Method for identifying antigen-reactive immune cells of human

Abstract

To provide a method of identifying human antigen-reactive immune cells employing an animal model where issues of graft-versus-host disease and rejection reaction are eliminated or alleviated.SOLUTION: A method for identifying antigen-reactive immune cells of human includes: a xenotransplant animal creation step of creating a xenotransplant animal by transplanting human-derived tissue and immune cells into an immunodeficient animal with mouse MHC deleted by gene editing; a human immune response step of inducing a human immune response within a first xenotransplant animal obtained in the xenotransplant animal creation step; and an isolation step of separating antigen-reactive immune cells from a second xenotransplant animal obtained in the human immune response step.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to a method for identifying antigen-reactive immune cells in humans. [Background technology]

[0002] Attempts have been made to generate xenografts (humanized mice) bearing human immune cells by transplanting human immune cells into severely immunodeficient mice (e.g., NOG mice). However, Non-Patent Documents 1 and 2 disclose that humanized mice were generated using immunodeficient mice with a normal major histocompatibility complex (MHC). Human immune cells recognize the MHC of the severely immunodeficient mice as foreign, resulting in graft-versus-host disease (GVHD). The transplanted human immune cells are nonspecifically activated and proliferate in the severely immunodeficient mice, resulting in failure to reproduce a physiological cancer immune environment and death within a few weeks (see Figure 1). Furthermore, Non-Patent Document 1 discloses that human CD34-positive hematopoietic stem cells were transplanted into NOG mice, and human T cells were matured in the mouse thymus. However, the human T cells selected in the mouse thymus possess antigen specificity not actually present in the original donor's body, and are therefore considered unphysiological as human immune cells.

[0003] Non-Patent Document 2 discloses the development, by gene editing, of NOG-dKO mice (strain name at current distributor: NOG-ΔMHC mice), which are severely immunodeficient mice lacking MHC class I and II. These mice exhibit significantly reduced GVHD following transplantation of human immune cells compared to normal NOG mice. Non-Patent Document 2 further discloses that attempts were made to generate humanized mice by transplanting peripheral blood mononuclear cells (PBMCs) from healthy individuals into the NOG-ΔMHC mice engrafted with commercially available human cancer cell lines. However, because the MHC of PBMCs from healthy individuals is typically different from the MHC of commercially available human cancer cell lines, it is believed that T cells in the PBMCs from healthy individuals undergo an alloreaction (rejection) against the commercially available cancer cell lines, preventing the recapitulation of physiological cancer immune responses (see Figure 2). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Saito R et al, Int J Clin Oncol; 25(5): p831-841, 2020 [Non-patent document 2] Ashizawa et al, Clin Can Res; 23(1): p149-158, 2017 Summary of the Invention [Problem to be solved by the invention]

[0005] In recent years, there has been a demand for the development of a xenotransplant animal model that eliminates or reduces the problems of graft-versus-host disease and rejection, and for a method for identifying human antigen-reactive immune cells using such xenotransplant animals.

[0006] The problem to be solved by the present invention is to develop a xenotransplant animal model that eliminates or reduces the problems of graft-versus-host disease and rejection, and to provide a method for identifying human antigen-reactive immune cells using the xenotransplant animal. [Means for solving the problem]

[0007] The present inventors have conducted extensive research in light of the above-mentioned problems and have found that when cancer tissue and peripheral blood immune cells collected from the same cancer patient are transplanted into NOG mice, the resulting humanized mice develop a patient-derived human cancer immune response. The present invention was completed based on these findings.

