged using a confocal optical sheet microscope. The images were processed using software (IMARIS) to quantify the tumor volume.

[0159] In both conditions, the presence of immune cells within the tumor was observed. The immune "contingency" was more robust in terms of cell count with the co-transplantation method. The results for condition (1) are shown in Figure 5. The "merge" images represent both types of populations.

[0160] The results for condition (2) are shown in Figures 6 and 7.

[0161] In Figure 6, the brighter halos (marked with dashed lines) indicate immune cells reorganized around tumors formed in bird embryos.

[0162] In Figure 7, individual tumor cells in a tumor transplanted into a chicken embryo are labeled (left image), and immune cells derived from PBMCs are labeled (right image). The immune cells are reorganized around the tumor.

[0163] Example 6. Measurement of BSA and tumor volume of transplanted chicken embryos with and without immune cells. The body size area (BSA), a key indicator, is measured to determine the toxicity of introducing PBMCs into chicken embryos.

[0164] The results shown in Figure 8 indicate that no statistically significant difference in BSA was observed between embryos transplanted with Pmela cells alone (Glo) and embryos co-transplanted with PBMCs (GLO-PBMC).

[0165] The results shown in Figure 9 represent the measured tumor volume when Pmela cells were transplanted alone (Glo) or in combination with human PBMCs (GLO-PBMC) according to condition (1).

[0166] The graph does not show any statistically significant difference between embryos with transplanted cancer cells and embryos with co-grafts of cancer cells and immune cells. The data was normalized to the body size area (BSA) of the embryos.

[0167] Example 7. Co-transplantation of tumor cells and PBMCs In chicken embryos, PBMCs were introduced into the following two tumor cell lines: -Pmela- Primary cells derived from metastatic BRAF V600 melanoma (PBMC-GLO) -MDAMB436 and MDAMB231-Immortalized cell lines derived from triple-negative breast cancer (PBMC-MDAM) It was co-transplanted with at least one of the following:

[0168] Two days after transplantation, PBMCs were purified from the embryos, and molecular markers for different cell populations—CD3+, CD4+, and CD8+—were analyzed by flow cytometry. The results are shown in Figure 10A.

[0169] As shown, PBMCs respond when transplanted into avian embryonic tissue compared to their pre-transplant resting state. The presence of tumor cells inhibits T cell activity, resulting in downregulation of CD69 and HLADR markers, returning their levels to those of fresh PBMCs. Higher levels of CTLA4 on CD4 and CD8 T cells are observed after PBMC transplantation. Keytruda administration results in minimization of PD1 expression / accessibility on CD8 T cells. PD1 blockade leads to very high levels of CTLA4 on the CD8 compartment, suggesting compensatory expression.

[0170] Figure 10B shows the marker expression patterns on the surface of immune cells after co-transplantation of PBMCs and MDAMB231 cells expressing high levels of PDL1 in chicken embryos (left side: CD8+, right side: CD4+).

[0171] In the presence of cancer cells, immune cells, In particular, increased expression of the activation markers CD69 and CD25 in the CD8+ pool. Increased expression of fatigue markers PD1 and TIM-3 (also known as immune checkpoint regulatory modulators) It is included in tumors that exhibit this characteristic.

[0172] These results demonstrate a two-step immune response to the presence of co-transplanted tumor cells. (i) MDAMB231 tumor cells activate T cells, as indicated by increased CD69 and CD25 expression compared to PBMCs transplanted alone. (ii) The inhibition of the immune response is already visible, as indicated by the increased expression of the immune checkpoint regulatory modulators PD1 and TIM-3. This expression pattern reflects negative immune regulation of lymphocytes by cancer cells (corresponding to tumor cell "escape").

[0173] Therefore, xenotransplantation of human immune cells into avian embryos induces their activation and "neutralization." This indicates that, when introduced into avian embryos, T cells remain functional and capable of recognizing allogeneic cells.

[0174] III. Demonstrating the feasibility of treating transplanted PBMCs with immunotherapy Example 8. Keytruda (anti-PD1) treatment This experiment was conducted to study the human T cell state of PBMCs introduced into avian embryos. The T cell compartment is a target for several currently approved checkpoint inhibitors, such as αCTLA4 and PD1 axis blockade.

[0175] First, we confirmed the expression of the PD1 receptor on transplanted PBMCs (Figure 11A) and the expression of PDL1 by cancer cells (Figure 11B). MDAMB436 cells were cultured in vitro and immunolabeled with an anti-PDL1 antibody to determine the expression of PDL1, a PD1 ligand. Similar experiments were performed on Pmela cells and MDAMB231 cells.

[0176] Next, MDAMB436 cells or Pmela cells were co-transplanted with human PBMC cells.

[0177] The anti-PD1 antibody Keytruda (Pemzolibrumab, Merk) was injected 24 hours after transplantation. Two days (48 hours) after transplantation, PBMCs were isolated from the transplanted embryo and analyzed by FACS.

[0178] Finally, the transplanted PBMCs were isolated by Percoll density separation, and the cells were stained for the activation marker (CD69 / HLADR) and the exhaustion marker (CTLA4 / PD1).

