KR102316506B1 - A method for promoting humanized activity of animal immune system - Google Patents

Abstract

It relates to a technology for promoting the humanization activity of the immune system of animals other than humans by using cells, lysates, or lysates thereof present in mixed peripheral blood obtained from a plurality of humans. It can play a role in building an in vivo functional platform that helps to rebuild and verify immune rejection of cell therapy products in the body in the future.

Description

A method for promoting humanized activity of animal immune system

A composition for promoting humanization activity of an animal's immune system other than humans, a humanized animal model in which humanization activity is promoted using the same, and a method for promoting humanization activity using the same.

Clinical trials are an indispensable step in the study of human diseases. Clinical trials are arguably the most effective test methods, but the reality is that they are only possible in a very limited way due to various risk factors and ethical issues. Several animal models are being used to replace them, but since they are different from the human body, even if they succeed in animal experiments, it is difficult to obtain certainty that they will be applied in the same way as the human body. Therefore, it is believed that the production of animal models with conditions similar to those of the human body will bring great development in research on the treatment of incurable diseases such as cancer and AIDS, so the development of excellent animal models continues to be active. Among them, the mouse, which is 97% similar to the human gene, is widely used in cancer research. In particular, in the 1960s, as it became known that the immune rejection response was weak due to the lack of activated T-lymphocytes in athymic nude mouse, research on how to transplant cancer cells into the nude mouse was active. And, in 1983, Severe Combined Immunodeficiency (SCID) Mouse was reported by Bosma. SCID Mouse combines V(D)J, a region that recognizes antibodies (Antigen) of B cells and T cells in the lymphocyte differentiation process. It is a model in which B cells and T cells do not function properly due to the lack of the break repair activity of the DNA involved in the process. Because of these defects in B and T cell function, SCID Mouse has almost no rejection reaction to accept human cells, so SCID Mouse is widely used in research in various fields such as cancer, immune diseases, organ transplantation, and infection. Non Obese Diabetic-Severe Combined Immunodeficiency (NOD/SCID) Mice is a mouse model derived from NOD Mouse lacking innate immunity and SCID Mouse lacking acquired immunity. Functional characteristics of NOD Mouse (Phenotype) decrease in activity of Natural Killer Cell, secretion dysfunction of monocytes to cytokine, lack of activity of complement 5 components (Complement5, C5), etc. SCID Mouse The functional characteristic (phenotype) of B-cells and T-cells is also shown at the same time. Human Peripheral Blood Mononuclear Cell (huPBMC) is a blood cell with circular nucleus such as Lymphocyte or Monocyte, and plays an important role in the immune system to adapt or resist infection by foreign substances. It can be separated from whole blood using Ficoll-Paque, and because it contains CD4+ cells, it can be used for HIV research and has the characteristic of providing an immune system similar to that of the human body in cancer research. is a cell

It was reported that the number of lymphocytes injected for a certain period of time was present in the mouse for a certain period of time by intraperitoneal injection (IP) of human peripheral blood mononuclear cells into SCID Mouse. was evaluated as a condition. However, according to a study by Hesselton in 1995, it was reported that human peripheral blood monocytes were accepted 5 to 10 times more well when transplanted into NOD/SCID mice than when human peripheral blood monocytes were transplanted into SCID. These results show that NOD-SCID mice are immunodeficient mice capable of significantly amplifying the proliferation of human cells and thus increasing the retention and engraftment rate of human cells.

Korean Patent Publication No. 1020150027752

An object of the present invention is to provide a technology for promoting humanization activity of an animal's immune system by using cells present in mixed peripheral blood obtained from a plurality of humans, a lysate thereof, or a lysate thereof.

In one aspect, there is provided a composition for promoting humanization activity of the immune system of animals other than humans, comprising cells present in mixed peripheral blood obtained from a plurality of humans, a lysate thereof, or a lysate thereof.

The animal may be a mouse, rabbit, dog, pig, monkey, etc., but is not necessarily limited thereto as long as it is a non-human animal that can be used as a humanized animal model by injecting the composition. Specifically, according to an embodiment, it may be a mouse.

