WO2024029561A1

WO2024029561A1 - Tumor-bearing immunodeficient nonhuman animal, and method for evaluating cancer immune response regarding test substance using same

Info

Publication number: WO2024029561A1
Application number: PCT/JP2023/028264
Authority: WO
Inventors: 美恵亀谷; 亮治伊藤
Original assignee: 学校法人東海大学; 公益財団法人実験動物中央研究所
Priority date: 2022-08-02
Filing date: 2023-08-02
Publication date: 2024-02-08

Abstract

Provided is a tumor-bearing immune nonhuman animal that enables suppression of the onset of graft-versus-host disease (GVHD) and engraftment of T cells and/or B cells.　Human peripheral blood monocytes are transplanted into a tumor-bearing immunodeficient nonhuman animal based on an immunodeficient nonhuman animal into which human IL-4 gene has been introduced.

Description

Cancer-bearing immunodeficient non-human animals, and cancer immune response evaluation methods for test substances using them

The present invention relates to a cancer-bearing, immunodeficient non-human animal obtained by transplanting cancer cells and/or cancer tissues into an immunodeficient non-human animal, a method for evaluating cancer immune response regarding a test substance using the same, and/or Or relates to a cancer immune response evaluation kit.

Immunodeficient non-human animals, typified by immunodeficient mice, are characterized by loss or decline in the function of one or more immune components such as B cells, T cells, and NK cells. For this reason, cells and tissues from foreign organisms such as humans can be transplanted into immunodeficient non-human animals, and immunodeficient non-human animals are used extensively in research fields such as immunology, infectious diseases, oncology, and stem cell biology. plays an important role in

For example, NOD-, which is capable of engrafting human cells, lacks both functional T cells and B cells, and exhibits a decrease in complement activity, macrophage function, and natural killer (NK) cell activity, etc. By backcrossing scid mice with mice in which the IL-2 receptor γ chain gene, which is a common domain of cytokine receptors, is knocked out (IL2RγKO mice), NK cells disappear, macrophage function declines, and dendritic cell function is reduced. NOG mice (also referred ^to as `` ^NOD ^. (For example, see Patent Document 1). NOG mice are considered to be immunodeficient mice with extremely high engraftment of human cells and tissues. In addition, by crossing NOD-scid mice with IL-2 receptor γ chain gene-deficient mice, NSG mice (also expressed as "NOD.Cg-Prkdc ^scid Il2rg ^tm1Wjl /SzJ"), which have similar characteristics to NOG mice ) is also known.

As a typical example of an immunodeficient non-human animal, severe immunodeficient mice such as those mentioned above have lost or decreased functions of T, B, NK cells, macrophages, dendritic cells, and complement, and therefore are not capable of human hematopoiesis. Highly efficient implantation of stem cells (HSC), human peripheral blood mononuclear cells (PBMC), human tumor cell line-derived xenografts (CDX), tumor patient-derived xenografts (PDX) or other adult stem cells and tissues. It has been widely applied to create humanized mice.

However,

Non-Patent Documents

1 and 2 point out that when HSCs and cancer cells are transplanted into NOG mice, it is impossible to engraft normally differentiated mature human T cells and B cells. This also applies to NSG mice and other severely immunodeficient mice.

Additionally, Non-Patent Document 3 discloses that graft-versus-host disease (GVHD) develops when PBMCs are transplanted into NOG mice. Similarly, it has been disclosed that NSG mice develop graft-versus-host disease (GVHD) when PBMCs are transplanted (Non-Patent Document 4). Furthermore, in Non-Patent Document 5, when human peripheral blood mononuclear cells and cancer cells were transplanted into NOG mice with double knockout of major histocompatibility complex class I/II, GVHD was avoided, but B. It is disclosed that B cell responses cannot be detected because cell engraftment is not possible.

By the way, improvements in which human genes are introduced into NOG mice or mouse genes are removed are aimed at improving the differentiation, engraftment, and functions of human cells that cannot be observed in humanized mice using NOG mice, or establishing human disease models. Various types of NOG mice (next generation humanized mice) have been developed. As an example, NOG mice expressing the human IL-4 gene under the control of the human cytomegalovirus promoter (CMV promoter) ("mouse NOG-hIL-4Tg" or "NOD.Cg-Prkdc ^scid Il2rg") ^tm1Sug Tg (CMV-IL4)/Jic") is known (Non-Patent Document 6). Mouse NOG-hIL-4Tg systemically expresses the human IL-4 gene, and when human PBMCs are transferred, a large amount of human T cells engraft and GVHD does not occur, making long-term experiments possible. have.

Regarding mouse NOG-hIL-4Tg, Non-Patent Document 7 analyzes the relationship between human lymphocyte subsets and glucocorticoid receptor (GR) expression levels and discloses humoral immunity when human PBMCs are transplanted. has been done. Furthermore, regarding mouse NOG-hIL-4Tg, see Non-Patent Document 8, PBMCs derived from breast cancer patients were transplanted, immunized with human epidermal growth factor receptor 2 (HER2) peptide, and the humoral immunity of breast cancer patients was analyzed. It is disclosed that what has happened.

Patent No. 3753321

By the way, when human PBMCs are further transplanted into a tumor-bearing non-human animal that has been transplanted with cancer cells and/or cancer tissue into an immunodeficient non-human animal, the development of graft-versus-host disease (GVHD) and T. Cells and/or B cells cannot engraft. For this reason, there has conventionally been a problem in that it has not been possible to test or evaluate immune responses to cancer using cancer-bearing non-human animals.

Therefore, in view of the above-mentioned circumstances, the present invention aims to provide a cancer-bearing, immune-immunized non-human animal that can suppress the onset of graft-versus-host disease (GVHD) and allow T cells and/or B cells to engraft. The purpose of the present invention is to provide a cancer immune response evaluation method and/or a cancer immune response evaluation kit for test substances.

In order to achieve the above-mentioned purpose, the present inventors conducted intensive studies and found that human The present invention was completed based on the discovery that when peripheral blood mononuclear cells (PBMCs) are transplanted, the onset of graft-versus-host disease (GVHD) can be suppressed and T cells and/or B cells can be engrafted. I ended up doing it.

The present invention includes the following.
(1) A mutation is introduced into the IL-2 receptor γ chain gene, resulting in a deficiency of the IL-2 receptor γ chain, and mutations in genes involved in the rearrangement of antigen receptor genes in T cells and B cells are biallelic. A tumor-bearing immune system in which cancer cells and/or cancer tissues and human peripheral blood mononuclear cells are transplanted into an immunodeficient non-human animal that is present in the human IL-4 gene and has been introduced with the human IL-4 gene. Insufficient non-human animals.
(2) The tumor-bearing, immunodeficient non-human animal according to (1), wherein the plasma concentration of human IL-4 encoded by the human IL-4 gene is 100 to 500 pg/ml.
(3) The tumor-bearing and immunodeficient non-human animal according to (1), wherein the human IL-4 gene is expressed under the control of a human cytomegalovirus promoter.
(4) The cancer-bearing, immunodeficient non-human animal according to (1), wherein the mutation in a gene involved in rearrangement of antigen receptor genes of T cells and B cells is a SCID mutation or a RAG mutation.
(5) The immunodeficient non-human animal described above lacks both functional T cells and B cells, has decreased macrophage function, has lost NK cells or NK activity, and has decreased dendritic cell function. The tumor-bearing immunodeficient non-human animal according to (1), which is characterized by having excellent adhesion to different types of cells.
(6) The immunodeficient non-human animal is produced by backcrossing the following non-human animal A with the non-human animal B; Immunocompromised non-human animals.
A: C. to NOD/Shi non-human animals. Non-human animal B obtained by backcrossing B-17-scid non-human animal: non-human animal in which the interleukin 2 receptor γ chain gene has been knocked out (7) The above immunodeficient non-human animal is NOG (NOD/ The tumor-bearing immunodeficient non-human animal according to (1), which is a Shi-scid, IL-2Rγ KO) mouse.
(8) The cancer-bearing, immunodeficient non-human animal according to (1), wherein the cancer cells and/or cancer tissues are human breast cancer cells and/or human breast cancer tissues.
(9) The cancer-bearing, immunodeficient non-human animal according to (1), wherein the number of transplanted human peripheral blood mononuclear cells is 1 to 5×10 ⁶ cells.
(10) The cancer-bearing, immunodeficient non-human animal according to (1), characterized in that human CD19-positive human B cells are engrafted therein.

