Preparation and application of macrophages

Abstract

The invention relates to the field of preparation of immune cells, in particular to preparation and application of macrophages. The invention provides a method for preparing macrophages, and relates to transdifferentiation of B cells into macrophages. According to the method disclosed by the invention, a B cell is taken as a starting cell, engineering transformation is carried out, an exogenous transcription factor is introduced, and the B cell is induced to generate cell reprogramming to prepare the macrophage. The method provided by the invention can also be used for preparing a modified macrophage (such as a CAR modified macrophage). By utilizing the preparation method provided by the invention, the macrophages can be rapidly and efficiently prepared, and the preparation period of the macrophages is greatly shortened. The macrophage produced by the invention has the due functions of normal macrophages, does not have multiplication capacity, still maintains the functions of the macrophages after ray irradiation, has safety in clinical use, and can be applied to clinical treatment.

Description

Technical Field

[0001] The present invention relates to the field of immune cell preparation, in particular to the preparation and application of macrophages. Background Art

[0002] Macrophages are a type of immune cell with phagocytic ability that are widely distributed in tissues and organs, performing a variety of physiological and pathological functions. They play a key role in innate immunity and also play an important role in adaptive immune responses.

[0003] As a powerful class of effector cells in the innate immune system, macrophages not only play a role in defending against pathogens and clearing aging, declining or dead cells, but also play an important role in tissue homeostasis and repair, such as wound healing and muscle regeneration. The classic functions of macrophages include cytotoxicity (e.g., targeted cytotoxicity), phagocytosis, antigen presentation (for activating T cells to coordinate adaptive immune responses), and the production and secretion of cytokines and chemokines. Like all immune cells, macrophages originate from hematopoietic stem cells in the bone marrow. Monocytes migrate from the blood circulation into tissues, and circulating monocytes are activated in response to various stimuli, promoting the differentiation of monocytes into macrophages.

[0004] During infection, inflammation, or tissue damage, macrophages can follow chemotactic signals and migrate to damaged tissues or sites of inflammation. There, they ingest pathogens and cellular debris through phagocytosis and digest them in phagolysosomes. Phagolysosomes are formed by the fusion of phagosomes with lysosomes, which contain a set of hydrolases capable of breaking down a variety of macromolecules. This degradation not only helps clear pathogens and sites of inflammation but also facilitates the presentation of antigenic peptides to specific immune system cells, such as T or B lymphocytes, thereby initiating antigen-specific immune responses.

[0005] Macrophages can enter tumor tissue and have a unique ability to infiltrate solid tumors, while other immune system cells, such as T cells, have very limited infiltration into tumor tissue. Within tumor tissue, macrophages can exhibit either a classically activated (M1, anti-tumor) or alternatively activated (M2, pro-tumor) phenotype. Macrophages are abundant in the tumor microenvironment (TME) of many cancers.

[0006] In summary, the functions of macrophages include: 1. phagocytosis and clearance of pathogens; 2. regulation of the direction and intensity of immune responses through secretion of cytokines and chemokines; 3. antigen presentation; and 4. macrophages also play a vital role in tissue repair and remodeling. Macrophages also possess remarkable plasticity, exhibiting distinct functions and phenotypes depending on microenvironmental signaling. Therefore, macrophages have important clinical applications, including: 1) tumor immunotherapy; 2) immunomodulation and anti-inflammatory effects; 3) treatment of infectious diseases; and 4) applications in tissue repair and regenerative medicine.

[0007] Existing macrophage preparation methods are mainly divided into two categories: 1) directly isolating primary cells and culturing / differentiating them in vitro; 2) preparing macrophages through the directed differentiation of induced embryonic stem cells / pluripotent stem cells. However, existing technologies all have certain shortcomings. For example, the number of peripheral blood mononuclear cells obtained from a single donor is limited, which is difficult to meet clinical treatment needs; and when obtained from different donors, the differences in cell sources between donors may lead to unpredictable differences in treatment effects. Cell differentiation methods, whether differentiated from bone marrow stem cells or embryonic stem cells / induced pluripotent stem cells, face shortcomings such as complex preparation conditions and techniques, time-consuming and high costs.

[0008] The source and preparation of macrophages are currently the primary issues restricting the clinical application of macrophages. How to efficiently and stably obtain macrophages with normal functions and a quantity that can meet clinical applications is an urgent problem that needs to be solved. Summary of the Invention

[0009] The present invention provides a new method for preparing macrophages, which overcomes the shortcomings of the existing technology. The method provided by the present invention can quickly prepare a large number of macrophages with the same functions as primary macrophages; for the convenience of description, this specification will name the macrophages prepared by the present invention as cell reprogrammed-macrophages (Cell Reprogrammed-Macrophage, REP-M). On the other hand, the method provided by the present invention can easily and quickly prepare modified macrophages to meet the needs of clinical medical use. For example, the method provided by the present invention can easily and quickly prepare macrophages expressing chimeric antigen receptors (CAR, Chimeric Antigen Receptor).

[0010] To achieve the above-mentioned purpose, the method provided by the present invention involves inducing the expression of one or more transcription factors in B cells and reaching a certain expression level, thereby inducing B cells to undergo cell reprogramming to generate macrophages. In this specification, the two terms "transdifferentiation" and "reprogramming" can be used interchangeably. "Transdifferentiation" refers to the process in which a differentiated functional cell (i.e., a mature somatic cell) is directly transformed into another terminally differentiated functional cell without going through an intermediate pluripotent state of a progenitor cell type, such as the transformation of a B cell into a macrophage. The term "transdifferentiation" was first proposed by Selman and Kafatos (Selman and Kafatos, "Transdifferentiation in the labial gland of silk moths: is DNA required for cellular metamorphosis?" Cell Differentiation, 1974, 3(2):81-94).

[0011] The transdifferentiation method of the present invention provides a simple, rapid and efficient method for producing macrophages. Once cell-reprogrammable B cells (REPable-B) are established, they can be transdifferentiated into macrophages under standard cell culture conditions in less than a week without the need for special conditions. The process is extremely efficient, and 100% of cell-reprogrammable B cells can be transdifferentiated into macrophages. Among them, the macrophages transdifferentiated by high expression of PU.1 as the main driving force for cell reprogramming highly express CD86 and CD80. In addition, the method described in the present invention can be used for mass production of macrophages, because B cells (especially immortalized B cells) are easy to expand in vitro and can provide a large number of starting cells for transdifferentiation. This has significant advantages in clinical applications, such as macrophage-mediated cell therapy.

[0012] In the present invention, "engineered" cells refer to cells into which nucleic acids that can achieve a certain purpose are introduced, such as nucleic acids encoding transcription factors, chimeric antigen receptors or cytokines. Engineered cells are different from natural cells that do not contain introduced nucleic acids. The nucleic acids in the present invention include nucleic acids obtained from natural sources, chemically synthesized nucleic acids or nucleic acids produced by recombinant technology. Therefore, macrophages that are transdifferentiated from B cells that can be cell reprogrammed are cells that have been reprogrammed or transdifferentiated. In the present invention, "transdifferentiated cells" refer to mature differentiated cells of one tissue type, whose phenotype is transdifferentiated or redirected to the phenotype of mature cells of another tissue type, and do not necessarily need to be restored to the pluripotent cell stage or multipotent phenotype. In the context of the present invention, cell reprogrammed macrophages (REP-M) are macrophages generated by transdifferentiation of B lymphocytes that can be cell reprogrammed (REPable-B), or any other macrophages that can be generated by transdifferentiation of cells that can generate macrophages by cell reprogramming methods.

[0013] The present invention also provides engineered macrophages, such as macrophages expressing therapeutic molecules (e.g., chimeric antigen receptor (CAR)-macrophages). Macrophages prepared using the methods of the present invention can be used for cell therapy. The diseases include cancer, autoimmune diseases, infectious diseases, or inflammatory diseases.

[0014] The present invention also provides B lymphocytes capable of cell reprogramming (REPable-B). Such engineered B lymphocytes capable of cell reprogramming (REPable-B) can be used for industrial mass production of macrophages.

[0015] In particular, the present invention relates to processes and methods for reprogramming B cells into macrophages, as well as cell reprogramming-macrophages (Rep-M), compositions comprising macrophages and uses thereof. The method of the present invention can be used to generate macrophages in vitro and in vivo. It should be noted that the preparation of macrophages by B cell transdifferentiation (cell reprogramming) is only one way to prepare cell reprogramming-macrophages. Other types of cells may also be induced to reprogram macrophages, such as T cells (including primary T cells, iPSC / ESC induced differentiation T cells and immortalized T-lymphocytes) or any other cells that can be reprogrammed to generate macrophages.

[0016] Transdifferentiation of B cells into macrophages

[0017] The present invention overcomes a major obstacle in this area by providing improved methods for producing macrophages both in vivo and in vitro.

[0018] The macrophage production method of the present invention involves directly transdifferentiating B cells into macrophages. The method of producing macrophages of the present invention includes preparing reprogrammable B cells (REPable-B cells), which express one or more transcription factors of PU.1, C / EBPα, C / EBPβ, C / EBPδ or C / EBPε, thereby inducing the transdifferentiation of these reprogrammable B cells (REPable-B cells) into macrophages ( Figure 1 ). In the present invention, macrophages obtained by cell reprogramming of REPable-B cells are referred to as "cell reprogrammed macrophages (REP-M)". The present invention has demonstrated that when B cells (e.g., immortalized B cell lines) express high levels of PU.1 or high levels of PU.1 superimposed with an appropriate amount of C / EBPα, B cells can be cell reprogrammed to generate macrophages. The present invention also found that more than 95% of the macrophages prepared by this scheme were CD86 and CD80 positive, that is, M1 polarized macrophages. The reprogrammed macrophages prepared by this scheme not only express CD86 and CD80, but also have the functions of M1 polarized macrophages, such as being activated when in contact with cancer cells and producing cytokines such as IL-1, IL-6, and TNF-α.

[0019] PU.1 is a transcription factor from the Ets family, primarily expressed in B cells and myeloid cells. Low levels of PU.1 in hematopoietic stem and progenitor cells induce B cell differentiation, while high levels promote myeloid differentiation. C / EBPα (CCAAT / enhancer binding protein α) is a leucine zipper-type transcription factor involved in regulating the differentiation of various cell types. Oevelen et al. (2015, Stem Cell Reports) also demonstrated that C / EBPα activates two myeloid-specific enhancers in pre-B cells during the transdifferentiation of pre-B cells to macrophages. Transcription factors can induce transdifferentiation in somatic cells. Activation or forced expression of key transcription factors can reprogram one somatic cell type into another. C / EBPα directly interacts with PU.1; the combination of C / EBPα and PU.1 can induce transdifferentiation of fibroblasts into macrophage-like cells. Although an experimental system for converting B cells into macrophages has been reported, for example, mature, antibody-producing B cells in mice can be induced to transdifferentiate into functional macrophages by forced expression of C / EBPα (Xie H, et al., Stepwise reprogramming of B cells into macrophages. Cell. 2004; 117: 663-676); human B lymphoma and leukemia cell lines can also be induced to transdifferentiate into functional macrophages by forced expression of C / EBPα (Rapino F. et al., C / EBPα induces highly efficient macrophage transdifferentiation of B lymphoma and leukemia cell lines and impairs their tumorigenicity. Cell Rep. 2017; 19: 1281). Human primary BCR-ABL1-positive B-ALL cells can also be induced to undergo cell reprogramming to generate macrophage-like cells by forced expression of C / EBPα (McClellan JS, et al., Reprogramming of primary human Philadelphia chromosome-positive B cell acute lymphoblastic leukemia cells into nonleukemic macrophages. Proc Natl Acad Sci USA. 2015; 112: 4074-4079).

[0020] The transdifferentiation method described in the present invention relates to establishing B cells that can be reprogrammed to generate macrophages. The preparation of cell-reprogrammable B cells (REPable-B cells) includes engineering them to have the ability to be induced to highly express the transcription factor PU.1, or to co-express the transcription factor PU.1 and C / EBPα. When the cell-reprogrammable B cells only express exogenous PU.1, the expression level of exogenous PU.1 that can induce cell reprogramming has been determined. When the cell-reprogrammable B cells co-express exogenous PU.1 and C / EBPα, the correlation between the expression levels of exogenous PU.1 and C / EBPα and the characteristics of cell-reprogrammed macrophages has been well determined. Therefore, the method provided by the present invention can not only induce cell-reprogrammable B cells to efficiently transdifferentiate into macrophages, but also control the characteristics of cell-reprogrammed macrophages, such as specific polarized types of macrophages, by controlling the expression levels of PU.1 and C / EBPα. In certain embodiments, these cell-reprogrammable B cells are reprogrammed into macrophages in vitro (e.g., in cell culture). In certain embodiments, these reprogrammable B cells can be transplanted into animals, and these engineered B cells can also be induced to undergo cell reprogramming to generate macrophages in vivo (e.g., in mice).

[0021] Based on the above-mentioned cell preparation method, the technical solutions adopted by the present invention include:

[0022] In a first aspect, the present invention provides a method for producing a macrophage population, comprising the following steps:

[0023] (1) Engineering a B cell population and introducing an exogenous transcription factor PU.1 to prepare a B cell population expressing the exogenous transcription factor;

[0024] (2) Inducing B cell populations to transdifferentiate into macrophages; the macrophages are CD86 and CD80 positive.

[0025] In certain embodiments, in step (1), after the exogenous transcription factor PU.1 is introduced into the B cell population, the expression level of the exogenous transcription factor PU.1 is more than 10 times higher than the expression level of endogenous PU.1 in the B cells.

[0026] The method for producing a macrophage population described herein comprises introducing an exogenous transcription factor, PU.1, into a B cell population to establish B cells expressing PU.1 and capable of cell reprogramming, and inducing the B cell population to transdifferentiate into macrophages, including macrophages positive for CD86 and CD80. The reprogrammable B cells are induced to express high levels of PU.1.

[0027] The cell-reprogrammable B cells can highly express PU.1. In certain embodiments, the expression level of PU.1 in the cell-reprogrammable B cells is at least 10 times higher than the expression level of endogenous PU.1. In certain embodiments, the expression level of PU.1 in the cell-reprogrammable B cells is 10 to 200 times, or 10 to 100 times, or 10 to 50 times, or 20 to 100 times, or 20 to 200 times, or 50 to 200 times, or 50 to 100 times, or 100 to 150 times, or 100 to 200 times, or 150 to 200 times the expression level of endogenous PU.1. In certain embodiments, the expression level of PU.1 in the cell-reprogrammable B cells is at least 200 times the expression level of endogenous PU.1. In certain embodiments, the expression level of PU.1 in the cell-reprogrammable B cells is at least 190 times the expression level of endogenous PU.1. In certain embodiments, the expression level of PU.1 in a cell-reprogrammable B cell is at least 180 times the expression level of endogenous PU.1. In certain embodiments, the expression level of PU.1 in a cell-reprogrammable B cell is at least 170 times the expression level of endogenous PU.1. In certain embodiments, the expression level of PU.1 in a cell-reprogrammable B cell is at least 160 times the expression level of endogenous PU.1. In certain embodiments, the expression level of PU.1 in a cell-reprogrammable B cell is at least 150 times the expression level of endogenous PU.1. In certain embodiments, the expression level of PU.1 in a cell-reprogrammable B cell is at least 140 times the expression level of endogenous PU.1. In certain embodiments, the expression level of PU.1 in a cell-reprogrammable B cell is at least 130 times the expression level of endogenous PU.1. In certain embodiments, the expression level of PU.1 in a cell-reprogrammable B cell is at least 120 times the expression level of endogenous PU.1. In certain embodiments, the expression level of PU.1 in a cell-reprogrammable B cell is at least 110 times the expression level of endogenous PU.1. In certain embodiments, the expression level of PU.1 in a cell-reprogrammable B cell is at least 100 times the expression level of endogenous PU.1. In certain embodiments, the expression level of PU.1 in a cell-reprogrammable B cell is at least 90 times the expression level of endogenous PU.1. In certain embodiments, the expression level of PU.1 in a cell-reprogrammable B cell is at least 80 times the expression level of endogenous PU.1. In certain embodiments, the expression level of PU.1 in a cell-reprogrammable B cell is at least 70 times the expression level of endogenous PU.1. In certain embodiments, the expression level of PU.1 in a cell-reprogrammable B cell is at least 60 times the expression level of endogenous PU.1. In certain embodiments, the expression level of PU.1 in a cell-reprogrammable B cell is at least 50 times the expression level of endogenous PU.1. In certain embodiments, the expression level of PU.1 in a cell-reprogrammable B cell is at least 40 times the expression level of endogenous PU.1.In certain embodiments, the expression level of PU.1 in the cell-reprogrammable B cells is at least 30 times the expression level of endogenous PU.1. In certain embodiments, the expression level of PU.1 in the cell-reprogrammable B cells is at least 20 times the expression level of endogenous PU.1. In certain embodiments, the expression level of PU.1 in the cell-reprogrammable B cells is at least 10 times the expression level of endogenous PU.1.