The animal may be an animal from which an intrinsic immune system has been removed, specifically, may be an animal lacking lymphocyte cells constituting the immune system, and may be an animal capable of establishing a human immune system. According to one embodiment, the animal may be a mouse deficient in all of T cells, B cells and NK cells.

Promotion of the humanization activity of the immune system may mean promoting the survival, differentiation, and proliferation of cells constituting the human lymphocyte system in the body of the humanized animal to successfully establish the human immune system in the body of the animal. . In this case, the cells constituting the living, differentiated, and proliferated human lymphocytes may be newly reconstructed by engraftment of human hematopoietic stem cells in the body of the animal. According to a specific example, when promoted by the composition, the expression of markers such as CD45, CD8, CD4, and CD19 indicating that the cells are human lymphocytes is significantly increased compared to the control group.

More specifically, the composition may include cells present in mixed peripheral blood obtained from a plurality of humans, a lysate thereof, and a lysate thereof.

The lysate may include fragments obtained by lysing cells present in the mixed peripheral blood by using a conventional technical means such as a spin or pressure, and the lysate is present in the mixed peripheral blood It may mean collectively a substance obtained by lysing cells.

The mixed peripheral blood obtained from a plurality of humans may mean that the peripheral blood is obtained from a plurality of humans, respectively, and mixed. This may be to overcome the limitation of targeting only one epitope by injecting only one epitope in the conventional method for preparing a humanized animal model. That is, by sensitizing and training animals with more human epitopes that cause various causes of immune rejection, the humanized animal model provides a diverse range of epitopes of human lymphocytes that are developed in the body, and at the same time increases the recognition of human epitopes. It can be expanded to promote the development and maturation of lymphocyte cells and contribute to the stable establishment of human and animal models using this.

The plurality of humans may mean 2 or more, 3 or more, 5 or more, 10 or more, 15 or more, or 20 or more humans instead of 1, and according to one embodiment, specifically, 10 or more people. It could mean human.

The cell is not particularly limited as long as it contains a human epitope in the mixed peripheral blood, but according to one embodiment, specifically, it may be a peripheral mononuclear cell present in the mixed peripheral blood.

The composition may include a plurality of different human antigenic determinants as it contains a substance derived from cells present in mixed peripheral blood obtained from a plurality of humans. As can be seen in one embodiment, this can be linked to the effect of producing a result in which all markers such as CD45, CD8, CD4, CD19 are significantly increased compared to before injection in the body of the injected animal model.

The cell, its lysate, or its lysate may or may not be filtered according to a specific particle size. If filtering is performed, it may be a technical measure to reduce side effects through embolism in the body of the animal during injection. It may be a technical measure to further increase the degree. Even when filtering was performed, the overall expression of the pan-human cell marker was significantly increased compared to before injection, but when no filtering was performed, the expression of the pan-human cell marker was significantly better than before the filtering compared to the case where the filtering was performed. elevation can be seen.

The composition may further comprise a conventionally acceptable carrier, and may be formulated together with the carrier. The 'normally acceptable carrier' refers to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound. Acceptable pharmaceutical carriers for compositions formulated as liquid solutions include sterile and biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostats may be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets.

In one aspect, a non-human animal model is provided in which the humanizing activity of the immune system is promoted by injecting the composition.

The above model overcomes the limitation that it was not easy to induce the development of human lymphocytes in a state in which cells engrafted in the bone marrow by injecting human hematopoietic stem cells in the prior art have almost no mature thymus, The humanization activity may be promoted.

In the model, the expression of a marker that can determine whether humanization and the degree of humanization in the thymus, spleen, peripheral blood or bone marrow may be significantly increased compared to a control group not injected with the composition, specifically, CD45+, CD45+, CD8+ , the number of CD4+ or CD19+ cells may be significantly increased compared to an animal model not injected with the composition, which is a material derived from cells in which the composition is obtained from a plurality of humans and is present in mixed peripheral blood. It may be based on the fact that it may include a plurality of different human antigenic determinants.

In addition, specific descriptions related to the animal, composition, and the like are the same as described above.