(11) The step of administering a test substance to the cancer-bearing immunodeficient non-human animal described in any of (1) to (10) above, and comparing the lymphocytes before and after administration of the test substance. A method for evaluating a cancer immune response regarding a test substance, the method comprising:
(12) The above test substance is an immune checkpoint inhibitor or a candidate substance for an immune checkpoint inhibitor, and it is possible to compare T cells and/or B cells among lymphocytes before and after administration of the above test substance. The cancer immune response evaluation method according to feature (11).
(13) If the number of human CD8-positive killer T cells included in the lymphocytes increases compared to before administration of the test substance, the cancer transplanted into the cancer-bearing immunodeficient non-human animal, The cancer immune response evaluation method according to (11), characterized in that the test substance is determined to have an immune checkpoint inhibitory effect.

(14) A cancer immune response evaluation kit comprising the cancer-bearing immunodeficient non-human animal according to any one of (1) to (10) above and a cell surface marker analysis reagent for peripheral blood mononuclear cells.
(15) The cancer immune response evaluation kit according to (14), further comprising a test substance to be evaluated.
This specification includes the disclosure content of Japanese Patent Application No. 2022-123595, which is the basis of the priority of this application.

The cancer-bearing immunodeficient non-human animal according to the present invention is transplanted with cancer cells and/or cancer tissues and human peripheral blood mononuclear cells, and the onset of graft-versus-host disease (GVHD) is suppressed, and , T cells and/or B cells engraft. Therefore, the cancer-bearing immunodeficient non-human animal according to the present invention can be used in research on cancer immunotherapy and the like.

Further, according to the cancer immune response evaluation method and cancer immune response evaluation kit regarding the test substance according to the present invention, the onset of graft-versus-host disease (GVHD) is suppressed, and T cells and/or B cells Because it uses cancer-bearing, immunodeficient non-human animals with engrafted cancer, the immune response to cancer can be evaluated with high precision.

FIG. 2 is a characteristic diagram showing the relationship between plasma IL-4 concentration and the number of engrafted B cells in NOG mice into which the human IL-4 gene has been introduced. Characteristic diagram showing the results of lymphocyte measurement after transplantation of human PBMC for NOG mice introduced with the human IL-4 gene expressed by the CMV promoter and NOG mice introduced with the human IL-4 gene expressed by the CAG promoter. It is. FIG. 3 is a characteristic diagram showing the results of measuring the tumor diameters of the PBS administration group and the atezolizumab administration group after breast cancer cell lines were transplanted into NOG mice into which the human IL-4 gene expressed by the CMV promoter was introduced. This is a characteristic diagram showing the results of measuring lymphocytes in the PBS administration group and atezolizumab administration group for NOG mice into which the human IL-4 gene expressed by the CMV promoter was introduced and mice to which breast cancer cell lines were transplanted. be. FIG. 2 is a photograph showing the results of histochemical staining of tumor tissue in a PBS administration group and an atezolizumab administration group in NOG mice into which a human IL-4 gene expressed by a CMV promoter has been introduced and which has been transplanted with a breast cancer cell line.

Hereinafter, the cancer-bearing immunodeficient non-human animal, the cancer immune response evaluation method using the same, and the cancer immune response evaluation kit according to the present invention will be explained in detail.

[Cancer-bearing – immunodeficient non-human animal]
Cancer-bearing immunodeficient non-human animals have a mutation introduced into the IL-2 receptor γ chain gene and are deficient in the IL-2 receptor γ chain, resulting in rearrangement of the antigen receptor genes of T cells and B cells. It is produced using an immunodeficient non-human animal in which the relevant gene mutations are present at both allelic loci and the human IL-4 gene has been introduced. The cancer-bearing immunodeficient non-human animal according to the present invention can be produced by transplanting cancer cells and/or cancer tissues and human peripheral blood mononuclear cells into this immunodeficient non-human animal. can.

Here, the non-human animal is not particularly limited as long as it is an animal other than human, but preferably means a mammal other than human. Examples of non-human animals include non-human primates (monkeys, chimpanzees, gorillas, etc.), rodents (mice, rats, guinea pigs, etc.), dogs, cats, rabbits, cows, pigs, horses, goats, sheep, etc. These include, but are not limited to: Among these, as the non-human animal, rodents are preferred, and mice are more preferred.

That is, the cancer-bearing immunodeficient non-human animal according to the present invention can particularly be a cancer-bearing immunodeficient non-human mammal, and is preferably a tumor-bearing immunodeficient rodent animal. , more preferably tumor-bearing immunodeficient mice.

In the following description including Examples, a tumor-bearing immunodeficient mouse will be described in detail as an example of a tumor-bearing immunodeficient non-human animal, but the technical scope of the present invention is not limited to a tumor-bearing immunodeficient mouse. It is not intended to be limited to. The present invention is not limited to tumor-bearing immunodeficient mice, but can be applied to cancer-bearing, immunodeficient non-human animals as described above.

In the present invention, tumor-bearing immunodeficient mice have a mutation introduced into the IL-2 receptor γ chain gene, resulting in deletion of the IL-2 receptor γ chain, and the human IL-4 gene is introduced. Produced using immunodeficient mice. This immunodeficient mouse has a wild-type gene base sequence that encodes a protein that controls specific immune functions, including substitution of one or more bases with other bases, deletion of one or more bases, and deletion of one or more bases. or a mouse in which the immune function exerted by the wild-type gene does not function due to genetic modification that introduces genetic mutations such as insertion of two or more bases, out-of-frame reading frame shift of amino acids, and/or combinations of these. . Note that the above-mentioned wild type can include mice that have the most common allele or polymorphism found in a population and have not been genetically modified by mutation or artificial manipulation.

More specifically, it is known in humans and mice that the IL-2 receptor is composed of three types of proteins called alpha (α) chain, beta (β) chain, and gamma (γ) chain. The above IL-2 receptor γ chain does not have the ability to bind to IL-2 when the γ chain alone has the ability to bind to IL-2, but the heterodimer of the β chain and the γ chain serves as a medium affinity receptor for IL-2. , a heterotrimer of α, β, and γ chains becomes a high-affinity receptor for IL-2.

The above-mentioned IL-2 is a type of cytokine, and the action of IL-2 is to transmit IL-2 signals from the cytoplasm into the nucleus by binding to the IL-2 receptor present on the cell surface. Examples include inducing proliferation of T cells and B cells and activation of NK cells. Therefore, when the IL-2 receptor γ chain is not functionally expressed, the IL-2 receptor loses its ability to bind to IL-2 due to a mutation introduced into the IL-2 receptor γ chain gene. cases in which signal transduction occurs, or cases in which signal transduction does not occur.

Additionally, cancer-immunodeficient mice are characterized by the introduction of the human IL-4 gene. The human IL-4 gene is a gene encoding human IL4 protein. The term "gene" refers to a polynucleotide that includes at least one open reading frame that encodes a specific protein, and may have a structure consisting only of exons or a structure containing both exons and introns.

Here, the introduction of the human IL-4 gene means that the human IL-4 protein is expressed in the body of the cancer-immunodeficient mouse. In particular, the expression of human IL-4 protein means that in the body of cancer-immunodeficient mice, B cells derived from transplanted human peripheral blood mononuclear cells engraft, and helper T cells and killer T cells are balanced. This means that it is expressed in a way that allows for good engraftment. The engraftment of B cells, helper T cells, and killer T cells is determined by, for example, the profile of lymphocytes engrafted in the spleen of cancer-immunodeficient mice, that is, CD19-positive cells (B cells) among CD45-positive cells, Evaluation can be made by detecting CD4-positive cells (helper T cells) and CD8-positive cells (killer T cells).

In addition, in order for the transplanted B cells derived from human peripheral blood mononuclear cells to engraft in the body of cancer-immunodeficient mice, and for the balance of helper T cells and killer T cells to engraft, it is necessary to It is preferable that the plasma concentration of human IL-4 protein is within a predetermined range. Specifically, when the plasma concentration of introduced human IL-4 protein is 100 to 500 pg/ml, B cells derived from transplanted human peripheral blood mononuclear cells will engraft, and helper T cells and Killer T cells can engraft in a well-balanced manner.