[0028] In certain embodiments, the expression level of the exogenous transcription factor PU.1 is 50-200 times the expression level of endogenous PU.1 in B cells.

[0029] As a preferred embodiment of the method of the present invention, after the exogenous transcription factor PU.1 is introduced into the B cell population in step (1), at least 80% of the B cells are transdifferentiated into macrophages.

[0030] In certain embodiments, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of PU.1-overexpressing B cells are transdifferentiated into macrophages. In certain embodiments, at least 80% of PU.1-overexpressing B cells are transdifferentiated into macrophages. In certain embodiments, at least 85% of PU.1-overexpressing B cells are transdifferentiated into macrophages. In certain embodiments, at least 90% of PU.1-overexpressing B cells are transdifferentiated into macrophages. In certain embodiments, at least 91% of PU.1-overexpressing B cells are transdifferentiated into macrophages. In certain embodiments, at least 92% of PU.1-overexpressing B cells are transdifferentiated into macrophages. In certain embodiments, at least 93% of PU.1-overexpressing B cells are transdifferentiated into macrophages. In certain embodiments, at least 94% of PU.1-overexpressing B cells are transdifferentiated into macrophages. In certain embodiments, at least 95% of PU.1-overexpressing B cells are transdifferentiated into macrophages. In certain embodiments, at least 96% of PU.1-overexpressing B cells are transdifferentiated into macrophages. In certain embodiments, at least 97% of PU.1-overexpressing B cells are transdifferentiated into macrophages. In certain embodiments, at least 98% of PU.1-overexpressing B cells are transdifferentiated into macrophages. In certain embodiments, at least 99% of PU.1-overexpressing B cells are transdifferentiated into macrophages. In certain embodiments, 100% of PU.1-overexpressing B cells are transdifferentiated into macrophages.

[0031] In certain embodiments, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the macrophages reprogrammed from PU.1 overexpressing B cells (REP-M) are CD80 positive. In certain embodiments, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the macrophages reprogrammed from PU.1 overexpressing B cells (REP-M) are CD86 positive. In certain embodiments, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the macrophages reprogrammed from PU.1 overexpressing B cells (REP-M) are CD80 and CD86 positive. In certain embodiments, at least 90% of the reprogrammed macrophages (REP-M) derived from PU.1 overexpressing B cells are CD86 positive. In certain embodiments, at least 90% of the reprogrammed macrophages (REP-M) derived from PU.1 overexpressing B cells are CD80 positive. In certain embodiments, at least 90% of the reprogrammed macrophages (REP-M) derived from PU.1 overexpressing B cells are CD86 and CD80 positive. In certain embodiments, at least 95% of the reprogrammed macrophages (REP-M) derived from PU.1 overexpressing B cells are CD86 positive. In certain embodiments, at least 95% of the reprogrammed macrophages (REP-M) derived from PU.1 overexpressing B cells are CD80 positive. In certain embodiments, at least 95% of the reprogrammed macrophages (REP-M) derived from PU.1 overexpressing B cells are CD86 and CD80 positive. In certain embodiments, 100% of the reprogrammed macrophages (REP-M) derived from PU.1 overexpressing B cells are CD86 positive. In certain embodiments, 100% of reprogrammed macrophages (REP-M) derived from PU.1 overexpressing B cells are CD80 positive. In certain embodiments, 100% of reprogrammed macrophages (REP-M) derived from PU.1 overexpressing B cells are CD86 and CD80 positive.

[0032] As a non-limiting example, the process described herein can transdifferentiate 100% of the B cells in a PU.1 overexpressing reprogrammable B cell population into reprogrammed macrophages (i.e., REP-M), wherein at least 95%, 96%, 97%, 98%, 99% or 100% of the REP-M express CD86 and CD80.

[0033] In certain embodiments, the method transdifferentiates PU.1-overexpressing B cells into macrophages within 1 to 7 days after induction (i.e., the B cells are cultured in a medium containing an inducer (e.g., doxycycline) for 1 to 7 days). In certain embodiments, the method transdifferentiates PU.1-overexpressing B cells into macrophages within 2 to 6 days after induction. In certain embodiments, the method transdifferentiates PU.1-overexpressing B cells into macrophages within 3 to 4 days after induction. In certain embodiments, the method transdifferentiates PU.1-overexpressing B cells into macrophages within 4 to 5 days after induction. In certain embodiments, the method transdifferentiates PU.1-overexpressing B cells into macrophages within 7 days after induction. In certain embodiments, the method transdifferentiates PU.1-overexpressing B cells into macrophages within 6 days after induction. In certain embodiments, the method transdifferentiates PU.1-overexpressing B cells into macrophages within 5 days after induction. In certain embodiments, the method transdifferentiates PU.1-overexpressing B cells into macrophages within 4 days after induction. In certain embodiments, the method transdifferentiates PU.1-overexpressing B cells into macrophages within 3 days of induction. In certain embodiments, the method transdifferentiates PU.1-overexpressing B cells into macrophages within 2 days of induction. In certain embodiments, the method transdifferentiates PU.1-overexpressing B cells into macrophages within 1 day of induction.

[0034] As a preferred embodiment of the method of the present invention, 100% of the B cell population is transdifferentiated into macrophages within 2-7 days.

[0035] As a preferred embodiment of the method of the present invention, 100% of the B cell population is transdifferentiated into macrophages within 5 days.

[0036] In certain embodiments, 100% of the B cell population transdifferentiates into CD80 and CD86 positive macrophages within 5 days.

[0037] According to the methods of the present invention, expression of PU.1 can be inducible. Any known inducible gene expression system can be used to induce expression of PU.1 in B cell populations. In certain non-limiting embodiments, expression of PU.1 is regulated by a doxycycline-inducible overexpression system.

[0038] As a preferred embodiment of the method of the present invention, in step (1), after the exogenous transcription factor PU.1 is introduced into the B cell population, the transdifferentiation of the B cell population into macrophages is induced by doxycycline; the concentration range of the doxycycline used is 0.05-1.0 μg / mL.

[0039] In certain embodiments, the method comprises inducing PU.1 expression in B cells using doxycycline at a concentration ranging from 0.05 μg / mL to 1.0 μg / mL, or from 0.4 μg / mL to 0.8 μg / mL, or from 0.5 μg / mL to 1.0 μg / mL. In certain embodiments, the method comprises inducing PU.1 expression in B cells using doxycycline at a concentration of 0.05 μg / mL, 0.1 μg / mL, 0.15 μg / mL, 0.2 μg / mL, 0.25 μg / mL, 0.3 μg / mL, 0.35 μg / mL, 0.4 μg / mL, 0.45 μg / mL, 0.5 μg / mL, 0.55 μg / mL, 0.6 μg / mL, 0.65 μg / mL, 0.7 μg / mL, 0.75 μg / mL, 0.8 μg / mL, 0.85 μg / mL, 0.9 μg / mL, 0.95 μg / mL, or 1.0 μg / mL.

[0040] As a preferred embodiment of the method of the present invention, after the exogenous transcription factor PU.1 is introduced into the B cell population in step (1), the B cell population is cultured in a cell culture medium containing cytokines during the transdifferentiation process; the cytokines include at least one of IL-3, M-CSF and GM-CSF.

[0041] As a preferred embodiment of the method of the present invention, during the transdifferentiation process, the B cell population is cultured in a cell culture medium containing IL-3 and M-CSF.

[0042] In certain embodiments, the reprogrammable B cells are cultured in a medium containing IL-3 and M-CSF. In some examples, the concentration of IL-3 ranges from 5 ng / mL to 100 ng / mL, or from 10 ng / mL to 50 ng / mL, or from 10 ng / mL to 25 ng / mL, or from 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, or 100 ng / mL. In some examples, the concentration of M-CSF ranges from 5 ng / mL to 100 ng / mL, or 10 ng / mL to 50 ng / mL, or 10 ng / mL to 25 ng / mL, or 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, or 100 ng / mL.

[0043] In a second aspect, the present invention further provides a method for producing a macrophage population, the method comprising:

[0044] Exogenous transcription factors PU.1 and C / EBPα are introduced into a B cell population, thereby inducing the transdifferentiation of the B cell population into macrophages. The present invention provides a method for producing a macrophage population, which primarily involves introducing exogenous transcription factors PU.1 and C / EBPα into a B cell population, thereby inducing the transdifferentiation of the B cell population into macrophages. Low levels of C / EBPα expression can further reduce the concentration of the transdifferentiation inducer. According to this method for producing macrophages, B cell transdifferentiation into macrophages can be induced using a low dose of the inducer.

[0045] As a preferred embodiment of the method of the present invention, the expression level of PU.1 is more than 10 times higher than the expression level of endogenous PU.1 in B cells; the expression level of C / EBPα is more than 0.05 times that of endogenous C / EBPα in M0 polarized macrophages.

[0046] B cells that co-express PU.1 and C / EBPα are reprogrammable. These reprogrammable B cells are designated REPable-B (PA). During the reprogramming process, REPable-B (PA) cells express exogenous PU.1 at a level that is at least 10-fold or higher than the endogenous PU.1 level, for example, 10 to 200-fold higher than the endogenous PU.1 level. During the reprogramming process, reprogrammable B cells express exogenous C / EBPα at a level that is 0.05 to 5-fold higher than the level of C / EBPα expressed in M0-polarized macrophages. The interaction between PU.1 and C / EBPα promotes the progress and efficiency of B cell reprogramming. Without being bound by theory, C / EBPα may induce PU.1 expression. Co-expression of PU.1 and C / EBPα increases the sensitivity of reprogrammable B cells to induction agents. Low doses of induction agents can effectively transdifferentiate reprogrammable B cells into macrophages.

[0047] In certain embodiments, reprogrammable B cells are designed to inducibly express PU.1 and C / EBPα. As a non-limiting example, B cells are engineered to inducibly express PU.1 and C / EBPα using a doxycycline-mediated inducible expression system. In certain embodiments, B cells are simultaneously infected with a viral vector expressing PU.1 and a viral vector expressing C / EBPα to prepare reprogrammable B cells. In certain embodiments, B cells are infected with a viral vector that can simultaneously express PU.1 and C / EBPα, for example, PU.1 and C / EBPα linked by a 2A polypeptide, which are inducible and split into two proteins, PU.1 and C / EBPα, during translation; or PU.1 and C / EBPα linked by an IRES element, which simultaneously express two independent proteins, PU.1 and C / EBPα, during translation; or the expression of the two proteins is controlled by two independent promoter elements in the same viral vector. In other embodiments, the PU.1-overexpressing reprogrammable B cells of the present invention are further infected with a viral vector expressing C / EBPa to prepare PU.1 and C / EBPa-overexpressing B cells.

[0048] In certain embodiments, overexpression of PU.1 and C / EBPa in reprogrammable B cells is achieved using a doxycycline-inducible overexpression system. In certain embodiments, the method comprises inducing expression of PU.1 and C / EBPa in B cells using a low concentration of doxycycline, thereby transdifferentiating the B cells into macrophages.

[0049] As a preferred embodiment of the method of the present invention, doxycycline at a concentration of at least 10 ng / mL is used to induce the transdifferentiation of the B cell population into macrophages.

[0050] In certain embodiments, the method comprises inducing expression of PU.1 and C / EBPa in B cells using doxycycline at a concentration of 10 ng / mL or greater (eg, 10 ng / mL to 200 ng / mL). In certain embodiments, the method comprises inducing expression of PU.1 and C / EBPa in B cells using doxycycline at a concentration of 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, or 200 ng / mL.

[0051] In certain embodiments, the method comprises inducing expression of PU.1 and C / EBPa in the cell-reprogrammable B cells using doxycycline at a concentration of 50 ng / mL.

[0052] In certain embodiments, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of B cells overexpressing PU.1 and C / EBPa are transdifferentiated into macrophages. In certain embodiments, at least 80% of B cells overexpressing PU.1 and C / EBPa are transdifferentiated into macrophages. In certain embodiments, at least 85% of B cells overexpressing PU.1 and C / EBPa are transdifferentiated into macrophages. In certain embodiments, at least 90% of B cells overexpressing PU.1 and C / EBPa are transdifferentiated into macrophages. In certain embodiments, at least 91% of B cells overexpressing PU.1 and C / EBPa are transdifferentiated into macrophages. In certain embodiments, at least 92% of B cells overexpressing PU.1 and C / EBPa are transdifferentiated into macrophages. In certain embodiments, at least 93% of B cells overexpressing PU.1 and C / EBPa are transdifferentiated into macrophages. In certain embodiments, at least 94% of PU.1 and C / EBPa-overexpressing B cells are transdifferentiated into macrophages. In certain embodiments, at least 95% of PU.1 and C / EBPa-overexpressing B cells are transdifferentiated into macrophages. In certain embodiments, at least 96% of PU.1 and C / EBPa-overexpressing B cells are transdifferentiated into macrophages. In certain embodiments, at least 97% of PU.1 and C / EBPa-overexpressing B cells are transdifferentiated into macrophages. In certain embodiments, at least 98% of PU.1 and C / EBPa-overexpressing B cells are transdifferentiated into macrophages. In certain embodiments, at least 99% of PU.1 and C / EBPa-overexpressing B cells are transdifferentiated into macrophages. In certain embodiments, 100% of PU.1 and C / EBPa-overexpressing B cells are transdifferentiated into macrophages.

[0053] In a preferred embodiment of the method of the present invention, at least 80% of the macrophages transdifferentiated from the B cell population are positive for CD86 and CD80. In certain embodiments, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the reprogrammed macrophages derived from PU.1 and C / EBPa-overexpressing B cells (REP-M(PA)) are positive for CD80. In certain embodiments, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the reprogrammed macrophages derived from PU.1 and C / EBPa-overexpressing B cells (REP-M(PA)) are positive for CD86. In certain embodiments, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the reprogrammed macrophages derived from PU.1 and C / EBPa overexpressing B cells (REP-M(PA)) are double positive for CD80 and CD86.

[0054] In certain embodiments, at least 90% of the reprogrammed macrophages derived from PU.1 and C / EBPa-overexpressing B cells (REP-M(PA)) are CD86 positive. In certain embodiments, at least 90% of the reprogrammed macrophages derived from PU.1 and C / EBPa-overexpressing B cells (REP-M(PA)) are CD80 positive. In certain embodiments, at least 90% of the reprogrammed macrophages derived from PU.1 and C / EBPa-overexpressing B cells (REP-M(PA)) are double positive for CD86 and CD80.