[0179] I will comment on the results below.

[0180] a) PD1 expression by transplanted immune cells Figure 11A shows the levels of PD1 expression on the cell surface of CD8+ and CD4+ T cells derived from two different donors. After treatment with Keytruda (diluted 5-fold), which antagonizes the PD1 receptor, the detection of PD1 is significantly reduced. These results indicate that Keytruda binds to the PD1 receptor on transplanted immune cells.

[0181] b) PDL1 expression by transplanted cancer cells The results are shown in Figure 11B. Approximately 10% of MDAMB436 cells cultured in vitro showed expression of the PDL1 ligand. PDL1 expression was not detected in Pmela cells. PDL1 expression was high in MDAMB231 cells.

[0182] c) T cell status Among the tumor cells, Pmela is a PDL1-negative cell lineage, while MDAMB436 is a weakly PDL1-positive cell lineage. Transplanted avian embryos were treated with or without the immunotherapy anti-human PD1 Keytruda(K).

[0183] Subpopulations of cells were identified by FACS after Percoll isolation of PBMCs.

[0184] The results are shown in Figure 11C. Activation markers (CD69 and HLADR, left) and exhaustion markers (CTLA-4 and PD1, right) were measured for each cell population: - Fresh PBMC (not introduced into any embryos) - Transplanted PBMC - The transplanted PBMC+K embryos were treated with Keytruda, an anti-PD1 antibody, for 24 hours. - Transplanted Pmela / PBMC: Co-transplantation of immune cells and Pmela, which are cancer cells that do not express PDL1. - Transplanted MDAM / PBMC: Co-transplantation of immune cells and MDAM, which are cancer cells expressing PDL1. - Treat the transplanted MDAM / PBMC+K embryos with Keytruda, an anti-PD1 antibody, for 24 hours. A decision was made regarding this.

[0185] The standard deviation represents data from a pool of PBMCs from various healthy donors. The value is expressed as the ratio of specific markers to the CD3+CD4+ population and the CD3+CD8+ population.

[0186] Generally, CD4 and CD8 T cells isolated after transplantation showed signs of activation, and while most CD4 and CD8 T cells were positive for CD69, CD4 T cells acquired HLADR expression.

[0187] Regarding inhibitory receptors, the inventors observed higher levels of CTLA4 on CD4 T cells and CD8 T cells after PBMC transplantation, but higher levels of PD1 were observed only on CD4 T cells.

[0188] Therefore, xenotransplantation of human immune cells into avian embryos induces their activation. Activation of human immune cells has also been reported in mouse models. This indicates that, when introduced into avian embryos, T cells remain functional and capable of recognizing allogeneic cells.

[0189] PD1 blockade resulted in very high levels of CTLA4 in the CD8 compartment, suggesting compensatory expression.

[0190] d) Another experiment was performed using MDAMB231 cells co-transplanted with PBMCs, and the transplanted embryos were subsequently treated with Keytruda (5 mg / ml).

[0191] The expression of activation and exhaustion markers on co-transplanted immune cells was quantified. The results are shown in Figure 11D.

[0192] The expression of PD1 and TIM-3, which are fatigue markers, decreases with Keytruda treatment. This confirms that Keytruda can restore the responsiveness of fatigued T lymphocytes.

[0193] Activation markers will likely be reconstituted later in the Keytruda treatment (24-hour administration is probably insufficient to reconstitute the expression of these activation markers). Furthermore, higher concentrations of Keytruda should lead to enhanced cell activation.

[0194] In summary, 24-hour Keytruda administration can initiate the immune cell reactivation process, as demonstrated by the reduction in PD1 and TIM-3 expression.

[0195] IV. Demonstration of the potential of the transplanted avian embryos of the present invention as a model for monitoring responses to immunotherapy treatments. Example 9. PBMCs co-transplanted with A375 cell lineage

[0196] To determine whether tumors composed of mixed human tumor cells and immune cells are sensitive to anti-PD1 antibody treatment, the human melanoma A375 cell line was selected for the experiment. Indeed, this cell line has recently been reported to express significant levels of PDL1 and respond to anti-PD1 immunotherapy (Kuryk L, Moller AW, Jaderberg M, 2019).

[0197] A375 melanoma cells were cultured and co-transplanted with human PBMC cells as described above.

[0198] The anti-PD1 antibody Keytruda or a negative control (excipient) was injected into the embryo 24 hours after transfer.

[0199] Two to three days after transplantation, the embryos were retrieved and processed for imaging using a confocal optical sheet microscope. Tumor volume was quantified using IMARIS software.

[0200] The results are shown in Figure 12A. In the presence of Keytruda, tumor volume is significantly reduced. This demonstrates that immune cells surrounding cancer cells respond positively to this immunotherapy treatment.

[0201] Example 10. PBMCs co-transplanted with MDAMB231 cells or patient cells The same experiment as in Example 9 was performed on other cancer cells: - MDAMB231 and are known to be responsive to immunotherapy. - Tumor samples from patients with metastatic melanoma I went along with it.