In general, the concentration of protein in plasma (in this case, human IL-4 protein) may vary depending on the measurement method and the calibration curve used (particularly, in this case, the type of anti-human IL-4 antibody used). . The plasma concentration of the introduced human IL-4 protein as defined herein is determined by using BD OptEIA Set Human IL-4 (manufactured by BD Biosciences, catalog number: 555194, rod number: 9189127). -4 using a calibration curve created from standard samples with concentrations of 500 pg/ml, 250 pg/ml, 125 pg/ml, 62.5 pg/ml, 31.3 pg/ml, 15.6 pg/ml, and 7.8 pg/ml. Calculate. Further, IL-4 contained in the standard sample or sample is measured at absorbance at 450 nm using Human IL-4 ELISA Set BD OptEIATM (manufactured by BD Biosciences).

The plasma concentration of human IL-4 protein can be regulated by the transcription control region introduced together with the human IL-4 gene. Examples of transcription control regions include promoters. That is, by changing the promoter operably linked to the human IL-4 gene, the plasma concentration of human IL-4 protein can be regulated. For example, but not limited to, by using the human cytomegalovirus promoter (CMV promoter), transplanted human peripheral blood mononuclear B cells derived from the bulb will engraft, and helper T cells and killer T cells will also engraft in a well-balanced manner. For example, by introducing the human IL-4 gene downstream of the CMV promoter into cancer-immunodeficient mice, the plasma concentration of human IL-4 protein in cancer-immunodeficient mice can be reduced to 100-500 pg/ ml.

Note that usable promoters are not limited to the above CMV, but include, for example, SV40 promoter, PGK promoter, SRα promoter, retrovirus LTR promoter, RSV (Rous sarcoma virus) promoter, and HSV-TK (herpes simplex virus thymidine kinase). Promoters include EF1α promoter, metallothionein promoter, heat shock promoter, and the like. For example, as described in PLOS ONE 2010, e10611, it is preferable to use a promoter known to moderately regulate the expression of a downstream gene in mammals, such as the CMV promoter. However, among known promoters, it is not preferable to use promoters that highly enhance expression, such as the CAG promoter (consisting of the chicken β-actin promoter hybridized with the CMV enhancer sequence). When these promoters that highly enhance expression are used, there is a possibility that the plasma concentration of human IL-4 protein exceeds 500 pg/ml.

As used herein, sequence identity (or homology) between amino acid sequences or base sequences refers to the insertion and deletion of two amino acid sequences or base sequences in such a way that the corresponding amino acids or bases match the most. It is determined as the ratio of matched amino acids or bases to the entire amino acid sequence or base sequence excluding the gaps in the resulting alignment. Sequence identity between amino acid sequences or base sequences can be determined using various homology search software known in the technical field. For example, the sequence identity value of an amino acid sequence can be obtained by calculation based on an alignment obtained using the known homology search software BLASTP, and the sequence identity value of a base sequence can be obtained by calculation based on the alignment obtained using the known homology search software BLASTP. It can be obtained by calculation based on the alignment obtained by the software BLASTN.

As used herein, "stringent conditions" refer to, for example, Molecular Cloning-A LABORATORY MANUAL THIRD EDITION (Sambrook et al., Cold Spring Harbor Laboratory Examples include the method described in Press). Stringent conditions include, for example, 6x SSC (composition of 20x SSC: 3M sodium chloride, 0.3M citric acid solution, pH 7.0), 5x Denhardt's solution (composition of 100x Denhardt's solution: 2% by mass). Bovine serum albumin, 2 wt% Ficoll, 2 wt% polyvinylpyrrolidone), 0.5 wt% SDS, 0.1 mg/mL salmon sperm DNA, and 50% formamide at 42-70°C. Conditions for hybridization include incubation for several hours to overnight. The washing buffer used for washing after incubation is preferably a 1×SSC solution containing 0.1% by mass of SDS, more preferably a 0.1×SSC solution containing 0.1% by mass of SDS.

As used herein, the term "operably linked" with respect to polynucleotides means that a first base sequence is located sufficiently close to a second base sequence, and the first base sequence is located close enough to the second base sequence or This means that it can affect the region under the control of the second base sequence. For example, a polynucleotide being "operably linked to a promoter" means that the polynucleotide is linked such that it is expressed under the control of the promoter.

As used herein, "linked in a functional manner" means that the gene (polynucleotide) inserted downstream of the promoter can be expressed in the cells of the target non-human animal. As used herein, "an expressible state" means that the polynucleotide is in a state in which it can be transcribed within a cell into which the polynucleotide has been introduced.

As used herein, the term "expression vector" refers to a vector that contains a polynucleotide of interest and is equipped with a system that enables expression of the polynucleotide of interest in cells into which the vector has been introduced.

Human IL-4 will be explained. Human IL4 is human interleukin 4. Among interleukins, IL-4 is classified into the hematopoietin family, which is involved in hematopoiesis, etc., and is a soluble protein composed of 129 amino acids. IL-4 is an anti-inflammatory glycoprotein produced in a variety of cells such as Th2 cells, mast cells, and basophils. IL-4 is known to promote class switching to IgE via the type I IL-4 receptor on hematopoietic cells, and is also involved in the differentiation of naive helper T cells into Th2 cells.

The gene sequence and amino acid sequence of human IL-4 are known, and their sequence information can be obtained from known databases such as GenBank. For example, the gene sequence and amino acid sequence of human IL-4 are listed in GenBank as Accession No. Examples include sequences registered as NM_000589.4, NM_001354990.2, and NM_172348.3. Note that these NM_000589.4, NM_001354990.2, and NM_172348.3 are sequences registered as transcriptional variants in human IL-4. Furthermore, human IL-4 is not limited to those having these sequences, but includes homologs (orthologs, paralogs) and variants thereof.

That is, the human IL-4 gene is listed in GenBank with accession No. Consists of an amino acid sequence that has 80% or more sequence identity to the amino acid sequences registered in NM_000589.4, NM_001354990.2, and NM_172348.3, and encodes a polypeptide that has human IL-4 activity. It's okay to have one. Note that the sequence identity is not particularly limited as long as it is 80% or more and the resulting polypeptide has human IL-4 activity. The sequence identity is particularly preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and particularly preferably 97% or more.

In addition, the human IL-4 gene is listed in GenBank with accession No. Consists of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequences registered in NM_000589.4, NM_001354990.2, and NM_172348.3, and has human IL-4 activity. It may also encode a polynucleotide encoding a polypeptide having the following. Note that the number of amino acids to be deleted, substituted, added, or inserted is not particularly limited as long as the resulting polypeptide has human IL-4 activity. The number of amino acids to be deleted, substituted, added, or inserted can be, for example, 1 to 15, preferably 1 to 13, more preferably 1 to 10, 1 to 8, and 1 to 7. Examples include 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2.

An immunodeficient mouse in which a mutation has been introduced into the IL-2 receptor γ chain gene defined as above, resulting in a deficiency in the IL-2 receptor γ chain and in which the human IL-4 gene has been introduced, is, for example, By introducing the human IL-4 gene into immunodeficient mice such as NOG mice (NOD.Cg-Prkdc ^scid Il2rg ^tm1Sug /ShiJic), NSG mice (NOD.Cg-Prkdc ^scid Il2rg ^tm1Wjl /SzJ), and NOD/ShiJcl mice. It can be made. Note that the immunodeficient mouse into which the human IL-4 gene is introduced is not limited to these, and any of these commercially available immunodeficient mice can be used without any particular restriction.

As an example, the NOG mouse (NOD.Cg-Prkdc ^scid Il2rg ^tm1Sug /ShiJic) is a mouse named NOD (Non Obese Diabetes) because its pathology is similar to human type 1 diabetes (insulin-dependent diabetes). For example, see Jikken Dobutsu. 29:1-13, 1980) and SCID mice, which lack T cell and B cell function and exhibit severe immunodeficiency due to mutations in the DNA-dependent protein kinase (Prkdc) gene. NOD/scid mice were combined with NOD/scid mice, and mice in which the IL-2 receptor γ chain was knocked out (IL-2RγKO), which is the causative gene for the immunodeficiency disease mouse) (Ohbo K et al., Blood 1996) using the Cross Intercross method (Inbred Strains in Biomedical Research, M.FW. Festing, 1979, The Macmillan Press, London and Basingstoke). Specifically, it can be produced by referring to Japanese Patent No. 3753321, and is also available from the Central Research Institute for Experimental Animals, a public interest incorporated foundation.