[0008] The present invention relates to a method for identifying human antigen-reactive immune cells, comprising a xenograft animal production step of transplanting tissues and immune cells collected from a human into an immunodeficient animal that has been genetically edited to delete mouse MHC to produce a xenograft animal; a human immune response step of generating a human immune response in a first xenograft animal obtained in the xenograft animal production step; and an isolation step of isolating antigen-reactive immune cells from a second xenograft animal obtained in the human immune response step. The tissue preferably includes at least one selected from the group consisting of cancer tissue and human normal tissue. The cancer tissue preferably includes at least one selected from the group consisting of kidney cancer tissue, urothelial cancer tissue, prostate cancer tissue, and liposarcoma tissue. The immunodeficient animal preferably comprises an immunodeficient mouse. The antigen-reactive immune cells preferably include at least one selected from the group consisting of antigen-specific T cells, antigen-specific B cells, antigen-specific plasma cells, and antigen-presenting cells that have taken up an antigen. [Effects of the Invention]

[0009] The method for identifying human antigen-reactive immune cells of the present invention provides the development of a xenotransplant animal model that eliminates or reduces the problems of graft-versus-host disease and rejection, and a method for identifying human antigen-reactive immune cells using the xenotransplant animal. [Brief explanation of the drawings]

[0010] [Figure 1] A schematic diagram showing the non-specific activation and proliferation of transplanted human immune cells in severely immunodeficient mice. [Figure 2] Schematic diagram showing the rejection of human cancer cell lines by transplanted human immune cells. [Figure 3] Schematic diagram showing the process of generating xenograft animals and the human immune response process. [Figure 4] FIG. 1 shows changes in body weight over time when human peripheral blood mononuclear cells were transplanted into NOG-ΔMHC mice and conventional mice. [Figure 5] FIG. 1 shows the time course of graft-versus-host disease scores when human peripheral blood mononuclear cells were transplanted into NOG-ΔMHC mice and conventional mice. [Figure 6] Figure 1 shows a comparison of the flow cytometry analysis results of human peripheral blood CD3-positive T cells before transplantation and CD3-positive T cells collected from the peripheral blood of mice 28 days after transplantation into NOG-ΔMHC mice. [Figure 7] Renal cancer tissue and human peripheral blood mononuclear cells were transplanted into NOG-ΔMHC mice, and the figure shows a comparison of the expression of the proliferation marker Ki67 in human T cells within the xenograft tumors after treatment with anti-human PD-1 antibody compared with control mice. [Figure 8] This figure shows that when an anti-human PD-1 antibody was administered to tumor tissue and an immune cell transplant model derived from the same patient, tumor-reactive T cells with the same T cell receptor β CDR3 sequence proliferated in the treated mice. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention will now be described in further detail. Unless otherwise specified, the symbol "to" in a numerical range indicates a range from above to below, and both ends of the range are included. Furthermore, when a numerical range is indicated, the upper and lower limits can be combined as appropriate, and the resulting numerical range is also considered to be disclosed.

[0012] The method of the present invention for identifying human antigen-reactive immune cells includes a xenograft animal production step in which tissue 1 and immune cells 2 collected from a human are transplanted into an immunodeficient animal to produce a xenograft animal (see Figure 1).

[0013] The tissue collected from a human is not limited to a specific tissue and may be any tissue collected from a human (including pluripotent stem cells such as iPS cells and ES cells, their differentiated cells and tissues, and mesenchymal stem cells). The tissue preferably includes cancer tissue, more preferably cancer tissue. The cancer tissue preferably includes at least one selected from the group consisting of kidney cancer tissue, urothelial cancer tissue, prostate cancer tissue, sarcoma tissue, head and neck cancer tissue, lung cancer tissue, gastric cancer tissue, esophageal cancer tissue, duodenal cancer tissue, small intestine cancer tissue, colon cancer tissue, rectal cancer tissue, liver cancer tissue, biliary tract cancer tissue, pancreatic cancer tissue, brain tumor tissue, breast cancer tissue, ovarian cancer tissue, uterine cancer tissue, other solid cancer tissue, and blood cancer, more preferably at least one selected from the group consisting of kidney cancer tissue, urothelial cancer tissue, prostate cancer tissue, and liposarcoma tissue.