[0055] In certain embodiments, at least 95% of the reprogrammed macrophages derived from PU.1 and C / EBPa-overexpressing B cells (REP-M(PA)) are CD86 positive. In certain embodiments, at least 95% of the reprogrammed macrophages derived from PU.1 and C / EBPa-overexpressing B cells (REP-M(PA)) are CD80 positive. In certain embodiments, at least 95% of the reprogrammed macrophages derived from PU.1 and C / EBPa-overexpressing B cells (REP-M(PA)) are double positive for CD86 and CD80.

[0056] In certain embodiments, at least 100% of the reprogrammed macrophages derived from PU.1 and C / EBPa-overexpressing B cells (REP-M(PA)) are CD86 positive. In certain embodiments, at least 100% of the reprogrammed macrophages derived from PU.1 and C / EBPa-overexpressing B cells (REP-M(PA)) are CD80 positive. In certain embodiments, at least 100% of the reprogrammed macrophages derived from PU.1 and C / EBPa-overexpressing B cells (REP-M(PA)) are double positive for CD86 and CD80.

[0057] As a preferred embodiment of the method of the present invention, the macrophages obtained by transdifferentiation of 100% PU.1 and C / EBPa overexpressing B cells are CD86 and CD80 positive.

[0058] In certain embodiments, the method transdifferentiates PU.1 and C / EBPα-overexpressing B cells into macrophages within 1 to 6 days after induction with an inducer (i.e., the B cells are cultured in a medium containing an inducer (e.g., doxycycline) for 1 to 6 days). In certain embodiments, the method transdifferentiates PU.1 and C / EBPα-overexpressing B cells into macrophages within 1 to 5 days after induction with an inducer. In certain embodiments, the method transdifferentiates PU.1 and C / EBPα-overexpressing B cells into macrophages within 2 to 5 days after induction with an inducer. In certain embodiments, the method transdifferentiates PU.1 and C / EBPα-overexpressing B cells into macrophages within 2 to 4 days after induction with an inducer. In certain embodiments, the method transdifferentiates PU.1 and C / EBPα-overexpressing B cells into macrophages within 3 to 4 days after induction with an inducer. In certain embodiments, the method transdifferentiates PU.1 and C / EBPα-overexpressing B cells into macrophages within 3 to 5 days after induction with an inducer. In certain embodiments, the method transdifferentiates PU.1 and C / EBPα-overexpressing B cells into macrophages within 6 days after induction with the inducer. In certain embodiments, the method transdifferentiates PU.1 and C / EBPα-overexpressing B cells into macrophages within 5 days after induction with the inducer. In certain embodiments, the method transdifferentiates PU.1 and C / EBPα-overexpressing B cells into macrophages within 4 days after induction with the inducer. In certain embodiments, the method transdifferentiates PU.1 and C / EBPα-overexpressing B cells into macrophages within 3 days after induction with the inducer. In certain embodiments, the method transdifferentiates PU.1 and C / EBPα-overexpressing B cells into macrophages within 2 days after induction with the inducer. In certain embodiments, the method transdifferentiates PU.1 and C / EBPα-overexpressing B cells into macrophages within 1 day after induction with the inducer.

[0059] As a preferred embodiment of the method of the present invention, the PU.1 and C / EBPα overexpressing B cell population transdifferentiates into macrophages within 2-5 days.

[0060] As a preferred embodiment of the method of the present invention, the PU.1 and C / EBPα overexpressing B cell population transdifferentiates into macrophages within 3 days.

[0061] As a preferred embodiment of the method of the present invention, during the transdifferentiation process, the B cell population is cultured in a cell culture medium containing cytokines; the cytokines include at least one of IL-3, M-CSF and GM-CSF.

[0062] In some embodiments, the cell-reprogrammable B cells expressing PU.1 or PU.1 and C / EBPα are cultured and transdifferentiated into macrophages in the presence of one or more cytokines that support macrophage development and survival (e.g., IL-3, M-CSF (macrophage colony stimulating factor) and / or GM-CSF (granulocyte-macrophage colony stimulating factor)). For example, the cell-reprogrammable B cells are cultured and transdifferentiated into macrophages in the presence of IL-3. In certain embodiments, the cell-reprogrammable B cells are cultured and transdifferentiated into macrophages in the presence of M-CSF. In certain embodiments, the cell-reprogrammable B cells are cultured and transdifferentiated into macrophages in the presence of GM-CSF. In certain embodiments, the cell-reprogrammable B cells are cultured and transdifferentiated into macrophages in the presence of IL-3 and M-CSF. In certain embodiments, the cell-reprogrammable B cells are cultured and transdifferentiated into macrophages in the presence of IL-3 and GM-CSF. In certain embodiments, the cell-reprogrammable B cells are cultured in the presence of M-CSF and GM-CSF and transdifferentiated into macrophages. In certain embodiments, the cell-reprogrammable B cells are cultured in the presence of IL-3, M-CSF, and GM-CSF and transdifferentiated into macrophages.

[0063] As a preferred embodiment of the method of the present invention, during the transdifferentiation process, the B cell population is cultured in a cell culture medium containing IL-3 and M-CSF.

[0064] In a third aspect, the present invention further provides a method for producing a macrophage population, the method comprising:

[0065] The B cell population is engineered to express at least one of the cytokines IL-3, M-CSF, and GM-CSF, and the engineered B cell population is then induced to transdifferentiate into macrophages.

[0066] Another method for producing macrophage populations provided herein comprises engineering a B cell population to express at least one of the cytokines IL-3, M-CSF, and GM-CSF; then inducing the engineered B cell population to transdifferentiate into macrophages. In this method, the cytokine-expressing B cells are further engineered to express one or any combination of the exogenous transcription factors PU.1, C / EBPα, C / EBPβ, C / EBPδ, and C / EBPε. This method further optimizes the transdifferentiation process and eliminates the need for cytokines during cell culture.

[0067] In certain embodiments, the cell-reprogrammable B cells of the present invention are further engineered to express one or more cytokines selected from IL-3, M-CSF, and GM-CSF. Such cell-reprogrammable B cells express one or more cytokines essential for macrophage survival and can transdifferentiate into macrophages without the need for the addition of cytokines to the culture medium. In some embodiments, the cell-reprogrammable B cells of the present invention are engineered to express IL-3. In some embodiments, the cell-reprogrammable B cells of the present invention are engineered to express M-CSF. In some embodiments, the cell-reprogrammable B cells of the present invention are engineered to express GM-CSF. In some embodiments, the cell-reprogrammable B cells of the present invention are engineered to express IL-3 and M-CSF. In some embodiments, the cell-reprogrammable B cells of the present invention are engineered to express IL-3 and GM-CSF. In some embodiments, the cell-reprogrammable B cells of the present invention are engineered to express M-CSF and GM-CSF. In some embodiments, the cell-reprogrammable B cells of the present invention are engineered to express IL-3, M-CSF, and GM-CSF.

[0068] As a preferred embodiment of the method of the present invention, the B cell population is engineered to express IL-3 and M-CSF.

[0069] As a preferred embodiment of the method of the present invention, the B cell population is engineered to express IL-13, M-CSF and GM-CSF.

[0070] As a preferred embodiment of the method of the present invention, the B cell population is further engineered to express any one of the following groups of transcription factors (i)-(x):

[0071] (i) transcription factor PU.1;

[0072] (ii) transcription factor C / EBPα;

[0073] (iii) transcription factors PU.1 and C / EBPα;

[0074] (iv) transcription factors PU.1 and C / EBPβ;

[0075] (v) transcription factors C / EBPα and C / EBPβ;

[0076] (vi) transcription factors PU.1, C / EBPα, and C / EBPβ;

[0077] (vii) transcription factor C / EBPδ;

[0078] (viii) transcription factor C / EBPε;

[0079] (ix) transcription factors PU.1 and C / EBPδ;

[0080] (x) Transcription factors PU.1 and C / EBPε.

[0081] In some embodiments, the method for producing macrophages comprises introducing the transcription factors PU.1 and C / EBPα into cytokine-expressing B cells, and inducing the transdifferentiation of these B cells into macrophages. In some embodiments, the method for producing macrophages comprises introducing the transcription factors PU.1 and C / EBPβ into cytokine-expressing B cells, and inducing the transdifferentiation of these B cells into macrophages. In some embodiments, the method for producing macrophages comprises introducing the transcription factors PU.1 and C / EBPδ into cytokine-expressing B cells, and inducing the transdifferentiation of these B cells into macrophages. In some embodiments, the method for producing macrophages comprises introducing the transcription factors PU.1 and C / EBPε into cytokine-expressing B cells, and inducing the transdifferentiation of these B cells into macrophages.

[0082] Those skilled in the art will appreciate that the cell-reprogrammable B cells can be engineered to express the transcription factors and cytokines described herein in any possible combination. As a non-limiting example, the cell-reprogrammable B cells described herein for producing macrophages can express one of the following combinations: PU.1 (high level) and IL-3; PU.1 (high level) and M-CSF; PU.1 (high level) and GM-CSF; PU.1 (high level), IL-3 and M-CSF; PU.1 (high level), IL-3 and GM-CSF; PU.1 (high level), M-CSF and GM-CSF; PU.1 (high level), IL-3 , M-CSF and GM-CSF; PU.1, C / EBPα and IL-3; PU.1, C / EBPα and M-CSF; PU.1, C / EBPα and GM-CSF; PU.1, C / EBPα, IL-3 and M-CSF; PU.1, C / EBPα, IL-3 and GM-CSF; PU.1, C / EBPα, M-CSF and GM-CSF; or PU.1, C / EBPα, IL-3, M-CSF and GM-CSF. In addition, the reprogrammable B cells used to produce macrophages may also express C / EBPδ or C / EBPε, or PU.1 and C / EBPβ, or PU.1 and C / EBPδ, or PU.1 and C / EBPε and one or more cytokines selected from IL-3, M-CSF and GM-CSF.

[0083] As a preferred embodiment of the method of the present invention, during the transdifferentiation process, the B cell population is cultured in a cell culture medium that does not contain IL-3, M-CSF or GM-CSF.

[0084] As a preferred embodiment of the method of the present invention, at least 80% of the engineered B cell population is transdifferentiated into macrophages.

[0085] As a preferred embodiment of the method of the present invention, 100% of the engineered B cell population is transdifferentiated into macrophages.

[0086] As a preferred embodiment of the method of the present invention, the transdifferentiated macrophages are CD86 and CD80 positive.

[0087] In the method of the present invention, the source of the B cell population is human B cells.

[0088] In some examples, the B cells are from humans (eg, healthy humans or patients).

[0089] As a preferred embodiment of the method of the present invention, the B cell population is derived from primary B cells, B cells of a cell line, or B cells derived from stem cells.

[0090] According to the content of the present invention, B cells from any source can be used to prepare the cell reprogrammable B cells described herein. In certain embodiments, the B cells are from established B cell lines. The B cells can be from any commercialized B cell lines, such as B cells derived from cancer cells. In certain embodiments, the B cells are immortalized B cells.

[0091] In certain embodiments, the B cells are obtained from a blood sample, lymphoid tissue, or hematopoietic tissue (eg, spleen cells or bone marrow cells).

[0092] In certain embodiments, B cells are derived from stem cells (eg, embryonic stem cells and induced pluripotent stem cells (iPSCs) and hematopoietic stem cells).

[0093] In certain embodiments of the methods of the present invention, the B cell population is further engineered to express a therapeutic substance.

[0094] Macrophage-based cell therapy has attracted attention due to its therapeutic effect or potential effect in many diseases. However, it is well known that macrophages are generally difficult to be modified. The cell reprogramming macrophages described herein have a significant advantage in that the cell reprogrammable B cells can be further modified to contain therapeutic substances intended to be introduced into macrophages, and then when the further modified cell reprogrammable B cells are transdifferentiated into macrophages, the cell reprogrammed macrophages (REP-M) will express the same therapeutic substances. Therapeutic substances include but are not limited to chimeric antigen receptors (CARs), immunomodulators, and therapeutic cytokines.

[0095] In certain embodiments of the methods described herein, the B cell population is further engineered to express a chimeric antigen receptor, an immunomodulatory agent, and / or a therapeutic cytokine.

[0096] In certain embodiments of the method of the present invention, the B cells are further modified and the macrophages obtained by inducing transdifferentiation express chimeric antigen receptors, immunomodulators and / or therapeutic cytokines.

[0097] In certain embodiments of the method of the present invention, the B cell population is modified to express a chimeric antigen receptor. The B cell population expressing the chimeric antigen receptor is induced to transdifferentiate into macrophages that express the chimeric antigen receptor.

[0098] In some embodiments, the cell reprogramming B cells of the present invention are further modified to express chimeric antigen receptors, so as to generate macrophages expressing the chimeric antigen receptors. In other embodiments, the reprogrammable B cells of the present invention are further modified to express therapeutic cytokines, so as to generate macrophages expressing the same cytokines. CAR can be expressed in immune cells (such as B cells) using a variety of methods. Retroviral vectors have been established and widely used because they can permanently and genetically express CAR between cell generations because they are integrated into genomic DNA. For example, lentiviral vectors have higher and more stable transgenic expression advantages. In some embodiments, recombinant AAV is used to express chimeric antigen receptors in reprogrammable B cells.

[0099] In one embodiment, the method for generating CAR-expressing macrophages comprises the following steps: i) modifying the cell-reprogrammable B cells of the present invention (e.g., B cells expressing high levels of PU.1 or B cells overexpressing PU.1 and C / EBPα) to express CAR; ii) culturing the CAR-expressing B cells in a culture medium containing doxycycline, IL-3, and M-CSF for 2 to 5 days, thereby transdifferentiating the CAR-expressing B cells into CAR-expressing macrophages.

[0100] In another embodiment, the method for generating CAR-expressing macrophages comprises the following steps: i) modifying the B cells of the present invention (e.g., expressing PU.1 (high level) or PU.1 and C / EBPα, or PU.1 and C / EBPβ, or PU.1 and C / EBPε, and one or more IL-3, M-CSF and GM-CSF) to express CAR; ii) inducing B cells expressing CAR to transdifferentiate into macrophages expressing CAR. In some embodiments, the transdifferentiation process occurs in vitro (e.g., in cell culture) or in vivo (e.g., by transplanting B cells expressing CAR into mice and inducing cells to transdifferentiate in vivo).

[0101] Any culture medium suitable for culturing mammalian cells in vitro can be used in the methods described herein. The definition of the term is not limited to any particular culture medium. For example, the definition encompasses growth medium and maintenance medium. Example culture media include DMEM, IMEM, MEM, or RPMI 1640.

[0102] According to the methods of the present invention, expression of the transcription factors and cytokines in reprogrammable B cells is inducible. The inducer (e.g., doxycycline) described herein is only one commonly used induction system. The methods of the present invention are not limited to this one inducible expression system. Any known gene inducible expression system can be used to induce expression of the transcription factors and cytokines in B cell populations.

[0103] According to the present invention, cell reprogrammed macrophages (REP-M) have been irreversibly transdifferentiated into macrophages. Removing inducers (such as doxycycline) from the culture medium does not affect the fate of the macrophages. Cell reprogrammed macrophages (REP-M) exhibit stable polarization characteristics and other characteristics of macrophages, such as the phagocytic function of macrophages. In some embodiments, cell reprogrammed macrophages can maintain a macrophage state for a long time in the absence of cytokines and inducers (such as doxycycline), for example, at least 3-5 weeks. In some embodiments, the process and method for producing macrophages described in this specification further include treating the transdifferentiated cell preparation with radiation to remove any possible residual B cells that have not undergone transdifferentiation. The present invention has demonstrated that appropriate doses of radiation irradiation can effectively kill B cells, but will not affect the viability and activity of cell reprogrammed macrophages.