Following the example of NOG mice, an immunodeficient non-human animal that can be used in the present invention can be produced as follows. That is, an immunodeficient non-human animal can be produced by backcrossing the following non-human animal A with the non-human animal B.
A: C. to NOD/Shi non-human animals. Non-human animal B obtained by backcrossing B-17-scid non-human animal: non-human animal in which the interleukin 2 receptor γ chain gene has been knocked out

Here, NOD/Shi in the non-human animal of A is applied to the non-human animal. B-17-scid non-human animals can be backcrossed using methods known to those skilled in the art, such as the Cross Intercross method (Inbred Strains in Biomedical Research, M.FW. Festing, 1979). ISBN 0-333- 23809-5, The Macmillan Press, London and Basingstoke), C. B-17-scid non-human animals are crossed with NOD/Shi non-human animals, and the F1 non-human animals are further crossed with each other, and the amount of immunoglobulin in the serum of the F2 non-human animals obtained is measured and cannot be detected. Select non-human animals. This non-human animal is bred again with a NOD/Shi non-human animal. This operation can be carried out by repeating this operation nine times or more (cross/intercross method).

In addition, knockout of the interleukin 2 receptor γ chain (IL-2Rγ) gene in a non-human animal in B can be performed using methods known to those skilled in the art, such as homologous recombination using non-human animal ES cells (Capecchi, M.R., Altering the genome by homologous recombination, Science, (1989) 244, 1288-1292), a specific gene derived from a non-human animal and, for example, neomycin. homologous genes including drug resistance genes such as After the stage replacement, the ES cells can be injected into fertilized eggs.

Methods of introducing the human IL-4 gene into an immunodeficient non-human animal include, for example, a method of introducing the human IL-4 gene into an immunodeficient non-human animal, a method of introducing the human IL-4 gene into an immunodeficient non-human animal, and a method of introducing the human IL-4 gene into an immunodeficient non-human animal. Examples include a method of transferring the immunoid to an immunodeficient non-human animal.

Whether or not an immunodeficient non-human animal has the human IL-4 gene in its body can be determined by collecting plasma from the immunodeficient non-human animal and measuring the human IL-4 concentration in the plasma. It can be confirmed. The method for measuring the human IL-4 concentration in plasma is not particularly limited, but includes an immunochemical method using an anti-human IL-4 antibody. Examples of such methods include ELISA, EIA, RIA, and Western blotting. For measuring human IL-4 in plasma, a commercially available ELISA kit for measuring human IL-4 or the like may be used.

Specifically, when introducing the human IL-4 gene into an immunodeficient non-human animal, human IL-4 is preferably operably linked downstream of the above-mentioned promoter. The human IL-4 gene is introduced into a non-human animal in an expressible state, for example, in the form of an expression vector. In addition to the human IL-4 gene and the above-mentioned promoter, the expression vector preferably has control sequences such as an enhancer, a polyA addition signal, and a terminator, and a marker gene such as a drug resistance gene.

The type of vector is not particularly limited, and commonly used expression vectors can be used without particular restrictions. The vector may be linear or circular, and may be a non-viral vector such as a plasmid, a viral vector (for example, a retroviral vector such as a lentivirus vector), or a transposon-based vector.

The method for introducing the human IL-4 gene into a non-human animal is not particularly limited, and methods commonly used for producing transgenic animals can be applied. As a method for introducing the human IL-4 gene into a non-human animal, for example, a method of introducing an expression vector containing the human IL-4 gene and the above-mentioned promoter into a fertilized egg of a non-human animal to be introduced by microinjection or the like. etc. When the non-human animal is a mouse, an example of the fertilized egg is a fertilized egg obtained by mating a NOG mouse (NOD.Cg-Prkdc ^scid Il2rg ^tm1Sug /ShiJic) with a NOD/ShiJcl mouse. However, it is not limited to this.

The fertilized eggs into which the human IL-4 gene has been introduced are cultured at 37°C for approximately 18 to 24 hours, and then transplanted and implanted into the uterus of a foster parent to give birth to an immunodeficient egg that has the human IL-4 gene. Non-human animals can be obtained.

Whether or not the immunodeficient non-human animal obtained as described above has the human IL-4 gene can be determined by extracting genomic DNA from a sample collected from the non-human animal and performing PCR, etc. , can be confirmed.

In addition, whether or not the immunodeficient non-human animal expresses the human IL-4 gene can be determined by extracting RNA from a sample collected from the non-human animal and performing RT-PCR, etc. This can be confirmed by performing in situ hybridization using a tissue sample collected from a non-human animal. Alternatively, by detecting human IL-4 in a sample collected from the non-human animal using an anti-human IL-4 antibody (for example, ELISA method, EIA method, RIA method, Western blotting method, EIA method, (RIA method, immunohistological staining, etc.).

Immunodeficient non-human animals into which the human IL-4 gene has been introduced secrete human IL-4, resulting in the engraftment of transplanted human peripheral blood mononuclear cell-derived B cells and helper T cells. and killer T cells can engraft in a well-balanced manner. Here, when an immunodeficient non-human animal secretes human IL-4, it mainly means that human IL-4 is released into body fluids (blood, tissue fluid, lymph, etc.) from the cells of the immunodeficient non-human animal. refers to Whether or not an immunodeficient non-human animal secretes human IL-4 should be confirmed by measuring the human IL-4 concentration in plasma collected from the immunodeficient non-human animal. I can do it. The method for measuring human IL-4 concentration in plasma is as described above.

When cells that have the human IL-4 gene and secrete human IL-4 (cells in which the human IL-4 gene is expressed) are transferred to an immunodeficient non-human animal, the cells in which the human IL-4 gene is expressed are Examples include cells derived from human organs and blood, cancer cells, and the like. Examples of cells derived from the human organ include cells derived from the spleen, thymus, liver, small intestine, large intestine, prostate, lung, heart, brain, kidney, testis, uterus, and the like. Examples of the cells derived from human blood include blood cells contained in the peripheral blood mononuclear cell fraction. For example, cells expressing the human IL-4 gene can be selected from cell lines established from these human cells and transferred to an immunodeficient non-human animal. Alternatively, cells of an immunized non-human animal into which the human IL-4 gene has been introduced may be used as cells in which the human IL-4 gene is expressed. The cells of the immunodeficient non-human animal are preferably those of an immunodeficient non-human animal belonging to the same species as the immunodeficient non-human animal to which the cells are transferred. For example, if the immunodeficient non-human animal to which the cells are transferred is a mouse, the cells into which the human IL-4 gene is introduced are preferably mouse cells. The cells of an immunodeficient non-human animal into which the human IL-4 gene is introduced are not particularly limited, and include, for example, cells derived from organs or blood, or cell lines thereof, hematopoietic stem cells, cancer cells, and the like. Examples of cells derived from the organs and blood of immunodeficient non-human animals include cells of non-human animals derived from the same organs and blood fractions as exemplified in the cells expressing the human IL-4 gene. It will be done. Similarly to the above, the human IL-4 gene is introduced into non-human animal cells in an expressible state, for example, in the form of an expression vector. The method for introducing the human IL-4 gene into non-human animal cells is not particularly limited, and methods commonly used for gene introduction can be applied. Examples of such methods include virus infection method, lipofection method, microinjection method, calcium phosphate method, DEAE-dextran method, electroporation method, method using transposon, particle gun method, etc. but not limited to.

Whether cells expressing the human IL-4 gene secrete human IL-4 can be confirmed by measuring the IL-4 concentration in the culture medium of the cells. The method for measuring human IL-4 concentration is as described above.

The method for transferring cells expressing the human IL-4 gene into an immunodeficient non-human animal is not particularly limited, and methods commonly used for transferring cells into immunodeficient non-human animals can be applied. As a method for transferring cells expressing the human IL-4 gene into an immunodeficient non-human animal, for example, depending on the type of cells used, the human IL-4 gene may be introduced into the spleen, liver, subcutaneously, intravenously, etc. Examples include a method of administering cells expressing the expression.

The immunodeficient non-human animal into which cells expressing the human IL-4 gene have been transferred secretes human IL-4, and as a result, the transplanted B cells derived from human peripheral blood mononuclear cells engraft. , helper T cells and killer T cells can be engrafted in a well-balanced manner. Here, when an immunodeficient non-human animal secretes human IL-4, it mainly means that human IL-4 is released into body fluids (blood, tissue fluid, lymph, etc.) from the cells of the immunodeficient non-human animal. refers to Whether or not an immunodeficient non-human animal secretes human IL-4 should be confirmed by measuring the human IL-4 concentration in plasma collected from the immunodeficient non-human animal. I can do it. The method for measuring human IL-4 concentration in plasma is as described above.