In one aspect, the method comprising: obtaining and mixing peripheral blood from a plurality of humans to obtain mixed peripheral blood; And it provides a method of promoting the humanization activity of the immune system of animals other than humans, comprising injecting the cells present in the mixed peripheral blood, a lysate thereof, or a lysate thereof into a subject.

The injecting into the subject may include injecting the cells present in the mixed peripheral blood, a lysate thereof, and a lysate thereof.

The step of obtaining the mixed peripheral blood may include obtaining and mixing peripheral blood from a plurality of humans, wherein the plurality of humans is not one but 2 or more, 3 or more, 5 or more, 10 or more, It may mean 15 or more or 20 or more humans, and according to an embodiment may specifically mean 10 or more humans.

The cell is not particularly limited as long as it contains a human epitope in the mixed peripheral blood, but according to one embodiment, specifically, it may be a peripheral mononuclear cell present in the mixed peripheral blood.

The method may or may not further include the step of filtering the cells, their lysates, or their lysates present in the mixed peripheral blood according to a specific particle size. When it includes the step of filtering, it may be a technical purpose to reduce side effects through embolism in the body of the animal during injection, and when the step of filtering is not included, the antigenic determinants obtained from a plurality of human peripheral blood as it is It may be for a technical purpose to further increase the degree of humanization by injection. Even when the filtering step is included, the overall expression of the pan-human cell marker is significantly increased compared to before injection, but when the filtering step is not included, the expression of the pan-human cell marker compared to the case before the injection is filtered A more remarkably superior elevation may be exhibited.

In addition, other detailed descriptions such as animals, peripheral blood, cells, lysates, and the like are the same as described above.

It relates to a technology for promoting the humanization activity of the immune system of animals other than humans by using cells, lysates, or lysates thereof present in mixed peripheral blood obtained from a plurality of humans. It can play a role in building an in vivo functional platform that helps to rebuild and verify immune rejection of cell therapy products in the body in the future.

1 is a result of measuring the expression level of human cells in the animal model prepared in Example (**: p<0.01, *: p<0.05).
2 to 5 are the results of measuring the expression level of human cells in each tissue of the spleen, peripheral blood, bone marrow and thymus of the animal model prepared in Example, and the results before and after filtering with a sieve and HLA-I (MHC- I) and the results of measuring the number of cells expressing HLA-II (MHC-II) are included.

Hereinafter, in order to explain in more detail, examples will be described in detail.

Example 1. Preparation of Humanized Animal Models

a) After obtaining and mixing from 10 adults, a vial of frozen peripheral blood (concentration is 2*10 ⁷ cells/composition) was thawed using a 37°C constant temperature water bath, and 2 with dPBS to remove DMSO. washed once. b) The washed peripheral mononuclear cells were immersed in 1 ml of dPBS and flash frozen in LN2 (Liquid Nitrogen) for 3 minutes. Cells confirmed to be cooled were transferred to a constant temperature water bath at 37° C. and thawed for 3 minutes. After repeating the process of b) three times, spin to mix the lysate with the lysate at the same time, and a dose of 10 adults was injected into the tail vein of a mouse (NSG Mouse). The final concentration was about 2*10 ⁷ cells/composition.

Example 2. Confirmation of the degree of humanization of the prepared animal model

In this method for using human peripheral blood-derived mixed epitope, broken pieces and lysates of cells were injected into the tail vein of humanized mice, where the frequency of human cells was maintained as low as 5%. Within a week after injection, CD45-positive cells, a marker of pan-human cells, significantly increased. Compared to before injection, 20.6 times in the thymus, 9.2 times in the spleen, 18.9 times in the bone marrow (BM), There was an 8.3-fold increase in peripheral blood (PB). In addition, CD8-positive cells increased 8.1-fold in the thymus and 2.8-fold in the spleen, and CD4-positive cells increased 3.3-fold in the thymus and 5.8-fold in the spleen. The number of CD19-positive cells was also increased, showing significant increases in spleen, bone marrow, and peripheral blood 9.9-fold, 19.7-fold, and 8.2-fold, respectively ( FIG. 1 ).