[0104] Methods for generating macrophages in vivo:

[0105] The method described in this specification can be used to generate macrophages in vitro, for example in cell culture. In addition, the transdifferentiation method described in this specification can also be used to generate macrophages in vivo. In some embodiments, the method for generating macrophages by cell reprogramming in vivo comprises the following steps: i) transplanting the cell reprogrammable B cells described in this specification into a mammal; ii) inducing the cell reprogrammable B cells to transdifferentiate into macrophages by administering an inducer (e.g., doxycycline) to the mammal. The dosage range of doxycycline is 10 to 200 mg / kg (body weight), for example, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 150 mg / kg and 200 mg / kg. In one example, the dosage of doxycycline is 100 mg / kg. Doxycycline is administered once a day by intraperitoneal or intravenous injection. In some cases, doxycycline can also be given by adding it to food or drinking water.

[0106] As a preferred embodiment of the method of the present invention, the method for producing macrophages comprises the following steps: i) infecting B cells with a viral vector expressing PU.1; optionally, repeatedly infecting B cells with a virus expressing PU.1 until PU.1 expression in the B cells reaches a high level; ii) culturing the infected B cells (i.e., reprogrammable B cells) in a medium containing doxycycline, thereby transdifferentiating the reprogrammable PU.1-overexpressing B cells into macrophages. Optionally, one or more selected cytokines, such as IL-3, M-CSF, and GM-CSF, may also be added to the culture medium.

[0107] As a preferred embodiment of the method of the present invention, the method for producing macrophages comprises the following steps: i) infecting B cells with an inducible viral vector expressing PU.1 and an inducible viral vector expressing C / EBPα; alternatively, infecting reprogrammable PU.1-overexpressing B cells with an inducible viral vector expressing C / EBPα to generate reprogrammable PU.1 and C / EBPα-overexpressing B cells; ii) culturing these reprogrammable PU.1 and C / EBPα-overexpressing B cells in a culture medium containing 50 ng / mL of doxycycline, thereby transdifferentiating the PU.1 and C / EBPα-overexpressing B cells into macrophages. Optionally, one or more selected cytokines, such as IL-3, M-CSF, and GM-CSF, may be added to the culture medium. Within 2 to 5 days, all B cells are transdifferentiated into macrophages.

[0108] Macrophages and their applications

[0109] In a fourth aspect, the present invention provides a macrophage population produced by the method of the present invention.

[0110] First, the macrophages obtained by B cell transdifferentiation provided by the present invention are irreversible and permanently possess the characteristics of macrophages.

[0111] The macrophages generated using the method of the present invention have the activity and function of natural macrophages. The present invention has demonstrated that cell reprogramming-macrophages (REP-M) are similar to primary macrophages, can perform phagocytosis, and can be activated when in contact with cancer cells, and have the function of killing cancer cells or inhibiting cancer cells in vitro and in vivo. In particular, cell reprogramming macrophages (REP-M) expressing chimeric antigen receptors (CARs) can engulf / gnaw cancer cells expressing antigens recognized by CARs, secrete cytotoxic factors to kill cancer cells and inhibit the progression of tumors in animals (such as mice).

[0112] As a preferred embodiment of the macrophage population of the present invention, the polarization state of the macrophage population can be stably maintained.

[0113] The macrophage population can stably maintain CD80 and CD86 positivity.

[0114] Reprogrammed macrophages can stably maintain their macrophage characteristics after the inducing agent is removed. Furthermore, after the withdrawal of the inducer, the macrophage identity is controlled by endogenous genes, allowing them to be further induced into macrophages with different characteristics to suit different application scenarios.

[0115] As a preferred embodiment of the macrophage population of the present invention, the macrophage population does not have proliferation ability.

[0116] As a preferred embodiment of the macrophage population of the present invention, the macrophage population has the same function as primary macrophages.

[0117] Cell-reprogrammed macrophages have the functions of normal macrophages and therefore have broad application prospects in biomedical research, biopharmaceutical development, and clinical treatment. Cell-reprogrammed macrophages have significant advantages in application scenarios where large cell quantities are required and cells need to be genetically modified, such as in macrophage-based immunotherapy, including anti-cancer, anti-inflammatory, and autoimmune disease treatment research and clinical applications, tissue repair and regeneration, and macrophage-based drug delivery. At the same time, human cell-reprogrammed macrophages that self-express human growth factor genes related to maintaining macrophage survival and function can better survive and perform their functions in ordinary severely immunodeficient mice, without the need for recipient mice to simultaneously carry transgenic expression of related growth factors that support macrophage survival function, thereby greatly reducing the cost of experimental animals.

[0118] In a fifth aspect, the present invention provides a composition comprising the macrophage population.

[0119] For example, the present invention provides M1 polarized macrophages and compositions thereof. In some embodiments, the present invention provides CD80 and CD86 positive macrophages and compositions thereof.

[0120] In some embodiments, the present invention provides a composition comprising a population of reprogrammed macrophages. In some embodiments, these macrophages are CD80 and / or CD86 positive. In some embodiments, these macrophages are M1 polarized. In other embodiments, these macrophages are M2 polarized.

[0121] M1 pro-inflammatory macrophages are activated upon exposure to certain molecules, such as lipopolysaccharide (LPS), interferon-gamma (IFN-γ), interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), and binding of toll-like receptors. M1 polarized macrophages are a potent weapon in the immune system used to fight infection. They are able to recognize pathogens directly (through pathogen pattern recognition receptors) or indirectly (through Fc receptors, complement receptors). They also have the ability to produce reactive oxygen species (ROS) to help kill pathogens. In addition, M1 polarized macrophages secrete pro-inflammatory cytokines and chemokines that attract other types of immune cells and integrate / coordinate the immune response. M1 activation is induced by IFN-γ, TNF-α, GM-CSF, LPS, and other toll-like receptor (TLR) ligands.

[0122] In this specification, the term "phagocytosis" generally refers to the process of engulfing cells or large particles (>0.5μm), which includes the attachment of target cells or particles, engulfment, and degradation of internalized target cells or particles. Phagocytosis includes the formation of a phagosome that surrounds the endocytosed target cell or particle, and the fusion of the phagosome with the lysosome to form a phagolysosome, the contents of which are degraded. Macrophages are important cells for phagocytosis. In some embodiments, the macrophages described in the present invention are capable of mediating phagocytosis of dangerous cells (including cancer cells) and pathogens. In other embodiments, the macrophages described in the present invention provide anti-inflammatory molecules. In other embodiments, the macrophages described in the present invention can be modified to provide anti-inflammatory molecules to promote tissue repair.

[0123] In some embodiments, the present invention provides M1 polarized macrophages and compositions thereof. Thus, the cell-reprogrammable B cells of the present invention can be induced to transdifferentiate into M1 polarized macrophages by one or more inducing agents. In other embodiments, the reprogrammed macrophages of the present invention can be polarized into M1 macrophages, for example, under stimulation by cancer cells.

[0124] In a sixth aspect, the present invention provides a genetically engineered macrophage, wherein the macrophage is produced by the following steps:

[0125] (i) Genetically engineered B cells;

[0126] (ii) inducing genetically engineered B cells to transdifferentiate into macrophages; the genetically engineered B cells are induced to undergo cell transdifferentiation by the expression of at least one transcription factor selected from the group consisting of PU.1, CEBPα, and CEBPβ.

[0127] Another advantage of the method of the present invention is the genetic engineering of macrophages. Macrophages are generally difficult to engineer, while B cells are more amenable to genetic engineering. The method of the present invention allows for genetic engineering of B cells before they transdifferentiate into macrophages, thereby producing large quantities of genetically engineered macrophages.

[0128] As a preferred embodiment of the macrophages of the present invention, the B cells are genetically engineered to express chimeric antigen receptors, immunomodulators and / or cytokines.

[0129] As a preferred embodiment of the macrophages of the present invention, the B cells are genetically engineered to express chimeric antigen receptors, thereby transdifferentiating into macrophages expressing chimeric antigen receptors.

[0130] As a preferred embodiment of the macrophages of the present invention, the CAR-expressing macrophages act on cancer cells expressing CAR-targeting antigens and kill cancer cells or inhibit cancer cell proliferation.

[0131] In some embodiments, the compositions described herein include genetically modified macrophages. In some embodiments, these genetically modified macrophages express chimeric antigen receptors. The chimeric antigen receptor comprises an external domain comprising a specific binding region capable of binding to one or more antigens including cancer-associated antigens; a connecting region, a transmembrane domain, and at least one intracellular signaling domain. As used herein, "binding region," "binding domain," and "binding portion" refer to molecules, such as peptides, oligopeptides, polypeptides, or proteins, that are capable of specifically and non-covalently binding, associating, binding, recognizing, or combining with a target molecule (such as a cancer-associated antigen). The binding domain of a chimeric antigen receptor includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced biological molecule or other binding partner for a target.

[0132] In some embodiments, the antigen binding domain is an antibody or an antigen binding fragment thereof. The antibody can be a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, an antibody containing only a heavy chain, a VHH (variant single domain antibody) and any fragment thereof. Non-limiting examples of antigen binding domains suitable for chimeric polypeptides in the present invention include antigen binding fragments (Fab), single-chain variable fragments (scFv), nanobodies, VH domains, VL domains, single domain antibodies (sdAb), VNAR domains, VHH, bispecific antibodies, diabodies or any functional fragments thereof.

[0133] In some embodiments, CAR (chimeric antigen receptor) includes a connecting domain between the extracellular antigen binding domain and the transmembrane domain. In some embodiments, CAR includes a transmembrane domain. The function of the connecting domain and the transmembrane domain is to stably anchor CAR in the cell membrane while providing a certain distance between the membrane and the binding element to allow the effective and activated formation of the CAR / target complex.

[0134] In some embodiments, CAR includes at least one intracellular signaling domain. In some embodiments, CAR includes a costimulatory domain and an intracellular activation domain. In some embodiments, CAR includes three intracellular signaling domains in series, including two costimulatory domains and an intracellular activation domain. In some embodiments, the intracellular signaling domain includes an immunoreceptor tyrosine activation motif (ITAMs). In some embodiments, the intracellular signaling domain is a CD3ζ signaling domain. In other embodiments, the intracellular signaling domain includes other intracellular domains containing ITAM, such as Fc receptor gamma subunit (FcRγ) and multiple epidermal growth factor-like domain protein 10 (Megf10). As a non-limiting example, the macrophages described in the present invention can be modified to express chimeric antigen receptors for phagocytosis, as described in U.S. Patent No. 11,041,023, the entire contents of which are incorporated herein by reference.

[0135] In some embodiments, CAR includes one or more additional signaling domains to enhance phagocytosis. In some embodiments, CAR also includes a signal peptide and / or one or more linkers. In some embodiments, the linker includes a peptide linker sequence. The linker peptide sequence can include 1 to 100 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, etc. amino acid residues). In some embodiments, the length and amino acid composition of the linker peptide sequence can be optimized to adjust the direction and / or proximity between the polypeptide domains and maintain the flexibility of the three-dimensional structure of each domain, thereby achieving the desired activity of the chimeric antigen receptor. In some embodiments, the direction and / or proximity between the polypeptide domains can be used as a "regulation" tool to adjust the biological activity of the chimeric antigen receptor to enhance or reduce its biological activity. In some embodiments, the direction and / or proximity between the polypeptide domains can be optimized to create a partial to complete phagocytic version of the chimeric antigen receptor. In some embodiments, the peptide linker is a GS linker. Examples of GS-containing linkers include (GS)n (n=2-5).

[0136] In some embodiments, the CAR targets a cancer-associated antigen, e.g., CD19, CD22, HER2 (ERBB2 / neu), mesothelin, PSCA, CD123, CD30, CD171, CD138, CS-1, CLECL1, CD33, CD79b, EGFRvIII, GD2, GD3, BCMA, PSMA, ROR1, FLT3, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3 (CD276), KIT (CD117), CD213A2, IL-11Ra, PRSS21, VEGFR2, CD24, MUC-16, PDGFR-beta, SSEA-4, CD20, MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, FAP, EphA2, GM3, TEM1 / CD248, TEM7R, CLDN6, TSHR, GPRC5D, CD97, CD179a, ALK and IGLL1.

[0137] In some embodiments, the macrophages expressed by CAR are M1 polarized macrophages. In some embodiments, the macrophages expressed by CAR are CD86 positive. In some embodiments, the macrophages expressed by CAR are CD80 positive. In some embodiments, the macrophages expressed by CAR are both CD86 positive and CD80 positive.

[0138] In some embodiments, the present invention provides engineered macrophages that express one or more cytokines.

[0139] In some embodiments, the present invention provides engineered macrophages that express an immunomodulatory agent.

[0140] In some embodiments, the engineered macrophages are used in cell therapy.

[0141] In some embodiments, a pharmaceutical composition comprising a macrophage population produced by the method of the present invention is provided, wherein the pharmaceutical composition is formulated for cell therapy.

[0142] In a seventh aspect, the present invention provides a use of the aforementioned macrophages in the preparation of a drug for treating a disease, wherein the use comprises infusing the macrophages into a subject in need thereof.

[0143] Macrophages are a type of antigen presenting cells. Therefore, without being limited by any particular theory, it is conceivable that in various embodiments of the present invention, by directing CAR-expressing macrophages to cancer cells, these antigen presenting cells may internalize and cross-present antigens to T cells, thereby inducing cytotoxic killing of cancer cells by T cells. Macrophages generated using the methods and processes of the present invention (e.g., reprogrammed macrophages) can be used for cancer immunotherapy. For example, the cell reprogrammed macrophages of the present invention, such as CAR-REP-M cells, have shown strong anti-tumor efficacy in both in vivo and in vitro experiments.

[0144] In certain embodiments, the engineered macrophages of the present invention, such as CAR-expressing macrophages, can be used in cell therapy. In certain embodiments, the CAR-expressing macrophages of the present invention can be used to treat solid tumors. Although CAR-T cells are effective in treating hematological malignancies, the lack of effective infiltration of solid tumors is a significant defect of T-cell-based cell therapy. Macrophage-based cell therapy may overcome this limitation because mononuclear macrophages easily enter tumors. Macrophage-based cell therapy strategies can use macrophages as Trojan horses to deliver cytokines or nanoparticles to the tumor microenvironment (TME), or equip macrophages with engineered receptors to enhance the targeted phagocytosis and killing ability of macrophages against tumor cells.

[0145] Macrophages and compositions thereof can be administered to an individual in need thereof by any known cell therapy administration route, including but not limited to intravenous injection and infusion.

[0146] In one embodiment, a method of treating solid cancers using macrophages comprises transdifferentiating cell-reprogrammable B cells into macrophages (ie, cell-reprogrammed macrophages); and administering these cell-reprogrammed macrophages to a subject in need of treatment.

[0147] As a preferred embodiment of the use of the present invention, the macrophages are pre-treated by radiation before being administered to a subject.

[0148] In some cases, the reprogrammed macrophages are pre-treated with radiation before administration to kill any remaining B cells.

[0149] As a preferred embodiment of the use of the present invention, the disease includes cancer, autoimmune disease, infectious disease or inflammatory disease.

[0150] As non-limiting examples, macrophages and compositions thereof can be used as drugs to treat the following cancers: glioblastoma (GBM), breast cancer, brain cancer, lung cancer, liver cancer, stomach cancer, spleen cancer, colon cancer, kidney cancer, pancreatic cancer, prostate cancer, uterine cancer, skin cancer, head and neck cancer, sarcoma, neuroblastoma or ovarian cancer.

[0151] As used herein, "subject" or "individual" includes animals, such as humans (e.g., human subjects) and non-human animals. In certain embodiments, a "subject" or "individual" is a patient under the care of a physician. Thus, a subject can be a patient who has, may have, or is suspected of having a disease (e.g., cancer) and / or one or more symptoms of the disease. A subject can also be an individual diagnosed as being at risk for the disease at the time of diagnosis or later.