Furthermore, in the immunodeficient non-human animal that can be used in the present invention, mutations in genes involved in rearrangement of antigen receptor genes of T cells and B cells are present at biallelic loci, such as SCID mutation or RAG mutations can be mentioned. The above SCID mutation is a DNA-dependent protein kinase (protein kinase, DNA activated, catalytic) found in mice exhibiting severe combined immunodeficiency (SCID). This is a mutation in the polypeptide (Prkdc) gene.

Examples of the above RAG mutations include mutations in the Rag (Recombination activating gene)-1 gene or the Rag-2 gene. These two genes are genes expressed in immature lymphocytes, have essential effects on the reconstitution of immunoglobulin genes and T cell receptors, and are essential for the maturation of T cells and B cells. .

Immunodeficient non-human animals that have mutations in genes involved in rearrangement of T cell and B cell antigen receptor genes, including the above SCID mutation and/or RAG mutation, at both allelic loci, have T cells due to abnormal DNA repair. This is a mutation in which T cells and B cells do not reach the maturation stage because gene rearrangement of cells and B cells is not possible, resulting in a loss of T cell and B cell function. All such mutations in immunodeficient non-human animals are at biallelic loci.

The cancer-bearing immunodeficient non-human animal according to the present invention is obtained by transplanting cancer cells or cancer tissues and human peripheral blood mononuclear cells into the above-described immunodeficient non-human animal. When using an immunodeficient mouse as an immunodeficient non-human animal to which cancer cells or cancer tissues and human peripheral blood mononuclear cells are to be transplanted, it is preferable to use an adult (e.g., 7 weeks of age or older) immunodeficient mouse. .

Here, the cancer cells and cancer tissues are preferably derived from mammals. Mammals include, for example, rodents such as mice, rats, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, horses, and sheep; feline animals such as dogs and cats; and humans. Examples include primates such as monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees. The mammal is preferably a rodent (such as a mouse) or a primate, more preferably a human.

Examples of cancers include, but are not limited to, stomach cancer, esophageal cancer, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, Epithelial cell carcinoma, basal cell carcinoma, adenocarcinoma, bone marrow cancer, renal cell carcinoma, ureteral cancer, liver cancer, bile duct cancer, cervical cancer, endometrial cancer, testicular cancer , small cell lung cancer, non-small cell lung cancer, bladder cancer, epithelial cancer, craniopharyngeal cancer, laryngeal cancer, tongue cancer, fibrosarcoma, mucosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma , angiosarcoma, lymphangiosarcoma, intralymphatic sarcoma, synovioma, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, seminoma, Wilms tumor, glioma, astrocytoma, bone marrow Examples include tissues such as blastoma, meningioma, melanoma, neuroblastoma, medulloblastoma, retinoblastoma, malignant lymphoma, and blood derived from cancer patients. The cancer cells may be cancer cells (primary culture) isolated using enzymes or the like from cancer tissues collected from cancer patients, or may be established cancer cell lines. Examples of cancer cell lines include, but are not limited to, human breast cancer cell lines such as HBC-4, BSY-1, BSY-2, MCF-7, MCF-7/ADR RES, HS578T, and MDA. -MB-231, MDA-MB-435, MDA-N, BT-549, T47D, HeLa as a human cervical cancer cell line, A549, EKVX, HOP-62, HOP-92, NCI- as a human lung cancer cell line H23, NCI-H226, NCI-H322M, NCI-H460, NCI-H522, DMS273, DMS114, human colon cancer cell lines Caco-2, COLO-205, HCC-2998, HCT-15, HCT-116, HT -29, KM-12, SW-620, WiDr, human prostate cancer cell lines DU-145, PC-3, LNCaP, human central nervous system cancer cell lines U251, SF-295, SF-539, SF -268, SNB-75, SNB-78, SNB-19, human ovarian cancer cell lines OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, SK-OV-3, IGROV-1, human kidney Cancer cell lines include RXF-631L, ACHN, UO-31, SN-12C, A498, CAKI-1, RXF-393L, 786-0, TK-10; human gastric cancer cell lines include AGS, MKN45, MKN28, St- 4. MKN-1, MKN-7, MKN-74, skin cancer cell lines LOX-IMVI, LOX, MALME-3M, SK-MEL-2, SK-MEL-5, SK-MEL-28, UACC- 62, UACC-257, M14, CCRF-CRM as leukemia cell line, K562, MOLT-4, HL-60TB, RPMI8226, SR, UT7/TPO, Jurkat, A431 as human epithelial cancer cell line, human melanoma cell line Examples include A375 as a human osteosarcoma cell line, HOS, MNMG, MNNG/HOS as a human osteosarcoma cell line, BxPC3, COLO-357HPAC, MIAPaCa-2, Panc-1 as a human pancreatic cancer cell line, and LS123 as a human colon cancer cell line. . Examples of cell lines include, but are not limited to, HEK293 (human embryonic kidney cells), MDCK, MDBK, BHK, C-33A, AE-1, 3D9, Ns0/1, NIH3T3, PC12, S2 , Sf9, Sf21, High Five (registered trademark), Vero, etc.

The cancer cells are preferably cancer cells that have the ability to form solid tumors when engrafted in vivo. Cancer cells that have the ability to form solid tumors include gastric cancer, esophageal cancer, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, and squamous cell cancer. Basal cell carcinoma, adenocarcinoma, renal cell carcinoma, ureteral cancer, liver cancer, bile duct cancer, cervical cancer, endometrial cancer, testicular cancer, small cell lung cancer, non-small Cellular lung cancer, bladder cancer, epithelial cancer, craniopharyngeal cancer, laryngeal cancer, tongue cancer, fibrosarcoma, mucosal sarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, lymphangiosarcoma , intralymphatic sarcoma, synoviomas, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, seminoma, Wilms tumor, glioma, astrocytoma, meningioma, melanoma, neuro Examples include, but are not limited to, cells such as blastoma, medulloblastoma, and retinoblastoma.

The cancer cells may be syngenic, allogenic, or xenogenic to the immunodeficient non-human animal to be transferred.

Transfer of cancer cells into an immunodeficient non-human animal usually involves suspending the cancer cells in a physiological aqueous composition and transferring the resulting suspension into the body of the non-human mammal. can. Cancer cells can be treated with proteins such as trypsin or chelating agents such as EDTA and dispersed into single cells, then suspended in a physiological aqueous composition and introduced into the body, or they can be introduced into the body in the form of spheroids. It may also be suspended in an aqueous composition and introduced into the body. Cancer tissue formation tends to be more accelerated when cells are transferred in the form of spheroids, while cancer cells tend to be more sensitive to anticancer drugs when transferred in the form of single cells. Physiological aqueous compositions include physiological aqueous solutions and physiological aqueous gels. Examples of the physiological aqueous solution include physiological buffers such as phosphate buffer, carbonate buffer, citrate buffer, Tris buffer, and borate buffer, and medium for cell culture. As an aqueous gel, extracellular matrix constituent factors (proteoglycans such as aggrecan; glycosaminoglycans such as hyaluronic acid; protein fibers such as collagen and elastin; or mixtures thereof (e.g., basement membrane preparations), etc.), polysaccharides (eg, agarose). A basement membrane preparation has the ability to control cell morphology, differentiation, proliferation, movement, functional expression, etc. when desired cells of the same or different type that have the ability to form a basement membrane are seeded and cultured thereon. , refers to isolated basement membrane. To prepare a basement membrane, for example, cells that have the ability to form a basement membrane that have adhered to the support via the basement membrane are removed from the support using a solution that has the ability to dissolve lipids in the cells, an alkaline solution, etc. It can be made by Basement membrane preparations may include Matrigel. By using an aqueous gel, it is possible to suppress the dispersion of transplanted cancer cells in vivo.

On the other hand, human peripheral blood mononuclear cells (human PBMC) can be obtained according to a standard method or using a commercially available kit. Furthermore, the cancer-bearing, immunodeficient non-human animal according to the present invention may be one in which human peripheral blood mononuclear cells derived from a healthy individual are transplanted, or human peripheral blood mononuclear cells derived from a cancer patient are transplanted into the cancer-bearing immunodeficient non-human animal. It may be a transplanted one. For example, when cancer cells or cancer tissues from a specific cancer patient are transplanted into the cancer-bearing, immunodeficient non-human animal according to the present invention, the human peripheral blood of the cancer patient may be transplanted.