This method for using human peripheral blood-derived mixed epitope showed differences between the group that was given after filtering the cell lysate fragments and lysate through a sieve with a particle size of 0.33 μm and the group that was given without filtering. In the case of CD45-positive cells, compared to before injection, the filtered case increased by 11.9%, whereas the non-filtered case increased by 48.2%. In the case of CD19-positive cells, compared to before injection, the filtered case increased by 21.1%, whereas the non-filtered case increased by 41.4%. In addition, it was confirmed through each tissue that the increase in cells showing sufficient HLA-I and II expression increased compared to before injection even after filtering and injecting considering the risk of embolism during tail vein injection by fragments of cell debris (Fig. 2 to 5).

Claims (20)

A composition for promoting humanization activity of the immune system of animals other than humans, comprising a lysate or a lysate of cells present in mixed peripheral blood obtained from a plurality of humans,
A composition that can increase the degree of humanization activity of the immune system depending on whether or not filtering is performed according to a specific particle size of the lysate or lysate of the cell.
The composition according to claim 1, comprising a lysate and a lysate of cells present in mixed peripheral blood obtained from a plurality of humans.
The composition according to claim 1, wherein the mixed peripheral blood is obtained from 10 or more humans and mixed.
The composition of claim 1, wherein the cells are peripheral mononuclear cells in the mixed peripheral blood.
The composition of claim 1 , wherein the composition comprises a plurality of different human antigenic determinants.
The composition of claim 1, wherein the lysate or lysate of the cell is not filtered according to a specific particle size,
The composition promotes humanizing activity of the immune system by increasing the number of CD45+ or CD19+ cells in the thymus, spleen, peripheral blood or bone marrow of animals other than humans, or by increasing the number of CD8+ or CD4+ cells in the thymus. A composition for
The composition of claim 1, wherein the lysate or lysate of the cell is filtered according to a specific particle size,
The composition is for promoting humanizing activity of the immune system by increasing the number of CD8+ or CD4+ cells in the spleen, peripheral blood or bone marrow of animals other than humans.
The composition of any one of claims 1 to 7 is injected to promote humanizing activity of the immune system, non-human animal model.
The model of claim 8 , wherein the animal is a mouse, rabbit or pig.
The model of claim 8, wherein the animal is an animal from which an intrinsic immune system has been removed.
The model according to claim 8, wherein the animal is an animal deficient in T cells, B cells and NK cells.
delete mixing peripheral blood separated from a plurality of humans to obtain mixed peripheral blood; and
A method for promoting humanization activity of the immune system of animals other than humans, comprising injecting a lysate or a lysate of cells present in the mixed peripheral blood into an individual other than a human,
The method is one that can increase the degree of humanization activity of the immune system depending on whether filtering is performed according to a specific particle size of the lysate or lysate of the cell.
The method according to claim 13, wherein the injecting into the subject comprises injecting the lysate and the lysate of the cells present in the mixed peripheral blood.
The method of claim 13 , wherein obtaining the mixed peripheral blood comprises mixing peripheral blood isolated from 10 or more humans.
The method of claim 13 , wherein the cells are peripheral mononuclear cells in mixed peripheral blood.
The method according to claim 13, wherein the method does not include filtering the lysate or lysate of cells present in the mixed peripheral blood according to a specific particle size,
The method promotes humanizing activity of the immune system by increasing the number of CD45+ or CD19+ cells in the thymus, spleen, peripheral blood or bone marrow of a non-human individual, or by increasing the number of CD8+ or CD4+ cells in the thymus. how to do it.
The method according to claim 13, wherein the method further comprises filtering the lysate or lysate of cells present in the mixed peripheral blood according to a specific particle size,
The method is for promoting humanizing activity of the immune system by increasing the number of CD8+ or CD4+ cells in the spleen, peripheral blood or bone marrow of a non-human individual.
The method of claim 13 , wherein the animal is a mouse, rabbit or pig, and wherein the injecting comprises injecting through the tail vein of the animal.
The method of claim 19 , wherein the animal is an animal with an intrinsic immune system depleted.

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