[0152] In certain embodiments, reprogrammed macrophages (REP-M) and compositions thereof can be used in combination with other anti-cancer therapies, such as other cell therapies, immunotherapy, chemotherapy, and radiation therapy.

[0153] In certain embodiments, macrophages can be used for drug delivery.

[0154] In certain embodiments, the macrophages described herein are used to treat infectious diseases. Macrophages can sense and migrate to sites of infection, engulfing and killing invading pathogens. Furthermore, as antigen-presenting cells, macrophages can stimulate adaptive immune responses by presenting antigens from engulfed pathogens.

[0155] In certain embodiments, the macrophages described herein are used to treat inflammatory diseases. Inflammatory diseases are diseases in which the immune system mistakenly attacks its own cells or tissues, leading to inflammation.

[0156] As a preferred embodiment of the use of the present invention, the disease is cancer.

[0157] As a preferred embodiment of the use of the present invention, the disease is a solid tumor.

[0158] In an eighth aspect, the present invention provides a cell-reprogrammable B cell for producing macrophages, wherein the cell-reprogrammable B cell is engineered to express at least one group of transcription factors selected from the following (i) to (x):

[0159] (i) exogenous PU.1 that is higher than the expression level of endogenous transcription factor PU.1 in B cells;

[0160] (ii) transcription factor C / EBPα;

[0161] (iii) transcription factors PU.1 and C / EBPα;

[0162] (iv) transcription factors PU.1 and C / EBPβ;

[0163] (v) transcription factors C / EBPα and C / EBPβ;

[0164] (vi) transcription factors PU.1, C / EBPα, and C / EBPβ;

[0165] (vii) transcription factor C / EBPδ;

[0166] (viii) transcription factor C / EBPε;

[0167] (ix) transcription factors PU.1 and C / EBPδ;

[0168] (x) Transcription factors PU.1 and C / EBPε.

[0169] In this specification, "cell-reprogrammable B cells" refer to genetically modified B cells that can be induced to undergo cell reprogramming to generate macrophages. In certain embodiments, B cells are engineered to overexpress the transcription factor PU.1. In order to achieve efficient transdifferentiation, the overexpression level of PU.1 needs to reach a very high level. For example, after induction, the PU.1 protein level in cell-reprogrammable B cells is at least 10 times higher than the expression level of endogenous PU.1 in B cells, such as 50-200 times. In certain embodiments, cell-reprogrammable B cells are engineered to express high levels of PU.1 and low to moderate levels of C / EBPα (such as 0.05 to 2 times the expression level of endogenous C / EBPα in M0 polarized macrophages).

[0170] In certain embodiments, the B cells of cell reprogramming can be engineered to stably express one or more transcription factors. In other embodiments, the B cells of cell reprogramming can be engineered to transiently express one or more transcription factors. In certain embodiments, the transcription factor is inducible in the B cells of cell reprogramming. As a non-limiting example, the expression of the transcription factor is regulated by an inducible promoter. Any system suitable for mammalian cell inducible gene expression can be used to express the transcription factor of inducible cell reprogramming, including but not limited to: an inducible expression system based on a lactose operator, a doxycycline inducible expression system, a Cumate inducible expression system, an insect ecdysone inducible expression system, a hormone inducible activation system (such as an estrogen receptor and a glucocorticoid receptor-mediated protein activity activation system), a heat shock protein promoter inducible expression system, and a light-sensitive induction system. Once a B cell line capable of cell reprogramming is established, by inducing the expression of transcription factors, the B cells capable of cell reprogramming can be transdifferentiated into macrophages.

[0171] As a preferred embodiment of the cell-reprogrammable B cells of the present invention, the cell-reprogrammable B cells are further engineered to express at least one cytokine among IL-3, M-CSF and GM-CSF. In some embodiments, cell-reprogrammable B cells that simultaneously express one or more cytokines (such as IL-3, M-CSF and GM-CSF) are used to generate macrophages in vivo. Cell reprogramming-macrophages that can secrete growth factors required for their own survival can survive and perform their normal functions in severely immunodeficient mice without human growth factor transgenes, reducing the dependence on expensive human growth factor transgenic severely immunodeficient mice in research.

[0172] As a preferred embodiment of the cell-reprogrammable B cells of the present invention, the cell-reprogrammable B cells are further engineered to express IL-3 and M-CSF. As a non-limiting example, the cell-reprogrammable B cells provided herein express exogenous PU.1, IL-3, and M-CSF at levels higher than the endogenous expression level of the B cell transcription factor PU.1. In some embodiments, the cell-reprogrammable B cells provided herein express the transcription factors PU.1, C / EBPα, IL-3, and M-CSF.

[0173] As a preferred embodiment of the cell-reprogrammable B cells of the present invention, the reprogrammable B cells can be induced to reprogram into macrophages; the macrophages are CD86 and CD80 positive.

[0174] In some embodiments, the reprogrammable B cells are used to generate macrophages. In some embodiments, the reprogrammable B cells are used to generate CD86 + CD80 + Macrophages. In other embodiments, the reprogrammable B cells are used to generate M2 polarized macrophages. In some embodiments, the reprogrammable B cells are used to prepare genetically modified macrophages, such as macrophages expressing CAR.

[0175] In a ninth aspect, the present invention provides a component kit for producing macrophages, comprising the cell-reprogrammable B cells and an inducer for inducing transcription factor expression.

[0176] As a preferred embodiment of the kit of parts of the present invention, the inducer is doxycycline. The inducer is used to induce the expression of transcription factors in reprogrammable B cells.

[0177] As a preferred embodiment of the component kit of the present invention, the component kit further comprises a cell culture medium and a cytokine; the cytokine is at least one of IL-3, M-CSF and GM-CSF.

[0178] The beneficial effects of the present invention are:

[0179] The present invention provides a novel cell reprogrammed-macrophage (REP-M) production technology. This new technology utilizes immortalized suspension cultured cells, such as immortalized suspension cultured B cells, as starting cells, and reprograms them to produce macrophages by introducing exogenous transcription factors. Immortalized suspension cultured cells (such as B-cell acute lymphoblastic leukemia cells) used as starting cells for cell reprogramming can be expanded indefinitely on a large scale, thus enabling the large-scale production of macrophages.

[0180] Immortalized suspension-cultured cells with proliferative capacity, such as B-cell acute lymphoblastic leukemia cells, are easily genetically manipulated. Through transgenic technology, B cells can be manipulated to express autocrine growth factor genes essential for macrophage differentiation and survival. Therefore, during the B-cell reprogramming process and after the cells are reprogrammed to generate macrophages, there is no need to add exogenous growth factors to the cell culture medium, significantly reducing the cost of macrophage production.

[0181] On the other hand, the method of the present invention can rapidly (eg, 3-6 days) and efficiently (100%) prepare macrophages by reprogramming B cells, greatly shortening the macrophage preparation cycle.

[0182] The cell reprogramming process is a process of changing cell fate. In the process of B cells being reprogrammed to generate REP-M, the cells not only acquire the functions of normal macrophages, but also lose their proliferation ability (one of the characteristics of macrophages), and are safe for clinical use.

[0183] Furthermore, the present invention demonstrates that B cells with cell reprogramming capabilities (REPable-B) used to prepare macrophages can be completely killed by a certain dose of radiation. However, the same dose of radiation has minimal effect on the function of the reprogrammed-macrophages. For example, radiation exposure has a very limited effect on the phagocytic ability of chimeric antigen receptor-reprogrammed-macrophages (CAR-REP-M) to phagocytose cancer antigen-positive cancer cells, with only a slight difference compared to unirradiated CAR-REP-M cells. This further demonstrates that REP-M is safe for clinical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0184] Figure 1 Flow chart for the preparation of reprogrammed macrophages (REP-M).

[0185] Figure 2 The graph shows the average GFP fluorescence intensity detection results of reprogrammable B cells that overexpress PU.1 alone.

[0186] Figure 3 The expression level of PU.1 protein in reprogrammable B cells that overexpress PU.1 was detected after treatment with doxycycline for different time periods (left), and the results were analyzed by grayscale scanning (right).

[0187] Figure 4 This is an analysis of the process of cell transdifferentiation induced by 1 μg / mL doxycycline in reprogrammable B cells that overexpress PU.1.

[0188] Figure 5 Flow cytometric analysis results of macrophage surface marker genes CD80 and CD86 produced by induced cell reprogramming of B cells obtained by overexpressing different driving transcription factors (PU.1 or C / EBPα).

[0189] Figure 6 The results show the response of reprogrammable B cells that overexpress PU.1 to different concentrations of doxycycline to induce cell reprogramming.

[0190] Figure 7 Flow cytometric sorting strategy for the preparation of reprogrammable B cells co-expressing PU.1 and C / EBPα.

[0191] Figure 8 Flow cytometry was used to analyze the sensitivity of reprogrammable B cells co-expressing PU.1 and C / EBPα to cell reprogramming induced by different concentrations of doxycycline.

[0192] Figure 9 These are the flow cytometry analysis results of cell reprogramming macrophage surface marker genes CD80 and CD86 generated by induced cell transdifferentiation of cell reprogrammable B cells overexpressing PU.1, C / EBPα, and co-expressing PU.1 and C / EBPα.

[0193] Figure 10 To construct reprogrammable-B cells (named: REPable-B (PA / CKs)) that can autosecrete IL-3 and M-CSF; Figure A shows the REPable-B (PA / CKs) cell line expressing human IL-3 and M-CSF obtained by flow cytometry sorting; Figure B shows the cell reprogramming process of REPable-B (PA / CKs) cells detected by flow cytometry after treatment with 50 ng / mL doxycycline without the addition of exogenous growth factors.

[0194] Figure 11Figure 2 shows the results of REP-M (PA / CKs) cells maintaining a macrophage state after doxycycline withdrawal. Figure A shows the results of flow cytometric analysis of the transdifferentiation status of REPable-B (PA / CKs) cells after induction of cell reprogramming with 50 ng / mL doxycycline, withdrawal of doxycycline after 1, 2, 3, 4, 5, and 6 days of induction, and continued culture in doxycycline-free medium for 7 days. Figure B shows the results of flow cytometric analysis of the transdifferentiation status of REPable-B (PA / CKs) cells after induction of cell reprogramming with 200 ng / mL doxycycline for 10 days, withdrawal of doxycycline from the culture medium, and continued culture for 4 and 10 days.

[0195] Figure 12 Figure 2 shows the results of REPable-B (PA / CKs) cells undergoing in vivo reprogramming to generate macrophages under the induction of doxycycline. Figure A shows the results of flow cytometry detection of the identity of REPable-B (PA / CKs) cells in the blood after mice were treated with PBS or 100 mg / kg doxycycline for 3 days, and then blood was collected from the cheek of the mice. Figure B shows the results of flow cytometry detection of the identity of REPable-B (PA / CKs) cells in the blood after 6 consecutive days of doxycycline treatment. The mice were sacrificed on the third day after the last doxycycline injection and the transplanted REPable-B (PA / CKs) cells (GFP + )’s identity status, and Figure C is the survival curve of REPable-B (PA / CKs) cell transplant recipient mice.

[0196] Figure 13 Comparative transcriptome analysis of macrophages differentiated from REP-M, CAR-REP-M, PMBMC, and iPSC.

[0197] Figure 14 This figure shows the results of the gradual loss of proliferation ability of REPable-B (PA) cells during reprogramming.

[0198] Figure 15 The preparation process of chimeric antigen receptor-cellular reprogramming-macrophages (CAR-REP-M).

[0199] Figure 16 The graph shows the results of CAR (EGFRVIII)-REP-M (PA / CKs) cells phagocytizing EGFRVIII-positive tumor cells.

[0200] Figure 17 For Figure 16 Laser confocal imaging results of cells sorted in the cytosol.

[0201] Figure 18Figure 1 is a graph showing the ability of CAR-REP-M cells to kill tumor cells; Figure A shows the results of CAR (EGFRVIII)-REP-M (PA / CKs) or CON-REP-M (PA / CKs) cells co-cultured with K562-EGFRVIII cells for 2 days, after which cells were collected and stained with Annexin V / DAPI, and apoptosis of cancer cells was detected by flow cytometry (the ratio of REP-M cells to cancer cells in the co-culture system was 10:1). Figure B is a bar graph showing the premature apoptosis of cancer cells in Figure A (Annex V + DAPI - ) and all apoptotic cancer cells (Annex V + )data.

[0202] Figure 19 Figure 1 is the result of activating CAR(HER2)-REP-M(PA / CKs) cells to express M1 polarized macrophage growth factor by co-culture with cancer cells; Figure A shows the GFP sorting after CAR(HER2)-REP-M(PA / CKs) cells were co-cultured with SW480-MiSi cells (expressing HER2) for 24 hours. + RFP - Cells (cells that did not undergo phagocytosis), total RNA was extracted from the sorted cells, and the results of quantitative PCR analysis were performed after reverse transcription; Figure B is the transcriptome sequencing comparison of CAR (EGFRVIII)-REP-M (PA / CKs) cells co-cultured with K562-EGFRVIII (flow cytometry sorting GFP + RFP - Transcriptome differences between CAR(EGFRVIII)-REP-M(PA / CKs) cells that were not co-cultured with cancer cells. The vertical axis represents the fold increase in the expression of M1 polarized macrophage-specific cytokines (IL-1β, IL-6, TNFα) after co-culture compared with the expression of the same genes in cells without co-culture.

[0203] Figure 20 Figure 3 is a graph showing the results of CAR(EGFRVIII)-REP-M(PA / CKs) cell elimination of co-cultured K562-EGFRVIII cells; Figure A shows the results of flow cytometry analysis of the proportion of surviving cancer cells after CAR(EGFRVIII)-REP-M(PA / CKs) or CON-REP-M(PA / CKs) cells were co-cultured with K562-EGFRVIII-LUC cells at a ratio of 10:1 for 3 days; Figure B is a bar graph analysis of the proportion of surviving cancer cells in Figure A.

[0204] Figure 21Figure 2 shows the results of CAR(EGFRVIII)-REP-M(PA / CKs) cell elimination of A549-EGFRVIII cells in the co-culture system; Figure A shows the results of flow cytometry detection of the proportion of surviving cancer cells after CAR(EGFRVIII)-REP-M(PA / CKs) or CON-REP-M(PA / CKs) cells were co-cultured with A549-EGFRVIII cells at an ET ratio of 30:1 for 3 days; Figure B is a bar graph analysis of the proportion of surviving cancer cells in Figure A.

[0205] Figure 22 Figure 2 shows the results of CAR(HER2)-REP-M(PA / CKs) cells eliminating SW480 cells in co-culture; Figure A shows the results of flow cytometry detection of the proportion of surviving cancer cells after CAR(HER2)-REP-M(PA / CKs) or CON-REP-M(PA / CKs) cells were co-cultured with SW480-Misi colorectal cancer cells (HER2-positive) at different effector-target ratios of 5:1, 10:1, or 30:1 for 24 hours; Figure B is a bar graph analysis of the proportion of surviving cancer cells in Figure A.

[0206] Figure 23 The figure shows the results of CAR (EGFRVIII)-REP-M (PA / CKs) cells suppressing A549-EGFRVIII tumor growth in vivo.

[0207] Figure 24 This figure shows the effect of radiation on the ability of CAR-REP-M (PA / CKs) cells to phagocytose cancer cells. DETAILED DESCRIPTION

[0208] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0209] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. In some cases, commonly understood terms are defined herein for clarity and / or ease of reference, and the connotations of these definitions should not be construed as being substantially different from those commonly understood in the art. Many of the techniques and procedures described or cited in this specification are widely understood and commonly employed by those skilled in the art using conventional methods. In addition, it should be understood that the terms used in this specification are intended to describe specific embodiments only and are not intended to be limiting.