The method for transplanting human peripheral blood mononuclear cells is not particularly limited, but may include a method in which the cells are adjusted to a predetermined cell number and then transplanted from the tail vein. The number of human peripheral blood mononuclear cells to be transplanted is not particularly limited, but can be 1 to 5 x 10 ⁶ or more. Most preferably, the number is 5×10 ⁶ . If the number of human peripheral blood mononuclear cells is less than this range, there is a risk that the engraftment ability of B cells will be low and that it will be difficult to engraft sufficient lymphocytes to evaluate the antitumor effect. Furthermore, if the number of human peripheral blood mononuclear cells exceeds this range, there is a possibility that the human clinical specimen will be wasted.

[Cancer-bearing cancer immune response evaluation method and kit using immunodeficient non-human animals]
The cancer-bearing immunodeficient non-human animal described above lacks both endogenous functional T cells and B cells, diminishes macrophage function, loses NK cells or NK activity, and has dendritic Cell function is reduced, and it has excellent heterogeneous cell adhesion. In addition, cancer-bearing, immunodeficient non-human animals may develop graft-versus-host disease (GVHD) despite transplantation of cancer cells, cancer tissues, and human peripheral blood mononuclear cells. Instead, cancer cells, cancer tissues, B cells, and T cells engraft. Furthermore, cancer-bearing immunodeficient non-human animals exhibit characteristics such as similar proportions of B cells and T cells as in cancer patients from which the engrafted cancer cells and cancer tissues are derived.

In this way, cancer-bearing and immunodeficient non-human animals can simulate the immune response to cancer in cancer patients, so they are useful for tests related to immune responses to cancer and cancer immunotherapy. It can be used for. In other words, adoptive immune cell therapy is cancer immunotherapy that enhances anti-tumor immunity by administering immune cells; it includes peptides that serve as cancer antigens, nucleic acids that encode the peptides, or dendritic cells that carry cancer antigens. Cancer vaccine therapy using cancer vaccines; mediated by antibody-dependent cellular cytotoxicity (ADCC) activity, in which the Fc portion of antibodies binds to Fc receptors expressed on immune cells such as monocytes and NK cells, and complement fixation. Antibody therapy whose mechanism of action is complement-dependent cytotoxicity (CDC) activity; therapy based on the immune response to cancer, such as therapy using inhibitors of immune checkpoints (immune checkpoint inhibitors) that suppress the immune system Cancer-bearing and immunodeficient non-human animals can be effectively used for various therapies.

As an example, a cancer-bearing immunodeficient non-human animal can be applied to a method of evaluating cancer immune response to a test substance. The test substance may be any known compound or a new compound. Examples of test substances include nucleic acids, carbohydrates, lipids, proteins, peptides, organic low-molecular compounds, compound libraries prepared using combinatorial chemistry techniques, random peptide libraries, random nucleic acid libraries, or microorganisms, animals and plants. Alternatively, natural ingredients derived from marine organisms and the like can be mentioned.

In this method, first, a test substance is administered to the above-mentioned cancer-bearing and immunodeficient non-human animal. Administration methods include, but are not particularly limited to, oral, subcutaneous, intraperitoneal, intravenous, intraportal vein, and the like. The test substance is administered for a period sufficient to evaluate its effects, and is usually 3 days or more, preferably 7 days or more, more preferably 14 days or more, and still more preferably 21 days or more.

Thereafter, lymphocytes in the cancer-bearing immunodeficient non-human animal are measured, and the lymphocytes before and after administration of the test substance are compared. Measuring lymphocytes means, for example, measuring B cells, helper T cells, killer T cells, NK cells, and monocytes contained in engrafted human peripheral blood mononuclear cells using a cell expression antigen test using flow cytometry. It means to measure. When measuring lymphocytes, a spleen or the like collected from a cancer-bearing, immunodeficient non-human animal can be used.

More specifically, the number of human CD45-positive white blood cells, human CD19-positive B cells, and human CD3-positive T cells in engrafted human peripheral blood mononuclear cells can be measured. Furthermore, human CD4-positive helper T cells and human CD8-positive killer T cells can also be measured. Furthermore, human CD56-positive NK cells may be measured. Furthermore, human CD2, human CD5, human CD7, and human TCR (T-Cell Receptor) may be measured as cell surface antigens for the T cell system. Furthermore, human CD10, human CD20, human CD22, human CD79a, and human κ/λ may be measured as cell surface antigens for B cell lines. Cell surface antigens are not limited to these representative examples, and human CD16, human CD57, human CD13, human CD33, human CD34, and human MPO (myeloperoxidase) can also be measured.

Based on the results of measuring lymphocytes in cancer-bearing immunodeficient non-human animals as described above, cancer immune responses can be evaluated for test substances. Evaluating cancer immune response means selecting a test substance as an effective candidate substance for adoptive immune cell therapy; selecting it as an effective candidate substance for cancer vaccine therapy; selecting it as an effective candidate substance for antibody therapy. ; This meaning includes selection as an effective candidate substance for immune checkpoint inhibitors.

More specifically, by comparing T cells and/or B cells among lymphocytes in cancer-bearing and immunodeficient non-human animals before and after administration of the above test substance, immune checkpoint inhibitors or immune checkpoint inhibition can be determined. Candidate substances for agents can be selected. Changes in the proportion of B cells and T cells when a known immune checkpoint inhibitor is applied to a specific cancer patient, and the prescribed test substance is administered to the cancer-bearing immunodeficient non-human animal. Compare the changes in the proportions of B cells and T cells when doing so, and if the latter proportion change is similar to the former proportion change, select the test substance as the known immune checkpoint inhibitor or its candidate substance. I can do it.

Atezolizumab can be mentioned as an example of an immune checkpoint inhibitor. Atezolizumab is an anti-PD-L1 antibody drug that inhibits programmed cell death-1 (PD-1) and its ligand PD-L1 (PD-L2) pathway. It has been shown that administration of atezolizumab causes reactivation of T cells, induces a cancer-specific immune response, and exhibits antitumor effects. It has been shown that in breast cancer patients, the proportion of T cells among CD45-positive cells decreases compared to healthy subjects, and the proportion of B cells increases. In cancer-bearing, immunodeficient non-human animals transplanted with breast cancer cell lines, administration of atezolizumab increases the proportion of T cells among CD45-positive cells and decreases the proportion of B cells, resulting in a state similar to that of healthy individuals. Become. Therefore, when a test substance is administered to a tumor-bearing, immunodeficient non-human animal transplanted with a breast cancer cell line, the proportion of B cells decreases, and T cells among CD45-positive cells decrease, similar to when atezolizumab is administered. In particular, when the proportion of CD8-positive killer T cells increases, the test substance can be selected as an immune checkpoint inhibitor of the PD-1/PD-L1 pathway or a candidate thereof.

Note that immune checkpoint inhibitors are not limited to immune checkpoint inhibitors of the PD-1/PD-L1 pathway, but include cytotoxic T lymphocyte antigen 4 (CTLA-4) receptors expressed on T cells. It may also inhibit the B7/CTLA4 pathway between B7 (B7-1:CD80, B7-2:CD86) molecules expressed on antigen-presenting cells. Such immune checkpoint inhibitors include ipilimumab.

In addition, in addition to measuring lymphocytes as described above, when evaluating cancer immune responses for test substances based on the results of measuring lymphocytes in tumor-bearing immunodeficient non-human animals, - The size of tumor tissue in immunodeficient non-human animals may be measured. Compare the size of tumor tissue in a tumor-bearing-immunodeficient non-human animal to which the test substance was administered with the size of tumor tissue in a control tumor-bearing-immunodeficient non-human animal to which the test substance was not administered. do. By considering the comparative results of tumor tissue size in addition to the above-mentioned lymphocyte measurement results, it is possible to evaluate the cancer immune response and determine the antitumor effect of the test substance.

In addition to measuring the size of tumor tissue in cancer-bearing, immunodeficient non-human animals as described above, it is also possible to measure the degree of lymphocyte infiltration into the tumor (density of tumor-infiltrating lymphocytes). good. The density of tumor-infiltrating lymphocytes in tumor-bearing-immunodeficient non-human animals administered with the test substance was compared with that of tumor-infiltrating lymphocytes in control tumor-bearing-immunodeficient non-human animals to which the test substance was not administered. Compare with density. By considering the results of comparing the density of tumor-infiltrating lymphocytes in addition to the above-mentioned lymphocyte measurement results and tumor tissue size, the cancer immune response of the test substance is evaluated and the antitumor effect is further determined. be able to.