[0014] The immunodeficient animal is an animal lacking immune function created using genetic engineering to reproduce human physiological phenomena and diseases, and is not limited to a specific animal. The immunodeficient animal includes at least one selected from the group consisting of immunodeficient mice, immunodeficient rats, immunodeficient pigs, immunodeficient monkeys, immunodeficient chimpanzees, immunodeficient gorillas, and immunodeficient fish. More preferably, it includes immunodeficient mice, even more preferably immunodeficient mice. Particularly preferred are immunodeficient mice with gene editing of MHC classes, not limited to class I and class II, and most preferably NOG-ΔMHC mice, such as NOG-ΔMHC mice with deletions of MHC classes I and II. Figure 1 illustrates an example of an immunodeficient animal. Furthermore, the immunodeficient animal may be at least one selected from the group consisting of animals with host MHC gene editing and animals with host MHC gene editing further subjected to gene editing.

[0015] The immune cells 2 may be transplanted into a first xenograft model constructed by transplanting the tissue 1 into the immunodeficient animal. The tissue 1 may be transplanted into a second xenograft model constructed by transplanting the immune cells 2 into the immunodeficient animal. Furthermore, the tissue 1 and immune cells 2 may be transplanted simultaneously into the immunodeficient animal to construct a third xenograft model.

[0016] The method of the present invention for identifying human antigen-reactive immune cells includes a human immune response step of generating a human immune response in a first xenograft animal obtained in the xenograft animal production step (see Figure 3). Tissue 1 collected from the same human and immune cells 2 derived from peripheral blood or the like react within the first xenograft animal to function as antigen-reactive immune cells, resulting in a second xenograft animal in which a third xenograft model is constructed. When tissue 1 includes cancer tissue, the antigen-reactive immune cells include at least one selected from the group consisting of antigen-specific T cells, antigen-specific gamma-delta T cells, antigen-specific B cells, antigen-specific plasma cells, and antigen-presenting cells that have taken up antigen.

[0017] The method for identifying human antigen-reactive immune cells of the present invention includes an isolation step of isolating antigen-reactive immune cells from the second xenograft animal obtained in the human immune response step. The method for isolating the antigen-reactive immune cells from the second xenograft animal is not limited to a specific method. Examples of the isolation method include a method of isolating the antigen-reactive immune cells by flow cytometry using tetramer staining followed by sequencing of the antigen-specific receptor, and a method of sequencing and cloning the antigen-specific receptor by single-cell sequencing.

[0018] The cancer-reactive immune cells include, for example, the antigen-specific T cells. The antigen-specific T cells are activated against cancer tissues specific to each individual cancer patient. Therefore, the antigen-specific T cells isolated by the method of identifying antigen-reactive immune cells derived from the same human of the present invention are very useful in the development of cancer immunotherapy and cancer immunology research. [Example]

[0019] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0020] [Cancer tissue transplantation into NOG-ΔMHC mice] Cancer tissues collected from each cancer patient were subcutaneously transplanted into NOG-ΔMHC mice (produced by Invivo Science Inc. or the Central Institute for Experimental Animals), and the NOG-ΔMHC mice were maintained under specific pathogen-free conditions. When tumor growth over time was observed, the cancer tissue was considered to have taken root, and subcutaneous tumors were harvested from the NOG-ΔMHC mice. Some of the tumors established by this method were passaged by retransplantation into other NOG-ΔMHC mice or cryopreserved. The mice transplanted with renal cancer, urothelial carcinoma, prostate cancer, and liposarcoma were maintained for one month. The results are shown in Table 1.

[0021] [Table 1]

[0022] It was found that various cancer tissues could be transplanted into NOG-ΔMHC mice.

[0023] [Human peripheral blood mononuclear cell transplantation] Human peripheral blood mononuclear cells (PBMCs) were subcutaneously transplanted into five of the NOG-ΔMHC mice, which were then maintained under specific pathogen-free conditions for 31 days. The mean body weight and mean GVHD score were calculated. The results are shown in Figure 2.

[0024] The GVHD score was measured by the method shown in Table 2 below.

[0025] [Table 2]

[0026] The mean body weight of the NOG mice transplanted with PBMCs (NOG in Figure 4) was significantly reduced compared to the mean body weight of the NOG-ΔMHC mice transplanted with PBMCs (DKO in Figure 4), while the mean body weight of the NOG-ΔMHC mice did not change significantly. Furthermore, the mean GVHD score of the NOG mice transplanted with PBMCs (NOG in Figure 5) was significantly increased compared to the mean GVHD score of the NOG-ΔMHC mice transplanted with PBMCs (DKO in Figure 5), while the mean GVHD score of the NOG-ΔMHC mice did not change significantly.