[0202] Figure 12B shows the effect of Keytruda administration on tumor volume in bird embryos co-transplanted with human PBMCs and MDAMB231 tumor cells (see center and right panels).

[0203] A significant reduction in tumor volume was observed under anti-PD1 treatment conditions compared to NaCl administration.

[0204] Figure 12C shows the effect of Keytruda administration to bird embryos co-transplanted with tumor samples from human PBMCs and patients with metastatic melanoma.

[0205] A significant reduction in metastatic volume was observed under Keytruda administration conditions.

[0206] Furthermore, the number of metastases formed by the patient's tumor cells in avian embryonic tissue was evaluated by tracking fluorescently labeled tumor cells and subsequently quantifying the fluorescence signal in the embryo.

[0207] After administration of Keytruda, the metastatic potential of tumor cells is significantly reduced, as shown in the histogram titled "Number of Metastases."

[0208] Both histograms indicate that anti-PD1 administration results in a significant reduction in the volume and number of metastases formed by the patient's tumor cells in avian embryonic tissue.

[0209] Example 11. Analysis of PBMCs at 48 and 72 hours post-transplantation Embryos were collected 48 hours (light gray) or 72 hours (dark gray) after transfer. Subgroups were observed across the living CD45+ population and compared with pre-transfer PBMCs (PBMC D0). The results are shown in Figure 13.

[0210] All major PBMC subpopulations are found to be viable. Furthermore, some populations show higher survival rates at 72 hours compared to 48 hours post-transplant (natural killer population). The allogeneic response of PBMCs to the avian host does not affect embryonic development at 72 hours. Therefore, 48-hour immunotherapy treatment can be achieved using this model.

[0211] References patent WO No. 2015 / 074050 US No. 2013 / 0171680 WO No. 2016 / 05398 WO No. 2017 / 103025 WO No. 2020 / 075168

[0212] Bibliographic reference [Table 1] TIFF2026136204000003.tif113161

Claims

1. It is a chimeric pheasant bird embryo, Both types of exogenous cells: a. A population of at least one type of cancer cell and b. A population of at least one type of immune cell Includes, Here, the exogenous cancer cells are present in at least one tissue of the embryo, and the exogenous immune cells are present in at least one tissue of the embryo and / or circulate in the blood vessels of the embryo. Chimeric pheasant bird embryo.

2. The chimeric pheasant bird embryo according to claim 1, wherein the group of immune cells consists of peripheral blood mononuclear cells (PBMCs).

3. The chimeric pheasant bird embryo according to claim 1, wherein the population of immune cells consists of lymphocytes, particularly genetically modified CAR-T cells.

4. A chimeric pheasant bird embryo according to any one of claims 1 to 3, wherein the cancer cells are derived from the patient's tumor.

5. A chimeric pheasant bird embryo according to any one of claims 1 to 4, wherein both cell populations are human cells.

6. The chimeric pheasant bird embryo according to any one of claims 1 to 5, wherein both types of exogenous cells are localized in the same at least one type of embryonic tissue, and in particular, immune cells are present in a tumor formed by cancer cells.

7. The chimeric pheasant bird embryo according to any one of claims 1 to 6, wherein the at least one embryonic tissue containing cancer cells is, in relation to the nature of the cancer cell population, a representative tissue / organ in which primary and / or secondary tumors are formed in cancer patients.

8. A chimeric pheasant bird embryo according to any one of claims 1 to 7, wherein at least one population of exogenous cells is labeled.

9. The following steps: - A process of introducing both a population of immune cells and a population of cancer cells into a pheasant bird embryo, - The process includes incubation of the chimeric embryo at a temperature between 35°C and 42°C for at least 6 hours. A method for obtaining a chimeric pheasant bird embryo according to any one of claims 1 to 8.

10. The following steps: - A process of co-transplanting both a population of immune cells and a population of cancer cells into at least one type of tissue of a pheasant bird embryo in the developmental stage between HH10 and HH30, The method according to claim 9, further comprising the step of incubating the chimeric embryo at a temperature in the range of 35°C to 42°C for at least 6 hours.

11. The following steps: - A process of transplanting a population of at least one type of cancer cells into the tissue of at least one type of pheasant bird embryo in the developmental stage between HH10 and HH30, - A step of injecting the population of at least one type of immune cells into the blood vessels of the embryo, The method according to claim 9, further comprising the step of incubating the transplanted embryo at a temperature in the range of 35°C to 42°C for at least 6 hours.

12. The method according to claim 10 or 11, wherein the transplantation of the population of cancer cells is performed in at least one embryonic tissue that is a representative tissue / organ in which primary and / or secondary tumors are formed in cancer patients, in relation to the nature of the population of cancer cells.

13. A chimeric pheasant bird embryo according to any one of claims 1 to 8, Animal models for human cancer research.

14. Use of the animal model according to claim 13 for screening at least one anti-cancer therapy selected from the following groups: immunotherapy, chemotherapy, targeted therapy, hormone therapy, anticancer drugs, and therapeutic enzymes.

15. Use of the animal model according to claim 13 for evaluating the activity of genetically modified CAR-T cells.

Images