Chimeric antigen receptor, human induced pluripotent stem cell, myeloid precursor cell-like macrophage and preparation method thereof

Abstract

The invention provides a chimeric antigen receptor, a human induced pluripotent stem cell, a myeloid precursor cell-like macrophage and a preparation method thereof, and relates to the technical field of biomedicine. According to the constructed chimeric antigen receptor, hiPSC expressing the chimeric antigen receptor can be differentiated into myeloid precursor cell-like macrophages. The myeloid precursor cell-like macrophage has the characteristics and advantages of rapid proliferation, high activity after cryopreservation and resuscitation, low immunogenicity, migration and infiltration to solid tumors and the like, shortens the period of preparation and clinical treatment, reduces the secondary risk of host anti-graft and the like, and has a wide application prospect. And a continuous and reliable cell quantity guarantee is provided for clinical treatment of tumors. Therefore, the cell product really becomes a spot type cell product. The myeloid precursor-like macrophage can be stimulated to be mature in an exogenous factor and tumor microenvironment, and then has stronger targeting activation ability, M1 polarization level and antigen presentation ability, and has more considerable clinical transformation prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a chimeric antigen receptor, human induced pluripotent stem cells, myeloid precursor cell-like macrophages and preparation methods thereof. Background Art

[0002] hiPSC (human induced pluripotent stem cells) have the potential to differentiate into all types of cells in the human body. Myeloid cells differentiated from iPSC, including macrophages (iMAC) and dendritic cells (iDC), are of great clinical significance for the realization of immune cell therapy and vaccine development for tumors. However, at present, iPSC-derived myeloid cells must be differentiated on demand according to the temporary needs of tumor labor treatment, which is time-consuming and labor-intensive, and cannot be effectively cryopreserved and have high recovery activity after cryopreservation, thus greatly prolonging the treatment cycle and may even miss the best treatment window. Therefore, the high viability and effective proliferation ability of immune cells after cryopreservation are the key to realizing the "spot type" concept of taking all kinds of immune cells on demand and seizing the treatment opportunity. At present, there is no research and patent on the differentiation of iPSC into myeloid precursor cells with rapid proliferation ability and high recovery activity after cryopreservation. For example, the myeloid cells obtained from patents for iPSC differentiation into macrophages (such as authorization announcement numbers CN109082411B, CN109266618B, CN116731967A, CN115433715A) and iPSC differentiation into DC cells (such as CN106852385A, CN110238265A, CN105272293A, US20110286965A1, WO2013142677A1, EP2612336A1) are all terminally differentiated cells, which can neither be effectively cryopreserved nor achieve rapid proliferation after differentiation. Therefore, developing spot myeloid immune cells with high value-added ability and high vitality after recovery will be the key to promoting iPSC-derived myeloid immune cells to achieve timely and effective treatment of corresponding diseases and shorten the treatment cycle.

[0003] In addition, the iPSC-derived chimeric antigen receptor (CAR) macrophages (CAR-iMAC) currently used in cell therapy face pain points such as poor targeted activation effect, low M1 polarization level, easy M2 polarization in the tumor microenvironment, insufficient antigen presentation ability, and low cytokine secretion level, which seriously hinder its clinical transformation progress.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The first object of the present invention is to provide a chimeric antigen receptor.

[0006] The second object of the present invention is to provide a nucleic acid.

[0007] The third object of the present invention is to provide a carrier.

[0008] A fourth object of the present invention is to provide a human induced pluripotent stem cell.

[0009] The fifth object of the present invention is to provide a method for preparing myeloid precursor cell-like macrophages.

[0010] The sixth object of the present invention is to provide a myeloid precursor cell-like macrophage to solve the above technical problems.

[0011] The seventh object of the present invention is to provide a mature macrophage.

[0012] In order to achieve the above objectives, the following technical solutions are adopted: In a first aspect, the present invention provides a chimeric antigen receptor, which includes, from N-terminus to C-terminus, an extracellular domain, a transmembrane domain, and an intracellular signaling domain; The extracellular domain includes a signal peptide, a single-chain variable region targeting an antigen, and a CD8α hinge from the N-terminus to the C-terminus; the nucleic acid sequence of the signal peptide is shown in SEQ ID NO.1; the nucleic acid sequence of the CD8α hinge is shown in SEQ ID NO.4; The transmembrane domain is the transmembrane domain of CD8α protein, and the nucleic acid sequence is shown in SEQ ID NO.5; The intracellular signaling domain includes the Box1 domain of GM-CSFRα and the GM-CSFRβ intracellular key activation domain from the N-terminus to the C-terminus; the nucleic acid sequence of the Box1 domain of GM-CSFRα is shown in SEQ ID NO.6; the sequence of the GM-CSFRβ intracellular key activation domain is shown in SEQ ID NO.7.