[0027] The spleens of the NOG-ΔMHC mice, which had been transplanted with PBMCs and maintained for 31 days, were immunostained to identify human T cells, B cells, and myeloid cells using anti-CD3, anti-CD4, anti-CD8, anti-CD20, and anti-CD11b antibodies. Flow cytometry analysis of the mouse peripheral blood confirmed the presence of human CD4+ T cells and CD8+ T cells in the mouse blood at rates comparable to those in the donor's peripheral blood before transplantation (Figure 6). These results demonstrate that PBMCs can be transplanted into NOG-ΔMHC mice.

[0028] Example 1 (Transplantation of kidney cancer tissue and PBMC into NOG-ΔMHC mice) Renal cancer tissue collected from one kidney cancer patient and PBMCs collected from that patient were subcutaneously transplanted into the NOG-ΔMHC mice, and a new patient-derived tumor tissue and immune cell transplant model (Patient-derived xenografts, PDX model) was constructed.

[0029] The PDX was treated with a commercially available human anti-Programmed Death-1 (PD-1) antibody (Bio X Cell), and tumors were excised and evaluated by immunohistochemistry (see Figure 7). Compared to control mice without the antibody, the proportion of human CD8+ T cells in the tumor was significantly increased (see bottom graph in Figure 7). Furthermore, among the human CD8+ cells, the proportion of cells positive for Ki67, a proliferation marker, was significantly higher (see bottom graph in Figure 7). These results indicate that a human immune response is generated within the PDX and that this immune response is enhanced by the anti-PD1 antibody. These results also suggest that various drugs, not just anti-PD1 antibodies, can induce any desired human immune response. Enlarged photographs of portions a and b of the photograph on the left side of Figure 7, which shows the immunohistochemical evaluation of the (PD-1) antibody-administered mice and the control mice, are shown on the right side of the photograph on the left side of Figure 7.

[0030] After administering the aforementioned human anti-PD-1 antibody to the PDX, the tumor was excised and dissociated into single cells. Human CD8+ T cells were isolated as single cells using a cell sorter. Sequencing of the TCRβ CDR3 region, which determines the antigen specificity of the T cell receptor β of these T cells, revealed that the most common TCRβ CDR3 sequences were different in each individual control mouse that did not receive anti-PD1 antibody (left side of Figure 8). In contrast, identical TCRβ CDR3 sequences were observed in all mice that received anti-PD1 antibody (right side of Figure 8). These results indicate that anti-PD1 antibody activated and proliferated cancer-reactive T cells in the PDX. Therefore, cancer-reactive T cells can be isolated using the above and similar methods. [Explanation of symbols]

[0031] 1 Cancer tissue, 2 Immune cells.

Claims

1. 1. A method for identifying antigen-reactive immune cells in a human, comprising: a xenotransplant animal production process in which tissues and immune cells collected from humans are transplanted into immunodeficient animals whose major histocompatibility complexes have been gene-edited to produce xenotransplant animals; a human immune response step of generating a human immune response in the first xenograft animal obtained in the xenograft animal production step; and A method for identifying human antigen-reactive immune cells, comprising an isolation step of isolating antigen-reactive immune cells from the second xenograft animal obtained in the human immune response step.

2. 2. The method for identifying antigen-reactive immune cells according to claim 1, wherein the tissue comprises cancer tissue.

3. 3. The method for identifying antigen-reactive immune cells according to claim 2, wherein the cancer tissue comprises at least one tissue selected from the group consisting of kidney cancer tissue, urothelial cancer tissue, prostate cancer tissue, and liposarcoma tissue.

4. 2. The method for identifying antigen-reactive immune cells according to claim 1, wherein the immunodeficient animal is an immunodeficient mouse.

5. The method for identifying antigen-reactive immune cells according to any one of claims 1 to 4, wherein the antigen-reactive immune cells include cancer-reactive T cells.

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