Furthermore, a cancer immune response evaluation kit can be provided to carry out the cancer immune response evaluation method described above. The cancer immune response evaluation kit according to the present invention includes the above-described cancer-bearing immunodeficient non-human animal and a cell surface marker analysis reagent for peripheral blood mononuclear cells. Here, the cell surface marker analysis reagent for peripheral blood mononuclear cells is used to detect lymphocytes contained in engrafted human peripheral blood mononuclear cells using a cell expression antigen test using flow cytometry, as described above. It is a reagent for Specifically, as described above, this kit contains an anti-CD45 antibody for detecting human CD45-positive leukocytes, an anti-CD19 antibody for detecting human CD19-positive B cells, and a human CD3-positive T cell. An anti-CD3 antibody for detection can be provided. Further, this kit can include an anti-CD4 antibody for detecting human CD4-positive helper T cells and an anti-CD8 antibody for detecting human CD8-positive killer T cells. Furthermore, this kit can also include an anti-CD56 antibody for detecting human CD56-positive NK cells. In addition, antibodies against human CD2, human CD5, human CD7, and human TCR (T-Cell Receptor) as cell surface antigens can be provided for detection of T cell lines. Furthermore, antibodies against human CD10, human CD20, human CD22, human CD79a, human CD38, CD27, Ig heavy chain, IgM, IgG subset, IgA subset, IgD, IgE, Ig light chain κ, λ were used as cell surface antigens in B cells. Provision may also be made for system detection. Furthermore, as antibodies against cell surface antigens, antibodies against human CD16, human CD57, human CD13, human CD33, human CD34, and human MPO (myeloperoxidase) can also be provided. Furthermore, antibodies such as various activation/exhaustion markers such as PD-1, PD-L1, CD25, and CTLA-4, and ligands for immune checkpoint antibodies can be provided.

Additionally, this kit can also contain the above-mentioned test substance. That is, this kit can be used as test substances such as nucleic acids, carbohydrates, lipids, proteins, peptides, small organic compounds, compound libraries prepared using combinatorial chemistry technology, random peptide libraries, and random nucleic acid libraries. It can contain natural components derived from lye or microorganisms, animals, plants, or marine organisms.

Hereinafter, the present invention will be explained in more detail with reference to Examples, but the technical scope of the present invention is not limited to the following Examples.

[Materials and methods]
(1) Tumor cells and mice The breast cancer cell line MDA-MB231 was stored at the Department of Molecular Life Science, School of Basic Medicine, Tokai University School of Medicine, and was incubated at 37°C using Leibovitz L-15 medium and 15% FCS. Cultured in CO ₂ free. NOG mice were purchased from INVIVO Science. CMV-NOG-hIL-4-Tg mice and CAG-NOG-hIL-4-Tg mice were produced by the Central Research Institute for Experimental Animals and maintained in an isolator at the Experimental Animal Facility, Tokai University School of Medicine, or the Central Laboratory for Experimental Animals. The one maintained at the research institute was used. The human IL-4 concentration was measured by DNA typing and ELISA and used for transplantation.
CMV-NOG-hIL-4-Tg mice are transgenic NOG mice into which an expression vector containing a CMV promoter, human IL-4 cDNA, and SV40poly(A) has been introduced. CAG-NOG-hIL-4-Tg mice are transgenic NOG mice into which an expression vector containing a CAG promoter, human IL-4 cDNA, and SV40poly(A) has been introduced.

(2) ELISA
Human IL-4 protein was quantified using Human IL-4 ELISA Set BD OptEIATM (manufactured by BD Biosciences, catalog number: 555194, rod number: 9189127). Quantification of IgG antibodies was performed according to a previously reported paper (Kametani Y, et al., Exp Hematol (2006) 34(9): 1240-1248). CH401MAP peptide dissolved in carbonate buffer (pH 9.5) was coated onto the wells of microtiter plates (manufactured by Sumiron), and the antigen was adsorbed onto the plate overnight at 4°C. The wells were then washed with PBS-Tween (0.05% v/v) and incubated with 3% BSA-PBS for 2 hours at room temperature. After washing with PBS-Tween three times, mouse plasma was added in a 10-fold dilution series and allowed to react at room temperature for 2 hours. The plate was washed three times, and biotin-conjugated mouse anti-human IgG mAb (manufactured by BD Pharmingen) (1:3,000) was added. After the plate was reacted for 2 hours at 37°C, it was washed three times and streptavidin-horseradish peroxidase (1:50,000 v/v; manufactured by BD Pharmingen) was added. After reacting the plate for 1 hour at room temperature, it was washed and EIA substrate kit solution (manufactured by Bio-Rad Laboratories) was added. The reaction was stopped with 10% HCl, and absorbance at 450 nm was measured. For quantitative determination, calibration samples prepared from standard samples with human IL-4 concentrations of 500 pg/ml, 250 pg/ml, 125 pg/ml, 62.5 pg/ml, 31.3 pg/ml, 15.6 pg/ml, and 7.8 pg/ml were used. I used a line.

(3) Preparation of human PBMC 30 mL of heparinized peripheral blood was collected from a healthy donor using Vacutainer ACD tubes (manufactured by NIPRO Corporation). The collected peripheral blood was immediately layered on Ficoll-Hypaque (manufactured by SIGMA-ALDRICH), and the mononuclear cell fraction was collected by density centrifugation (500 x g, 30 min, 20°C). The cells were washed with PBS by centrifugation at 300 x g, 5 min, 4°C, and the number of cells was counted before use.

(4) Transplantation into PBMC-NOG-hIL-4-Tg mice and analysis of engrafted human cells. 5×10 ⁶ MDA-MB231 was subcutaneously administered to the ventral region. After one week, the diameter of the tumor at 90 degrees from the ventral side was measured every two days using a caliper, and the product of the measurements was used to determine the tumor size. Two weeks after tumor transplantation, 5×10 ⁶ PBMCs were intravenously transplanted into tumor-bearing mice and non-tumor-bearing mice as a control group. Measurement of tumor diameter continued in the same way after PBMC transplantation. These mice were divided into two groups and administered PBS or atezolizumab (450ug/head/time/3 times (every 10 days)).

Two weeks later, the mice were anesthetized, heparinized blood was collected, and euthanized. After collecting cells from various lymphoid tissues, red blood cells were removed using hemolysis buffer to prepare a cell suspension. After counting these cells, flow cytometry (FCM) analysis was performed on the human leukocyte fraction. Tumor, lung, and liver tissues were used for immunohistochemical staining.

(5) Flow cytometry The antibodies shown in Table 1 were used for human immune cell staining.

The cells were reacted with an appropriate amount of various fluorescently labeled antibodies for 15 minutes at 4°C, and then washed with PBS containing 1% BSA. These cells were analyzed using FACS Fortessa or Verse (manufactured by BD Biosciences). After gating on live cells, human CD45-positive cells were further gated to obtain a human leukocyte fraction. These cells were further divided into lymphocyte subsets using cell surface markers. FlowJo (BD) was used for data analysis.

(6) Histochemical staining Mouse tissues were fixed with 20% formalin (Wako Pure Chemical Industries) and embedded in paraffin. Paraffin blocks were sliced, deparaffinized, mounted on glass slides, and stained with hematoxylin and eosin (HE). For immunohistochemical staining with anti-human CD45 antibody, slides were heat-treated at 97°C for 20 minutes and treated with endogenous peroxidase by reacting with 0.3% H ₂ O ₂ /MetOH at room temperature for 10 minutes. Thereafter, blocking was performed with 1% goat serum, anti-human CD45 (manufactured by Dako) was added, and the slides were reacted overnight at 4°C. The slides were washed three times for 5 min with 0.01M PBS, and colored with DAB solution. After washing with running water for 3 minutes, each stain was performed with hematoxylin, followed by dehydration, clearing, and mounting.

(7) Statistical processing Statistical processing was performed using Microsoft Excel (manufactured by Microsoft). Data are expressed as mean ± SD. Significant differences were tested using one-way ANOVA or two-sided Student's t-test analysis.

〔result〕
(1) hIL-4-Tg with a CMV promoter reproduces the human immune environment that maintains a balance of Th, Tc, and B cells by PBMC transplantation, whereas hIL-4-Tg with a CAG-promoter has a predominance of Th cells. It becomes an immune environment.

When plasma IL-4 concentrations were measured in NOG-hIL-4-Tg mice with a CMV promoter and NOG-hIL-4-Tg mice with a CAG-promoter, it was found that in CAG-NOG-hIL-4-Tg mice, IL-4 It was revealed that -4 is more than 5 times more expensive on average.