[0210] Although the present invention is explained in more detail through the following examples, these are only illustrative and do not limit the present invention.

[0211] Example 1: Expression vector preparation

[0212] In this example, expression vectors were constructed (the expression vectors used in this example were all lentiviral or retroviral vectors) for subsequent cell line construction and experimental exploration.

[0213] (1) L237 pCW57-hPU.1-P2A-MCS-hPGK-GFP expression vector:

[0214] The pCW57-MCS1-P2A-MCS2(GFP) vector was obtained from Addgene (#80924). The human PU.1 coding sequence was amplified by PCR using cDNA from human peripheral blood leukocytes as a template. NheI / AgeI restriction sites were added to the amplification primers. The PCR product and the pCW57-MCS1-P2A-MCS2(GFP) vector (Addgene, #80924) were digested with NheI / AgeI and then ligated using ligase to generate L237 pCW57-hPU.1-P2A-MCS-hPGK-GFP.

[0215] (2) L541 pCW57-CEBPA-CMV-mCD19 expression vector:

[0216] The pCW57-MCS1-P2A-MCS(GFP) (Addgene #80924) vector was digested with AgeI / MluI and blunt-ended at both ends. The vector was then ligated with the CEBPA coding sequence amplified by PCR using a blunt-end ligation to generate pCW57-MCS1-P2A-CEBPA(GFP). The CMV promoter sequence was obtained by PCR amplification. The pCW57-MCS1-P2A-CEBPA(GFP) plasmid was digested with MluI / MauBI to remove the PGK promoter sequence and ligated with the MluI / MauBI-digested PCR product to generate pCW57-MCS1-P2A-CEBPA(GFP). CMV-mCD19 was generated by PCR amplification and the PGK-GFP element was replaced with pCW57-MCS1-P2A-CEBPA(GFP) using a Gibson ligation to generate L541pCW57-CEBPA-CMV-mCD19.

[0217] (3) L560 MimCD5-hIL-3 expression vector:

[0218] The MSCV-IRES-GFP (MIG) retroviral vector was opened with NcoI / SalI endonucleases to remove the GFP sequence. The mouse mCD5 coding sequence, containing only the extracellular and transmembrane coding sequences (excluding the intracellular domain), was amplified by PCR, using primers containing NcoI / SalI cleavage sites. The PCR primers were digested with NcoI / SalI and then ligated with the MSCV-IRES-GFP fragment, which had been digested with NcoI / SalI to remove the GFP sequence, to create the MSCV-IRES-mCD5 (MimCD5) vector. The DNA sequence encoding human interleukin-3 (IL-3) was synthesized by Beijing Qingke Biotechnology Co., Ltd. (DNA sequence referenced NM_00588) and flanked by BglII / XhoI restriction sites. The IL-3 sequence was digested with BglII / XhoI and ligated into the BglII / XhoI sites of MimCD5 to generate L560MimCD5-hIL3.

[0219] (4) L547 MImCD4-M-CSF:

[0220] The DNA sequence encoding human macrophage colony-stimulating factor (M-CSF) was synthesized by Beijing Qingke Biotechnology Co., Ltd. (sequence reference NM_000757.6). The M-CSF sequence was inserted into the BglII / XhoI restriction site region of the MImCD5 vector using Gibson ligation to construct MImCD5-M-CSF. The mouse mCD4 coding sequence, containing only the extracellular and transmembrane domains (excluding the intracellular domain), was synthesized by Beijing Qingke Biotechnology Co., Ltd. The mCD4 coding sequence, excluding the intracellular domain, was amplified using mouse peripheral blood leukocyte cDNA as a template. The MimCD5 vector sequence, excluding the mCD5 sequence, was amplified, and the two PCR products were then ligated using Gibson ligation to create L547 MImCD4-M-CSF.

[0221] (5)MISi:

[0222] The mScarlet-i fragment was amplified by PCR using the pmScarlet-i_C1 plasmid (Addgene #85044). The PCR product was treated with NcoI / PacI endonucleases to create cohesive-end adapters. The MIG (MSCV-IRES-GFP) vector was digested with NocI / PacI to remove the GFP sequence and then ligated with the mScarlet-i sequence to create MiSi.

[0223] (6) L340 MISi-LUC:

[0224] The firefly enzyme gene (LUC) coding sequence was amplified by PCR using the pGL2-Basic plasmid as a template. The PCR product and the MiSi plasmid were digested with BglII / XhoI and ligated to generate L340 MiSi-LUC.

[0225] (7)L504 MSCV-EGFRVIII-P2A-mCherry:

[0226] The EGFR VIII coding sequence was amplified by PCR using cDNA from GL261 (hEGFR VIII-GFP) cells as a template. P2A-mCherry was also amplified by PCR using the LentiCRISPR v2-mCherry (Addgene #99154) plasmid as a template. The MIG plasmid was digested with XhoI / PacI to remove the IRES-GFP sequence, and then the two PCR products were ligated using a Gibson ligation to generate L504MSCV-EGFR VIII-P2A-mCherry.

[0227] (8)L507 MSCV-EGFRVIII-P2A-mCherry-T2A-LUC:

[0228] Primers were designed to amplify LUC-encoding DNA using the L340 Misi-LUC plasmid as a template. A linearized plasmid was prepared by PCR amplification using the L504MSCV-EGFRVIII-P2A-mCherry plasmid as a template. The two PCR products were then ligated by Gibson ligation to generate L507MSCV-EGFRVIII-P2A-mCherry-T2A-LUC.

[0229] (9)MImCD2:

[0230] The MSCV-IRES-GFP retroviral vector was opened with NcoI / SalI endonucleases to remove the GFP sequence. The mouse mCD2 coding sequence, containing only the extracellular and transmembrane coding sequences (excluding the intracellular region), was generated by PCR amplification using primers containing NcoI / SalI endonuclease cleavage sites. The PCR primers were digested with NcoI / SalI and then ligated with the MSCV-IRES-GFP fragment, which had been digested with NcoI / SalI to remove the GFP sequence, to create the MSCV-IRES-mCD2 (MImCD2) vector.

[0231] (10) L510 MImCD2-CAR (HER2):

[0232] The anti-HER2 single-chain chimeric antigen receptor sequence was synthesized by Beijing Qingke Biotechnology Co., Ltd. The DNA sequence was obtained from GenBank: AAB08080.1 and amplified by PCR to generate Gibson junction fragments. The MImCD2 vector was also amplified by PCR to generate Gibson junction fragments. The two fragments were generated by Gibson ligation: L510 MImCD2-CAR (HER2).

[0233] (11)L502 MImCD2-CAR(EGFRVIII):

[0234] The anti-EGFRVIII single-chain chimeric antigen receptor sequence was synthesized by Beijing Qingke Biotechnology Co., Ltd., with BglII / XhoI restriction sites added at both ends. The DNA sequence is referenced to Hum Gene Ther. 2012 Oct;23(10):1043-1053. The MSCV-IRES-GFP retroviral vector was opened with NcoI / SalI endonucleases to remove the GFP sequence. The MImCD2 and EGFRVIII fragments were digested with BglII / XhoI, respectively, and then ligated with T4 DNA ligase to prepare L502 MImCD2-CAR (EGFRVIII).

[0235] The primer sequences and vector nucleotide sequences involved in this example are shown in Table 1.

[0236] Table 1

[0237]

[0238]

[0239]

[0240] Example 2: Construction of PU.1-highly expressed RCH-ACV cell line 501

[0241] 1. RCH-ACV cells (a human acute B-lymphoblastic leukemia cell line) were infected with L237 pCW57-hPU.1-P2A-MCS-hPGK-GFP doxycycline-inducible PU.1-expressing lentivirus (based on the expression vector constructed in Example 1, co-transfected with lentiviral packaging-related gene expression plasmids into 293T cells to prepare the corresponding lentivirus. The preparation of other lentiviruses was similar). The 10% of positive cells with the highest GFP expression were enriched by flow cytometry and named 501 cells.

[0242] 2. The average GFP fluorescence intensity of the 501 cells obtained in the above steps was detected by flow cytometry. The results showed that ( Figure 2), the average GFP fluorescence intensity of 501 cells reached 190 times that of RCH-ACV cells.

[0243] 3.501 cells were induced with 800 ng / mL doxycycline. The cells were harvested after 0, 2, 4, 8, 12, 24, and 48 hours of induction, and the total cell protein was extracted for SDS-PAGE gel electrophoresis and western blotting analysis (PU.1 antibody: Cell Signaling CST2258s, ACTIN antibody: Santa Cruz, SC-47778) to detect the expression of PU.1 protein in cells after doxycycline treatment for different time periods.

[0244] The results are as follows Figure 3 As shown, after 8 hours of doxycycline treatment, the expression level of PU.1 in 501 cells reached more than 70 times that of PU.1 in untreated cells.

[0245] Example 3: Doxycycline induces cell reprogramming

[0246] 1. Set the quantity to 1×10 5 501 cells were cultured in complete medium supplemented with 10 ng / mL IL-3 and 10 ng / mL M-CSF, and 1 μg / mL doxycycline was used to induce cell reprogramming. Cells were collected daily from day 0 to day 6 to analyze the cell reprogramming status by flow cytometry. The results showed that 501 cells were able to complete 100% cell reprogramming within 6 days ( Figure 4 ).

[0247] 2. 501 cells and BlaER cells (RCH-ACV cells infected with a virus expressing the CEBPα-ER fusion protein) were cultured in medium supplemented with 10 ng / mL IL-3 and M-CSF. 501 cells were reprogrammed with 0.4 μg / mL doxycycline, while BlaER cells were reprogrammed with 1 μM β-Est to generate REP-M. After 6 days of induction, the reprogrammed cells were analyzed by flow cytometry for expression of CD80 / CD86, the surface markers of M1-polarized macrophages. Transdifferentiated cells without antibody staining served as a negative control.

[0248] The results are as follows Figure 5 As shown, compared with REP-M cells generated by C / EBPα (BlaER cells)-induced cell reprogramming, REP-M cells reprogrammed from 501 cells can express higher levels of M1 polarized macrophage surface marker genes CD80 and CD86. 501 cells are also named REPable-B(P) cells.

[0249] 3. 501 cells were cultured in culture medium supplemented with 10 ng / mL IL-3 and M-CSF and induced with 0.2, 0.4, 0.8, and 1.0 μg / mL doxycycline for 6 days. After the induction was completed on the 6th day, the efficiency of cell reprogramming was detected by flow cytometry.

[0250] The present invention investigated the induction concentration of doxycycline and found that a certain concentration of doxycycline was required to induce efficient cell reprogramming of 501 cells. When the concentration of doxycycline dropped to 0.8 μg / mL or lower, the efficiency of 501 cell reprogramming to generate macrophages began to decline, and 100% cell reprogramming could not be achieved within 6 days ( Figure 6 ).

[0251] Example 4: Preparation of REPable-B (PA) cells co-expressing PU.1 and C / EBPa

[0252] 1. Infect 501 cells with L541 pCW57-mCEBPAHA-CMV-mCD19 lentivirus. Flow cytometry was used to sort cells with medium to high expression intensity of the viral infection marker gene (mCD19 without the intracellular segment), accounting for about 1 / 4 of the total positive cells (total positive cells 26.8%, cells in the sorting gate 6.37%) ( Figure 7 ), named REPable-B (PA) cells.

[0253] 2. REPable-B (PA) cells obtained in 4.1 were cultured in 12-well plates. 10 ng / mL IL-3 and M-CSF were added to the culture medium. Doxycycline was added to final concentrations of 1 ng / mL, 5 ng / mL, 10 ng / mL, and 50 ng / mL, respectively, in the experimental groups. A control group without doxycycline was designated (induced). Cells were harvested 1, 3, and 5 days after induction for antibody staining and flow cytometry analysis of the reprogramming process and efficiency.

[0254] The results showed that REPable-B (PA) cells could complete 100% cell reprogramming within 5 days under the induction of 50ng / mL doxycycline to generate CD19 - CD11B + macrophages (REP-M), indicating that co-expression of C / EBPα reduces the expression level of PU.1 required for inducing cell reprogramming; in other words, it can increase the sensitivity of B cells to Dox-induced cell reprogramming ( Figure 8 ).

[0255] 3. REPable-B(P) cells (expressing PU.1), REPable-B(PA) cells (co-expressing PU.1 and C / EBPα), and BlaER cells (expressing C / EBPαER) were cultured in a medium containing 10 ng / mL IL-3 and M-CSF. REPable-B(P) and REPable-B(PA) cells were then induced to undergo reprogramming with 0.4 μg / mL doxycycline, while BlaER cells were induced to undergo reprogramming with 1 μM β-Est. Macrophages reprogrammed from REPable-B(P), REPable-B(PA), and BlaER cells were designated REP-M(P), REP-M(PA), and REP-M(A), respectively. Six days after induction, the expression of CD80 / CD86, the surface markers for M1-polarized macrophages, in the reprogrammed cells was assessed by flow cytometry. Transdifferentiated cells without antibody staining served as a negative control.

[0256] The results showed that the expression levels of CD80 and CD86 in REP-M(PA) cells were slightly lower than those in REP-M(P) cells, but much higher than those in macrophages reprogrammed by BlaER( Figure 9 ).

[0257] Example 5: Construction of REPable-B (PA / CKs) cells with the ability to autocrine myeloid cell growth factors

[0258] 1. Preparation of retrovirus expressing secretory human growth factor IL-3 and M-CSF (carrying mCD4 and mCD5 marker genes with intracellular segment deletion): L560 MimCD5-hIL3 and L547 MimCD4-M-CSF were prepared into lentivirus.

[0259] 2. The above-obtained lentivirus was used to infect REPable-B (PA) cells (either simultaneously or separately). The infected cells were sorted twice to enrich the cells expressing both IL-3 (the viral vector carried the extracellular segment of the mouse CD5 gene as a marker) and M-CSF (the viral vector carried the extracellular segment of the mouse CD4 gene as a marker) to 100%, thereby obtaining a REPable-B (PA) cell line expressing autocrine IL-3 and M-CSF, named REPabl-B (PA / CKs) ( Figure 10 A). Flow cytometry analysis of the reprogramming process of REPable-B (PA / CKs) cells treated with 50 ng / mL doxycycline without the addition of exogenous growth factors.

[0260] The results showed that under the induction of 50 ng / mL doxycycline and without the provision of exogenous human growth factors, REPable-B (PA / CKs) cells completed cell reprogramming within 5 days and generated CD19 - CD11B + macrophages, named REP-M (PA / CKs) ( Figure 10 B).

[0261] 3. Induce REPable-B (PA / CKs) cells to undergo cellular reprogramming with 50 ng / mL doxycycline. Doxycycline was withdrawn after 1, 2, 3, 4, 5, and 6 days of induction, and the cells were cultured in doxycycline-free medium for up to 7 days. The cells were then harvested and labeled with CD19 and CD11B antibodies, and flow cytometry was performed to assess transdifferentiation status.

[0262] The results showed that all cells were reprogrammed to CD19 after treatment with 50 ng / mL doxycycline for 5 days or longer. - CD11B + REP-M (PA / CKs), CD19 was still maintained when tested on day 7 - CD11B + Macrophage status ( Figure 11 A).

[0263] 4. Induce REPable-B (PA / CKs) cells to undergo cell reprogramming with 200 ng / mL doxycycline. Remove doxycycline from the culture medium after 10 days and continue culturing for 4 and 10 days. Flow cytometry is used to detect cell transdifferentiation status.

[0264] The results showed that REP-M (PA / CKs) cells could still stably maintain CD19 10 days after doxycycline withdrawal. - CD11B + REP-M (PA / CKs) status ( Figure 11 B).