[0013] As a further technical solution, the antigen-targeting single-chain variable region includes a single-chain variable region targeting EGFRvIII or a single-chain variable region targeting GPC3; The nucleic acid sequence of the single-chain variable region targeting EGFRvIII is shown in SEQ ID NO.2; The nucleic acid sequence of the single-chain variable region targeting GPC3 is shown in SEQ ID NO.3.

[0014] As a further technical solution, the Box1 domain of GM-CSFRα and the key intracellular activation domain of GM-CSFRβ are connected via a Linker; The linker nucleic acid sequence is shown in SEQ ID NO.8.

[0015] In a second aspect, the present invention provides a nucleic acid encoding the chimeric antigen receptor.

[0016] In a third aspect, the present invention provides a vector carrying the above-mentioned nucleic acid.

[0017] In a fourth aspect, the present invention provides a human induced pluripotent stem cell, which carries the above-mentioned nucleic acid, or contains the above-mentioned vector, or expresses the above-mentioned chimeric antigen receptor.

[0018] In a fifth aspect, the present invention provides a method for preparing myeloid precursor cell-like macrophages, comprising the following steps: The human induced pluripotent stem cells are suspended and cultured in MI medium containing ROCK inhibitor for 2-4 days to form embryoid bodies, and then cultured in MI medium for 1-2 days, HS medium for 5-7 days, and ME-1 medium for 3-4 days, and then continue to be cultured in ME-2 medium and harvest myeloid precursor cell-like macrophages; The MI medium is an APEL II medium containing 30-80 ng / mL BMP4 and 30-50 ng / mL Activin A; The HS medium is an APEL II medium containing 30-80 ng / mL BMP4, 50-100 ng / mL VEGF, 100-150 ng / mL SCF, 20-50 ng / mL IL-3, 25-50 ng / mL IL-6, 25-50 ng / mL IL-11 and 25-50 ng / mL Flt-3L; The ME-1 medium is an APEL II medium containing 10-20 ng / mL bFGF, 50-100 ng / mL VEGF, 50-100 ng / mL SCF, 10-20 ng / mL IGF1, 25-50 ng / mL IL-3, 30-50 ng / mL TPO, 25-50 ng / mL M-CSF and 25-50 ng / mL GM-CSF; The ME-2 medium is X-VIVO containing 20-40 ng / mL bFGF, 50-100 ng / mL VEGF, 50-80 ng / mL SCF, 10-20 ng / mL IGF1, 25-50 ng / mL IL-3, 25-50 ng / mL M-CSF and 25-50 ng / mL GM-CSF. TM -15 medium or StemSpan™ H3000 medium.

[0019] As a further technical solution, in the MI medium containing the ROCK inhibitor, the concentration of the ROCK inhibitor is 8-10 nM; The ROCK inhibitors include Y27632.

[0020] In a sixth aspect, the present invention provides a myeloid precursor cell-like macrophage prepared by the above-mentioned preparation method.

[0021] In a seventh aspect, the present invention provides a mature macrophage, which is obtained by incubating the myeloid progenitor-like macrophage in X-VIVO containing 20-50 ng / mL GM-CSF and 50-100 ng / mL M-CSF. TM -15 medium.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The chimeric antigen receptor constructed by the present invention can make hiPSC expressing the chimeric antigen receptor differentiate into myeloid precursor cell-like macrophages. The myeloid precursor cell-like macrophages have the characteristics and advantages of rapid proliferation and high activity after cryopreservation and resuscitation, as well as low immunogenicity and migration and infiltration into solid tumors, which shortens the cycle of its preparation and clinical treatment, reduces secondary risks such as host-versus-graft, and also provides a continuous and reliable cell quantity guarantee for the clinical treatment of tumors. It makes it a truly "spot-type" cell product. In addition, the myeloid precursor-like macrophages can be stimulated to mature in exogenous factors and tumor microenvironment, and then have stronger targeted activation ability and M1 polarization level, as well as stronger antigen presentation ability, and have more impressive clinical transformation prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 It is a schematic diagram of the gene expression sequence structure of CAR; Figure 2 G-CAR-iMACs differentiated from hiPSCs expressing G-CAR have phenotypic characteristics similar to myeloid progenitor cells; Figure 3 Detection of the proliferation capacity of G-CAR-iMAC in myeloid precursor cell-like state; Figure 4 To detect the activity of G-CAR-iMAC in myeloid precursor cell-like state after cryopreservation and resuscitation; Figure 5 Detection of the induction of G-CAR-iMAC in myeloid precursor cell-like state into mature G-CAR-iMAC; Figure 6 To detect the targeted anti-tumor ability of G-CAR-iMAC; Figure 7 To detect the polarization level of G-CAR-iMAC targeting EGFRvIII; Figure 8 To detect the anti-inflammatory presentation ability of G-CAR-iMAC; Fig. 9 Verification of the anti-tumor efficacy of G-CAR-iMAC. DETAILED DESCRIPTION