Human PBMC were transplanted into these mice, and 4 weeks later, the profile of lymphocytes that had engrafted in the spleen was analyzed using flow cytometry. As a result, the B cell engraftment rate was higher in CMV-NOG-hIL-4-Tg mice than in normal NOG mice, with over 70% of CD45-positive human leukocytes exceeding 10% in mice. Ta. On the other hand, almost no B cells engrafted in mice with plasma IL-4 concentrations of 500 pg/ml or higher, including CAG-hIL-4-Tg mice (see Figure 1). In Figure 1, the white dots indicate the results of measuring the plasma IL-4 concentration of CMV-NOG-hIL-4-Tg using the method described in [Materials and Methods] (2) ELISA above, and the black dots indicate the results of measuring the plasma IL-4 concentration of CMV-NOG-hIL-4-Tg using the same method. The results of measuring the plasma IL-4 concentration of CAG-NOG-hIL-4-Tg are shown. Figure 2 shows the results of measuring the plasma IL-4 concentration of CMV-NOG-hIL-4-Tg using

From this result, NOG mice transfected with human IL-4 are able to engraft B cells derived from human PBMC, and especially when the human IL-4 concentration in plasma is 100 to 500 pg/ml, B cells can be engrafted. It became clear that the cell survival rate was excellent.

Furthermore, as controls, CD3-positive T cells, CD19-positive B cells, CD4-positive helper T cells, and CD8-positive The results of measuring killer T cells are shown in Figure 2. As shown in FIG. 2, it was found that CD4-positive helper T cells and CD8-positive killer T cells were engrafted in a well-balanced manner in CMV-NOG-hIL-4-Tg mice. On the other hand, in CAG-NOG-hIL-4-Tg mice, most of the cells were CD4-positive helper T cells, and almost no CD8-positive killer cells were detected. Furthermore, in CMV-NOG-hIL-4-Tg mice, individuals with low plasma IL-4 concentrations had low B cell engraftment ability, resulting in a CD8-dominated GVHD-like profile.

From the above results, we found that immunodeficient mice in which the human IL-4 gene was introduced into NOG mice, especially immunodeficient mice with plasma IL-4 concentrations of approximately 100-500 pg/ml (approximately 100-1000 pg/ml depending on the measurement method). By transplanting human PBMC into mice, it was possible to confirm the engraftment of B cells and obtain a lymphocyte profile close to physiological conditions.

(2) The human breast cancer cell line MDA-MB-231 engrafts on CMV-PBMC-NOG-hIL-4-Tg, mimicking the patient's immune environment, and tumor growth suppression by atezolizumab is observed.

A human triple-negative breast cancer cell line MDA-MB231 expressing PD-L1 was transplanted into NOG-hIL-4-Tg, which has a CMV promoter. Figure 3 shows the results of measuring tumor diameters for the PBS administration group and the atezolizumab administration group. In addition, PBS or atezolizumab was administered to CMV-NOG-hIL-4-Tg mice transplanted with breast cancer cell line MDA-MB231 and CMV-NOG-hIL-4-Tg mice not transplanted with breast cancer cell line MDA-MB231. Figure 4 shows the results of measuring the lymphocyte profile when administered. In Figure 4, CMV-NOG-hIL-4-Tg mice transplanted with breast cancer cell line MDA-MB231 are described as "tumor-bearing mice," and CMV-NOG-hIL-4-Tg mice that are not transplanted with breast cancer cell line MDA-MB231 are referred to as "tumor-bearing mice." -4-Tg mice were described as "non-tumor-bearing mice."

From the results of the PBS administration group shown in Figure 3, it was observed that tumor cells were engrafted and the tumor mass increased over time. Almost no human lymphocytes were infiltrated into this tumor mass. In addition, among the results shown in Figure 4, CMV-NOG-hIL-4-Tg mice transplanted with breast cancer cell line MDA-MB231 and CMV-NOG-hIL-4-Tg mice without transplantation with breast cancer cell line MDA-MB231. Based on the results of PBS administration to mice, the percentage of T cells among CD45-positive cells was lower in CMV-NOG-hIL-4-Tg mice transplanted with MDA-MB231 compared to mice not transplanted with the MDA-MB231 strain. However, it was revealed that the proportion of B cells increased. This phenomenon has also been observed in peripheral blood mononuclear cells of breast cancer patients, and CMV-NOG-hIL-4-Tg mice transplanted with MDA-MB231 were shown to mimic the in vivo results.

On the other hand, when the anti-PD-L1 antibody atezolizumab was administered to these mice, it was shown that tumor growth tended to be suppressed, as shown in Figure 3. Furthermore, as shown in Figure 4, administration of atezolizumab to CMV-NOG-hIL-4-Tg mice transplanted with MDA-MB231 decreased B cell engraftment and increased the proportion of T cells, especially CD8. The percentage of positive killer T cells is increasing.

Additionally, the results of histochemical staining of tumor tissue are shown in FIG. As shown in Figure 5, no infiltration of lymphocytes into the tumor mass was observed in the PBS administration group, whereas clear infiltration of human T cells into the tumor mass was observed in the atezolizumab administration group.

Based on the above results, CMV-NOG-hIL-4-Tg mice transplanted with MDA-MB231 mimic the immune status of human breast cancer, and further show the antitumor effect of atezolizumab, an immune checkpoint inhibitor, on human breast cancer patients. It became clear that it was possible to reproduce.
All publications, patents, and patent applications cited herein are incorporated by reference in their entirety.

Claims

A mutation has been introduced into the IL-2 receptor γ chain gene, resulting in a deficiency of the IL-2 receptor γ chain, and mutations in genes involved in rearrangement of T cell and B cell antigen receptor genes are present at both allelic loci. , and a cancer-bearing immunodeficient non-human animal into which cancer cells and/or cancer tissues and human peripheral blood mononuclear cells have been transplanted into an immunodeficient non-human animal into which the human IL-4 gene has been introduced. animal.
The tumor-bearing immunodeficient non-human animal according to claim 1, wherein the plasma concentration of human IL-4 encoded by the human IL-4 gene is 100 to 500 pg/ml.
The tumor-bearing and immunodeficient non-human animal according to claim 1, wherein the human IL-4 gene is expressed under the control of a human cytomegalovirus promoter.
The tumor-bearing immunodeficient non-human animal according to claim 1, wherein the mutation in a gene involved in rearrangement of antigen receptor genes of T cells and B cells is a SCID mutation or a RAG mutation.
The immunodeficient non-human animal described above lacks both functional T cells and B cells, has decreased macrophage function, has lost NK cells or NK activity, and has decreased dendritic cell function, and has excellent 2. The cancer-bearing immunodeficient non-human animal according to claim 1, which has a property of adhesion to different types of cells.
The cancer-bearing non-immuno-deficient non-human animal according to claim 1, wherein the immuno-deficient non-human animal is produced by backcrossing the following non-human animal A with the non-human animal B. human animal.
A: C. to NOD/Shi non-human animals. Non-human animal B obtained by backcrossing B-17-scid non-human animal: non-human animal in which the interleukin 2 receptor γ chain gene has been knocked out
The tumor-bearing immunodeficient non-human animal according to claim 1, wherein the immunodeficient non-human animal is a NOG (NOD/Shi-scid, IL-2Rγ KO) mouse.
The cancer-bearing immunodeficient non-human animal according to claim 1, wherein the cancer cells and/or cancer tissues are human breast cancer cells and/or human breast cancer tissues.
The tumor-bearing, immunodeficient non-human animal according to claim 1, wherein the number of human peripheral blood mononuclear cells transplanted is 1 to 5×10 6 cells.
The tumor-bearing immunodeficient non-human animal according to claim 1, wherein human CD19-positive human B cells are engrafted.
A step of administering a test substance to the cancer-bearing immunodeficient non-human animal according to any one of claims 1 to 10;
A method for evaluating cancer immune response regarding a test substance, comprising the step of comparing lymphocytes before and after administration of the test substance.
The test substance is an immune checkpoint inhibitor or a candidate substance for an immune checkpoint inhibitor, and is characterized in that T cells and/or B cells among lymphocytes are compared before and after administration of the test substance. The cancer immune response evaluation method according to claim 11.
If the number of human CD8-positive killer T cells included in lymphocytes increases compared to before administration of the test substance, the test substance 12. The cancer immune response evaluation method according to claim 11, wherein the substance is determined to have an immune checkpoint inhibiting effect.
A cancer immune response evaluation kit comprising the cancer-bearing immunodeficient non-human animal according to any one of claims 1 to 10 and a cell surface marker analysis reagent for peripheral blood mononuclear cells.
The cancer immune response evaluation kit according to claim 14, further comprising a test substance to be evaluated.