[0265] Example 6: REPable-B (PA / CKs) cells undergo in vivo cell reprogramming to generate CD19 under doxycycline induction - CD11B + macrophages

[0266] In this example, severely immunodeficient NCG mice without human growth factor transgenes were used as experimental subjects (purchased from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.). REPable-B (PA / CKs) cells successfully transplanted into NCG mice underwent cell reprogramming to produce CD19 under the induction of doxycycline. - CD11B +The experimental process is as follows: REPable-B (PA / CKs) cells (number 5×10 6 ) were transplanted into NCG mice via the tail vein. Starting on day 17 after transplantation (when REPable-B (PA / CKs) cells were detectable in the peripheral blood of mice), PBS or 100 mg / kg doxycycline were injected intraperitoneally once a day.

[0267] 1. After mice were treated with PBS or doxycycline for 3 days, blood was collected from the cheeks of the mice and the status of transplanted REPable-B (PA / CKs) cells in the peripheral blood of the mice was detected by flow cytometry.

[0268] Flow cytometry results showed that all REPable-B (PA / CKs) cells in the doxycycline-treated group had been reprogrammed into CD19 - CD11B + Macrophages, while cells in the PBS-treated group maintained CD19 + CD11B - B cell status ( Figure 12 A).

[0269] 2. After 6 consecutive days of doxycycline treatment, the mice were stopped. On the third day after the last doxycycline injection, the mice were sacrificed and their organs were extracted. The status of REPable-B (PA / CKs) cells infiltrating into different organs was detected by flow cytometry.

[0270] The results showed that the transplanted REPable-B (PA / CKs) cells detected in the bone marrow, spleen, liver and lungs had been reprogrammed into macrophages ( Figure 12 B). Meanwhile, REPable-B (PA / CKs) transplanted mice treated with PBS eventually died due to the overgrowth of REPable-B (PA / CKs) cells ( Figure 12 C). However, no non-reprogrammed REPable-B (PA / CKs) cells were found in the doxycycline-treated NCG mice during the 3-month observation period, indicating that all REPable-B (PA / CKs) cells had undergone transdifferentiation.

[0271] Example 7: The transcriptome of REP-M cells and PBMC-differentiated macrophages are highly similar

[0272] The transcriptome sequencing data of REP-M (PA / CKs) or CAR-REP-M (PA / CKs) cells were compared with the transcriptome data of primary PBMC-differentiated macrophages and iPSC-differentiated macrophages (iMAC) downloaded from the public database (GSE55536).

[0273] Transcriptome data of REP-M (PA / CKs) and CAR-REP-M (PA / CKs) cells, M0, M1, and M2 macrophages derived from PBMCs, and M0, M1, and M2 macrophages derived from human iPSCs were normalized using DESeq2 and corrected for batch effects using SVA. The human macrophage cell line THP-1 was also used as a control in this comparison. Data for PMBCs and iPSC-derived macrophages were obtained from GSE55536 (Circ Res 2015 Jun 19;117(1):17-28).

[0274] The results showed that the transcriptomes of REP-M (PA / CKs) / CAR-REP-M (PA / CKs) cells and primary macrophages were highly similar ( Figure 13 ).

[0275] Example 8: REP-M (PA / CKs) cells lose their proliferation ability during cell reprogramming

[0276] RCH-ACV is a type of B-cell acute lymphoblastic leukemia cell (B-ALL cell) with unlimited proliferation capacity. After transplantation into immunodeficient NCG mice, RCH-ACV can expand and generate B-ALL in the mouse body; mature primary macrophages completely lose their proliferation capacity. This example investigates the changes in the proliferation capacity of REPable-B (PA / CKs) cells during the cell reprogramming process. The method is: 5×10 5 REPable-B (PA / CKs) cells were seeded in 12-well plates and treated with 50 ng / mL doxycycline for 3.5 and 9 days. 5-Ethynyl-2'-deoxyuridine (Edu) was then added to a final concentration of 10 μM. After incubation at 37°C for 1 hour, cells were harvested and stained for Edu. DNA incorporation of Edu was then detected by flow cytometry. Edu is a nucleotide analog that is incorporated into DNA during DNA synthesis in proliferating cells.

[0277] The results are as follows Figure 14As shown, more than 70% of the unreprogrammed REPable-B (PA / CKs) cells stained positive for Edu, indicating a highly proliferative state. By day 3.5 of reprogramming, the proportion of Edu-positive cells decreased significantly. By day 9 of reprogramming, all cells had lost their ability to proliferate, becoming Edu-negative. These results indicate that the proliferation capacity of REPable-B (PA / CKs) cells gradually decreases during reprogramming to generate REP-M (PA / CKs) cells, and that mature REP-M (PA / CKs) cells, generated after complete reprogramming, completely lose their ability to proliferate.

[0278] Example 9: Preparation of CAR-REP-M(PA / CKs) cells by expressing chimeric antigen receptors using REPable-B(PA / CKs) cells

[0279] In this example, the driving transcription factor for cell reprogramming was constructed into a doxycycline-inducible expression lentiviral vector and infected with an immortalized B-cell line to prepare cell reprogrammable-B cells (REPable-B). Then, the REPable-B cells were infected with a retrovirus expressing a chimeric antigen receptor (CAR) to obtain chimeric antigen receptor-cell reprogrammable B cells (CAR-REPable-B). Doxycycline was used to induce transdifferentiation of CAR-REPable-B cells to obtain chimeric antigen receptor-cell reprogramming-macrophages (CAR-REP-M) ( Figure 15 ).

[0280] The preparation method of CAR-REP-M (PA / CKs) cells includes:

[0281] 1. Prepare MImCD2, L510 MImCD2-CAR (HER2), L502 MImCD2-CAR (EGFRVIII), MISi, L340 MiSi-LUC, L504 MSCV-EGFRVIII-P2A-mCherry, and L507 MSCV-EGFRVIII-P2A-mCherry-T2A-LUC expression vectors and prepare them into lentivirus.

[0282] 2. Preparation of CAR-REPable-B (PA / CKs) cells

[0283] Preparation of CAR(HER2)-REPable-B(PA / CKs), CAR(EGFRVIII)-REPable-B(PA / CKs), and CON-REPable-B(PA / CKs) cells:

[0284] REPable-B (PA / CKs) cells were infected with L510 MImCD2-CAR (HER2) virus, and the 10% cells with the highest mCD2 expression were sorted by flow cytometry to obtain CAR (HER2)-REPable-B (PA / CKs) cells.

[0285] REPable-B (PA / CKs) cells were infected with L502 MImCD2-CAR (EGFRVIII) virus and the 10% cells with the highest mCD2 expression were sorted by flow cytometry to obtain CAR (EGFRVIII)-REPable-B (PA / CKs) cells.

[0286] REPable-B (PA / CKs) cells were infected with MImCD2 empty virus and the 10% cells with the highest mCD2 expression were sorted by flow cytometry to obtain CON-REPable-B (PA / CKs) cells.

[0287] 3. Preparation of CAR-REP-M (PA / CKs) cells

[0288] Preparation of CAR(HER2)-REP-M(PA / CKs), CAR(EGFRVIII)-REP-M(PA / CKs), and CON-REP-M(PA / CKs) cells:

[0289] CAR(HER2)-REPable-B(PA / CKs), CAR(EGFRVIII)-REPable-B(PA / CKs), and CON-REPable-B(PA / CKs) cells were induced to reprogram with 50 ng / mL doxycycline. After 6-9 days, 100% of the cells were reprogrammed, resulting in CAR(HER2)-REP-M(PA / CKs), CAR(EGFRVIII)-REP-M(PA / CKs), and CON-REP-M(PA / CKs) cells, respectively.

[0290] 4. Preparation of target antigen-positive tumor cells

[0291] (1) Preparation of K562-EGFRVIII-LUC / K562-MiSi cells:

[0292] K562 cells were infected with L507 MSCV-EGFRVIII-P2A-mCherry-T2A-Luc virus, and the top 10% of cells expressing mCherry were sorted by flow cytometry to obtain K562-EGFRVIII-LUC cells. K562 cells were infected with MISi virus, and the top 10% of cells expressing mScarlet-i were sorted by flow cytometry to obtain K562-MiSi cells.

[0293] (2) Preparation of A549-EGFRVIII-LUC / A549-MiSi cells:

[0294] A549 cells were infected with the L507 MSCV-EGFRVIII-P2A-mCherry-T2A-Luc virus, and the top 10% of cells expressing mCherry were sorted by flow cytometry to obtain A549-EGFRVIII-LUC cells. A549 cells were infected with the MISi virus, and the top 10% of cells expressing mScarlet-i were sorted by flow cytometry to obtain A549-MiSi cells.

[0295] (3) Preparation of SW480-LUC cells:

[0296] SW480 cells were infected with L340 MISi-LUC virus and the 10% cells with the highest mScarlet-i expression were sorted by flow cytometry, which were SW480-LUC cells.

[0297] Example 10: In vitro anticancer function experimental verification of CAR-REP-M (PA / CKs) cells

[0298] This example investigates the ability of CAR-REP-M (PA / CKs) cells to phagocytose, kill, and eliminate cancer cells through in vitro cell experiments.

[0299] 1. CAR-REP-M (PA / CKs) cells efficiently phagocytose cancer cells expressing corresponding tumor antigens

[0300] 6 × 10 cells were seeded per well in a 6-well plate. 5 After the cells adhered to the wall, 2.5×10 6 K562-EGFRVIII-LUC or K562-MiSi cells were co-cultured in a 37°C CO2 incubator for 24 hours. After the co-culture, the cells were collected and the phagocytosis of cancer cells by CAR(EGFRVIII)-REP-M(PA / CKs) cells was analyzed on a flow cytometer, and GFP was sorted. +RFP + Double positive cells (GFP is a CAR (EGFRVIII) -REP-M (PA / CKs) cell marker gene, RFP (mCherry or mScarleti) is a cancer cell marker gene). + RFP + Double positive cells can be distinguished as GFP + CAR(EGFRVIII)-REP-M(PA / CKs) cells phagocytized RFP + The cancer cells or CAR (EGFRVIII) -REP-M (PA / CKs) cells only adhered to the cancer cells, and the sorted cells were imaged by laser confocal microscopy. During the flow cytometry sorting process, cells are encapsulated in tiny droplets and collide with the liquid in the cell collection tube during the sorting process. When the collision occurs, the two adhered cells will separate. + If the cancer cells are engulfed by CAR(EGFRVIII)-REP-M(PA / CKs) cells, cell separation will not occur when the droplets encapsulating the cells hit the liquid in the collection tube.

[0301] The analysis results are as follows Figure 16 As shown, the proportion of double-positive cells in the group co-cultured with K562-EGFRVIII-LUC was significantly higher than that in the group co-cultured with K562-MiSi cells (which do not express the cancer antigen EGFRVIII). At the same time, when the cells after flow cytometry sorting were analyzed again by flow cytometry, GFP expression was observed in the group co-cultured with K562-EGFRVIII-LUC. + RFP + Most of the double-positive cells maintained the double-positive status after sorting, while only a small part of the cells in the co-culture group with K562-MiSi maintained the double-positive status after sorting.

[0302] The sorted cells were seeded into a cell culture plate suitable for laser confocal imaging. After the cells adhered to the wall, they were imaged using a Zeiss LSM-800 laser confocal microscope. The results showed that CAR (EGFRVIII)-REP-M (PA / CKs) cells (green) indeed phagocytosed EGFRVIII-positive tumor cells - K562-EGFRVIII-LUC cells ( Figure 17 ).

[0303] 2. CAR-REP-M (PA / CKs) cells kill co-cultured tumor cells expressing corresponding tumor antigens

[0304] (1) 5×10 5CAR (EGFRVIII)-REP-M (PA / CKs) or CON-REP-M (PA / CKs) cells were added to each well after the cells adhered to the wall. 4 K562-EGFRVIII-LUC cells were co-cultured in a 37°C CO2 incubator for 24 hours. After the co-culture, cells were collected and stained with Annexin V, and apoptosis was detected by flow cytometry.

[0305] The results showed that the proportion of cancer cells undergoing apoptosis in co-culture with CAR (EGFRVIII) -REP-M (PA / CKs) cells was significantly higher than that in co-culture with CON-REP-M (CKs) cells, suggesting that REP-CAR-M secretes cytokines with cancer cell killing ability during co-culture with cancer cells ( Figure 18 ).

[0306] (2) CAR(HER2)-REP-M(PA / CKs) cells were co-cultured with SW480-MiSi cells (expressing HER2) for 24 hours and GFP was sorted + RFP - Total RNA was extracted from the sorted cells and reverse transcribed before quantitative PCR analysis of the expression of three M1-polarized macrophage-specific growth factors: IL-1β, IL-6, and TNF-α. CAR(HER2)-REP-M(PA / CKs) cells (0 h) not co-cultured with cancer cells were used as a control.

[0307] RNAseq comparison of CAR(EGFRVIII)-REP-M(PA / CKs) cells co-cultured with K562-EGFRVIII (flow cytometry sorting of GFP + RFP - Transcriptome differences between CAR(EGFRVIII)-REP-M(PA / CKs) cells that were not co-cultured with cancer cells.

[0308] The results showed that co-culture with cancer cells enhanced the expression levels of M1 macrophage growth factors such as TNF-α, IL-1β and IL-6 in CAR-REP-M (PA / CKs) cells, which may be related to the killing of cancer cells ( Figure 19 ).

[0309] 3. CAR-REP-M cells eliminate co-cultured cancer cells expressing corresponding tumor antigens

[0310] (1) CAR(EGFRVIII)-REP-M(PA / CKs) cells eliminate co-cultured K562-EGFRVIII-LUC cells:

[0311] 6×10 5 CAR(EGFRVIII)-REP-M(PA / CKs) or CON-REP-M(PA / CKs) cells were co-cultured with 6×10 4 K562-EGFRVIII-LUC cells were mixed and co-cultured in a 37°C CO2 incubator for 3 days. After co-culture, cells were harvested and the proportion of surviving cancer cells was determined by flow cytometry.

[0312] The results showed that K562-EGFRVIII-LUC cells co-cultured with CON-REP-M (PA / CKs) cells significantly expanded within 3 days of culture, accounting for 62.4% of the total cells in the co-culture system. In contrast, K562-EGFRVIII-LUC cells in the co-culture group with CAR (EGFRVIII) -REP-M (PA / CKs) were phagocytosed / killed by CAR (EGFRVIII) -REP-M (PA / CKs) during the co-culture process, and K562-EGFRVIII-LUC cells only accounted for 3.58% of the total cells at the time of detection ( Figure 20 ).

[0313] (2) CAR(EGFRVIII)-REP-M(PA / CKs) cells eliminate co-cultured A549-EGFRVIII-LUC cancer cells:

[0314] 6×10 5 CAR (EGFRVIII)-REP-M (PA / CKs) or CON-REP-M (PA / CKs) cells were plated in a single well of a 6-well plate, and 2×10 4 A549-EGFRVIII-LUC cells were mixed and co-cultured in a 37°C carbon dioxide incubator for 3 days. After the co-culture, the cells were collected and the proportion of surviving cancer cells in the total number of cells was detected by flow cytometry. A549-EGFRVIII-LUC cells co-cultured with CON-REP-M (PA / CKs) cells expanded nearly 2 times within 3 days of culture, accounting for 5.5% of the total cells in the co-culture system. A549-EGFRVIII-LUC cells in the co-culture group with CAR (EGFRVIII) -REP-M (PA / CKs) were phagocytosed / killed by CAR (EGFRVIII) -REP-M (PA / CKs) during the co-culture process. At the time of detection, A549-EGFRVIII-LUC cells accounted for only 0.33% of the total cells ( Figure 21 ).

[0315] (3) CAR(HER2)-REP-M(PA / CKs) cells eliminate co-cultured SW480-LUC cancer cells (SW480 expresses endogenous HER2 gene):

[0316] 6×10 5 CAR(HER2)-REP-M(PA / CKs) or CON-REP-M(PA / CKs) cells were plated in a single well of a 6-well plate, and 1.2×10 5 , 6×10 4 , 2×10 4 SW480-LUC cells were mixed and co-cultured in a 37°C CO2 incubator for 24 hours. After co-culture, cells were harvested and the proportion of viable cancer cells to total cells was determined by flow cytometry.

[0317] The results showed that the proportion of SW480-LUC cells in the co-culture system of the CAR(HER2)-REP-M(PA / CKs) and SW480-LUC cells decreased with the increase of the effector-target ratio (CAR(HER2)-REP-M(PA / CKs):SW480-LUC). When the effector-target ratio was 30:1, SW480-LUC cells accounted for only 0.15% of the total cells. The CON-REP-M(PA / CKs) and SW480-LUC cells co-culture group also showed a downward trend in the proportion of SW480-LUC cells with the increase of the effector-target ratio. The ability of CAR(HER2)-REP-M(CKs) cells to eliminate cancer cells was significantly better than that of CON-REP-M(CKs) cells ( Figure 22 ).

[0318] These results indicate that REP-M cells have the characteristics of M1 macrophages and also have certain effects in inhibiting / killing cancer cells.

[0319] Example 11: In vivo infusion of CAR (EGFRVIII)-REP-M (PA / CKs) cells significantly inhibits tumor progression

[0320] Experimental subjects: NCG severely immunodeficient mice (purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.).

[0321] On day 0, each mouse received an intraperitoneal transplant of 5 × 10 5 A549-EGFRVIII-LUC cells were injected into the 4 groups and randomly divided into 4 groups, with half male and half female in each group, and each group received 2 therapeutic infusions.

[0322] The experimental mice received therapeutic intraperitoneal injections and were divided into the following groups:

[0323] (1) Intraperitoneal injection of PBS on days 1 and 4;

[0324] (2) intraperitoneal injection of CON-REP-M(CKs) on days 1 and 4;

[0325] (3) intraperitoneal injection of CAR(EGFRVIII)-REP-M(CKs)#1 on days 1 and 4;

[0326] (4) Intraperitoneal injection of CAR-REP-M#2 on days 4 and 7.

[0327] The first infusion dose of CON-REP-M (PA / CKs) and CAR (EGFRVIII)-REP-M (PA / CKs) cells was 1.5×10 7 cells / mouse, the second infusion dose was 3.0×10 7 All mice received 150 mg / kg D-luciferin sodium salt intraperitoneal injection on days 1, 4, 7, 10, 14, 19, and 24, and bioluminescence imaging was performed to monitor tumor progression. After day 24, bioluminescence imaging was performed every 7-8 days to continue monitoring tumor progression until the end of the experiment. Figure 23 shown.

[0328] Example 12: Radiation irradiation does not affect the ability of CAR-REP-M (PA / CKs) cells to phagocytose cancer cells

[0329] Macrophages hold great promise for clinical application, and the present invention proposes the clinical use of REP-M cells. Safety is a primary consideration for any substance intended for clinical treatment. REP-M cells possess the functions of normal macrophages and offer several advantages over primary macrophages and other macrophage preparation methods, thus possessing significant potential for clinical application. However, REP-M cells are derived from immortalized cells reprogrammed from REP-B cells. During the reprogramming process to produce REP-M cells, the cells gradually lose their ability to proliferate, ultimately completely losing their ability to proliferate upon reaching maturity. This suggests that REP-M cells may meet the safety requirements for clinical use. However, clinical treatment requires the infusion of a large number of REP-M cells. In extreme cases, if a very small percentage of cells, such as one in a million or even one in ten million, fail to undergo reprogramming, they may still proliferate in vivo, posing potential risks that must be considered. NK-92 cells are a type of NK cell lineage and also immortalized cells. However, NK-92 cells have already been used clinically as carriers for CAR-NK cells. Before being infused into the human body, CAR-NK cells carrying NK-92 cells will be irradiated with radiation to eliminate the cells' ability to proliferate and minimize the possibility of NK-92 cells proliferating in the body and generating leukemia.

[0330] This example investigates the safety of REP-M cells in clinical use. REP-M cells are irradiated to test whether an appropriate dose of radiation can completely kill CAR-REPable-B (CKs) cells while preserving the tumor phagocytic ability of CAR-REP-M (CKs) cells.

[0331] (1) First, an irradiation dose test was performed to determine the sensitivity of CAR-REP-B(CKs) to X-ray irradiation.

[0332] The results showed that all CAR-REP-B(CKs) cells could be killed by 8 Gy of radiation (dose rate of 7.67 Gy / min).

[0333] (2) CAR(EGFRVIII)-REP-M(PA / CKs) cells were irradiated at a dose higher than 8 Gy (10 Gy and 20 Gy) and the effect of irradiation on the energy of CAR(EGFRVIII)-REP-M(PA / CKs) phagocytosis of K562-EGFRVIII-LUC cells was examined. The irradiated CAR(EGFRVIII)-REP-M(PA / CKs) cells were plated in 6-well plates with 6×10 cells per well. 5 After the cells adhered to the wall, 6×10 4K562-EGFRVIII-LUC cells. The mixed cells were co-cultured in a 37°C carbon dioxide incubator for 24 hours. The cells were then harvested and flow cytometry was used to examine the effect of CAR(EGFRVIII)-REP-M(PA / CKs) cells on the phagocytic ability of K562-EGFRVIII-LUC cells. The effect of different doses of radiation on the phagocytic ability of CAR(EGFRVIII)-REP-M(PA / CKs) cells on K562-EGFRVIII-LUC cells was determined by comparing the phagocytic ability of unirradiated and irradiated CAR(EGFRVIII)-REP-M(PA / CKs) cells on K562-EGFRVIII-LUC cells.

[0334] The results showed that 10 Gy irradiation had a very limited effect on the phagocytic ability of CAR (EGFRVIII) -REP-M (PA / CKs) cells, and there was no significant difference in their phagocytic ability compared with non-irradiated cells; 20 Gy irradiation had a significant effect on the phagocytic ability of CAR (EGFRVIII) -REP-M (PA / CKs) cells, and their phagocytic ability decreased by 18% compared with non-irradiated cells ( Figure 24 The above results indicate that an appropriate dose of radiation can kill potential CAR-REP-B (PA / CKs) cells without affecting the phagocytic ability of CAR-REP-M (PA / CKs) cells, thereby improving the safety of clinical use.

[0335] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for producing a macrophage population, characterized in that, Comprising the following steps: (1) Engineering a population of B cells to introduce the exogenous transcription factor PU.1 to prepare a population of B cells expressing the exogenous transcription factor; (2) Inducing the population of B cells to transdifferentiate into macrophages; the macrophages are positive for CD86 and CD80.

2. The method according to claim 1, characterized in that, In step (1), after introducing the exogenous transcription factor PU.1 into the population of B cells, the expression level of the exogenous transcription factor PU.1 is more than 10 times higher than the expression level of endogenous PU.1 in B cells.

3. The method according to claim 2, characterized in that, The expression level of the exogenous transcription factor PU.1 is 20 - 200 times the expression level of endogenous PU.1 in B cells.

4. The method according to claim 1, characterized in that After introducing the exogenous transcription factor PU.1 into the population of B cells in step (1), at least 80% of the B cells transdifferentiate into macrophages.

5. The method according to claim 4, characterized in that, The population of B cells is 100% transdifferentiated into macrophages within 2 - 7 days.

6. The method according to claim 5, characterized in that, The population of B cells is 100% transdifferentiated into macrophages within 5 days.

7. The method according to claim 1, wherein After introducing the exogenous transcription factor PU.1 into the population of B cells in step (1), the transdifferentiation of the population of B cells into macrophages is induced by doxycycline; the concentration range of doxycycline used is 0.05 - 1.0 μg / mL.

8. The method according to claim 1, wherein After introducing the exogenous transcription factor PU.1 into the population of B cells in step (1), during the transdifferentiation process, the population of B cells is cultured in a cell culture medium containing cytokines; the cytokines include at least one of IL-3, M-CSF, and GM-CSF.

9. The method according to claim 8, wherein During the transdifferentiation process, the population of B cells is cultured in a cell culture medium containing IL-3 and M-CSF.

10. A method for producing a macrophage population, characterized in that, The method includes: Introducing the exogenous transcription factors PU.1 and C / EBPα into the population of B cells, and then inducing the population of B cells to transdifferentiate into macrophages.

11. The method according to claim 10, characterized in that, The expression level of PU.1 is more than 10 times higher than the expression level of endogenous PU.1 in B cells; the expression level of C / EBPα is more than 0.05 times the endogenous C / EBPα in M0 polarized macrophages.

12. The method according to claim 10, wherein At least 80% of the macrophages transdifferentiated from the population of B cells are positive for CD86 and CD80.

13. The method according to claim 12, wherein 100% of the macrophages transdifferentiated from the population of B cells are positive for CD86 and CD80.

14. The method according to claim 10, characterized in that, Using doxycycline at a concentration of at least 10 ng / mL to induce the population of B cells to transdifferentiate into macrophages.

15. The method according to claim 10, characterized in that, The population of B cells transdifferentiates into macrophages within 2 - 5 days.

16. The method according to claim 15, characterized in that, The population of B cells transdifferentiates into macrophages within 3 days.

17. The method according to claim 10, characterized in that, During the transdifferentiation process, the population of B cells is cultured in a cell culture medium containing cytokines; the cytokines include at least one of IL-3, M-CSF, and GM-CSF.

18. The method according to claim 17, wherein During the transdifferentiation process, the population of B cells is cultured in a cell culture medium containing IL-3 and M-CSF.

19. A method for producing a macrophage population, characterized in that, The method includes: Engineering a population of B cells to express at least one of the cytokines IL-3, M-CSF, and GM-CSF, and then inducing the engineered population of B cells to transdifferentiate into macrophages.

20. The method according to claim 19, characterized in that, The population of B cells is engineered to express IL-3 and M-CSF.

21. The method according to claim 19, wherein The population of B cells is engineered to express IL-13, M-CSF, and GM-CSF.

22. The method according to claim 19, wherein The B cell population is further engineered to express any one set of transcription factors among the following (i)-(x): (i) The transcription factor PU.1; (ii) The transcription factor C / EBPα; (iii) The transcription factors PU.1 and C / EBPα; (iv) The transcription factors PU.1 and C / EBPβ; (v) The transcription factors C / EBPα and C / EBPβ; (vi) The transcription factors PU.1, C / EBPα and C / EBPβ; (vii) The transcription factor C / EBPδ; (viii) The transcription factor C / EBPε; (ix) The transcription factors PU.1 and C / EBPδ; (x) The transcription factors PU.1 and C / EBPε.

23. The method according to claim 19, wherein During the transdifferentiation process, the B cell population is cultured in a cell culture medium without IL-3, M-CSF or GM-CSF.

24. The method according to claim 19, wherein, At least 80% of the engineered B cell population transdifferentiates into macrophages.

25. The method according to claim 24, wherein 100% of the engineered B cell population transdifferentiates into macrophages.

26. The method according to claim 19, wherein The transdifferentiated macrophages are positive for CD86 and CD80.

27. The method according to claim 1, 10 or 19, characterized in that, The source of the B cell population is human B cells.

28. The method according to claim 27, wherein The B cell population is derived from primary B cells, B cells of a cell line, or B cells derived from stem cells.

29. The method according to claim 1, 10 or 19, characterized in that The B cell population is further modified to express a therapeutic substance.

30. The method according to claim 29, wherein The B cell population is further modified to express a chimeric antigen receptor, an immunomodulator, and / or a therapeutic cytokine.

31. The method according to claim 30, wherein After the B cells are further modified, the macrophages obtained by induced transdifferentiation express a chimeric antigen receptor, an immunomodulator, and / or a therapeutic cytokine.

32. The method according to claim 30, wherein The B cell population is modified to express a chimeric antigen receptor.

33. The method according to claim 32, wherein After the B cell population is further modified, the macrophages obtained by induced transdifferentiation express a chimeric antigen receptor.

34. A macrophage population produced by the method according to any one of claims 1-33.

35. The macrophage population according to 34, characterized in that, The polarization state of the macrophage population can be stably maintained.

36. The macrophage population according to 34, characterized in that, The macrophage population does not have the ability to proliferate.

37. The macrophage population according to 34, characterized in that, The macrophage population has the same functions as primary macrophages.

38. A composition comprising the macrophage population according to claim 34.

39. A genetically engineered macrophage, characterized in that, The macrophages are produced by the following steps: (i) Genetically engineering B cells; (ii) Inducing the genetically engineered B cells to transdifferentiate into macrophages; the genetically engineered B cells are induced to undergo cell transdifferentiation by the expression of at least one transcription factor including PU.1, CEBPα, and CEBPβ.

40. The genetically engineered macrophage according to claim 39, wherein, The B cells are genetically engineered to express a chimeric antigen receptor, an immunomodulator, and / or a cytokine.

41. The genetically engineered macrophage according to claim 40, wherein, The B cells are genetically engineered to express a CAR, thereby transdifferentiating into macrophages expressing the CAR.

42. The genetically engineered macrophage according to claim 41, wherein The macrophages expressing the CAR act on cancer cells expressing the CAR-targeted antigen and kill the cancer cells or inhibit the proliferation of the cancer cells.

43. Use of the macrophage according to claim 34 or any one of claims 39-42 in the preparation of a medicament for treating a disease, characterized in that, The use includes infusing macrophages into a subject in need.

44. The use according to claim 43, characterized in that, The macrophages are pretreated by radiation before being administered to the subject.

45. The use according to claim 43, characterized in that, The diseases include cancer, autoimmune diseases, infectious diseases, or inflammatory diseases.

46. The use according to claim 45, characterized in that, The disease is cancer.

47. The use according to claim 46, characterized in that, The disease is a solid tumor.

48. A cell-reprogrammable B cell for producing macrophages, characterized in that, The reprogrammable B cells are engineered to express at least one set of transcription factors from the following (i)-(x): (i) exogenous PU.1 at a level higher than the endogenous transcription factor PU.1 expression level in B cells; (ii) transcription factor C / EBPα; (iii) transcription factors PU.1 and C / EBPα; (iv) transcription factors PU.1 and C / EBPβ; (v) transcription factors C / EBPα and C / EBPβ; (vi) transcription factors PU.1, C / EBPα and C / EBPβ; (vii) transcription factor C / EBPδ; (viii) transcription factor C / EBPε; (ix) transcription factors PU.1 and C / EBPδ; (x) transcription factors PU.1 and C / EBPε.

49. The cell-reprogrammable B cell according to claim 48, wherein The reprogrammable B cells are further engineered to express at least one cytokine from IL-3, M-CSF and GM-CSF.

50. The cell-reprogrammable B cell according to claim 49, wherein The reprogrammable B cells are further engineered to express IL-3 and M-CSF.

51. The cell-reprogrammable B cell according to claim 48 or claim 49, characterized in that, The reprogrammable B cells can be induced to reprogram into macrophages; the macrophages are positive for CD86 and CD80.

52. A set of components for producing macrophages, characterized in that, Comprising the cell-reprogrammable B cells according to any one of claims 48-51 and an inducer for inducing transcription factor expression.

53. The component set according to claim 52, characterized in that, The inducer is doxycycline.

54. The component set according to claim 52, characterized in that, The kit of components further comprises a cell culture medium and a cytokine; the cytokine is at least one of IL-3, M-CSF and GM-CSF.

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