[0025] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified, proceed according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0026] In a first aspect, the present invention provides a chimeric antigen receptor, which includes, from N-terminus to C-terminus, an extracellular domain, a transmembrane domain, and an intracellular signaling domain; The extracellular domain includes a signal peptide, a single-chain variable region targeting an antigen, and a CD8 αhinge in sequence from the N-terminus to the C-terminus; The nucleic acid sequence of the signal peptide is shown in SEQ ID NO.1.

[0027] The nucleic acid sequence of the CD8 αhinge is shown in SEQ ID NO.4.

[0028] The transmembrane domain is the transmembrane domain of CD8α protein, and the nucleic acid sequence is shown in SEQ ID NO.5.

[0029] The intracellular signaling domain includes the Box1 domain of GM-CSFRα and the key intracellular activation domain of GM-CSFRβ from the N-terminus to the C-terminus; The nucleic acid sequence of the Box1 domain of GM-CSFRα is shown in SEQ ID NO.6.

[0030] The sequence of the GM-CSFRβ intracellular key activation domain (GM-CSFRβ 461-897 aa) is shown in SEQ ID NO.7.

[0031] Based on the concept of synthetic biology, the present invention synthesized a new type of CAR (G-CAR) containing the key intracellular activation domain of GM-CSFRα and the key intracellular activation domain of GM-CSFRβ by genetic engineering. iPSCs expressing this new type of G-CAR can differentiate into macrophage precursor cells with high proliferation capacity and high recovery activity after cryopreservation, and can further induce differentiation into functionally enhanced G-CAR macrophages. This strategy has achieved the following breakthroughs in the application of CAR macrophages in clinical treatment: 1. The high activity after cryopreservation and cryopreservation recovery makes the myeloid precursor-like macrophages derived from iPSC have the advantage of being available for use, shortening the cycle of macrophage preparation and clinical treatment; 2. Its high value-added ability also provides a continuous and reliable cell quantity guarantee for the clinical treatment of tumors; 3. The myeloid precursor-like macrophages of the present invention can be stimulated to mature in exogenous factors and tumor microenvironment, and then have stronger targeted activation ability and M1 polarization level, as well as stronger antigen presentation ability, so the new generation of G-CAR macrophages has a more promising clinical transformation prospect; 4. The myeloid precursor-like macrophages of the present invention have the characteristics of low immunogenicity, which greatly enhances its versatility in clinical treatment applications and further gives it a "spot type" feature. 5. The myeloid precursor-like macrophages of the present invention have a small cell body and highly express tumor chemokine receptors, so they have a stronger ability to migrate to tumors and infiltrate into solid tumors, and have an innate advantage in treating solid tumors.

[0032] In some optional embodiments, the antigen-targeting single-chain variable region includes a single-chain variable region targeting EGFRvIII or a single-chain variable region targeting GPC3; The nucleic acid sequence of the single-chain variable region targeting EGFRvIII is shown in SEQ ID NO.2.

[0033] The nucleic acid sequence of the single-chain variable region targeting GPC3 is shown in SEQ ID NO.3.

[0034] In some optional embodiments, the Box1 domain of GM-CSFRα and the key intracellular activation domain of GM-CSFRβ are connected via a Linker; The linker nucleic acid sequence is shown in SEQ ID NO.8.

[0035] In a second aspect, the present invention provides a nucleic acid encoding the chimeric antigen receptor.

[0036] The nucleic acid is capable of expressing the chimeric antigen receptor of the present invention.

[0037] In a third aspect, the present invention provides a vector carrying the above-mentioned nucleic acid.

[0038] The vector can be used for targeted delivery of target genes to achieve expression of the chimeric antigen receptor of the present invention in host cells.

[0039] In some optional embodiments, the vector comprises a lentiviral vector.

[0040] In a fourth aspect, the present invention provides a human induced pluripotent stem cell, which carries the above-mentioned nucleic acid, or contains the above-mentioned vector, or expresses the above-mentioned chimeric antigen receptor.

[0041] The human induced pluripotent stem cells can express the chimeric antigen receptor of the present invention and can differentiate into macrophages expressing the chimeric antigen receptor of the present invention.

[0042] In a fifth aspect, the present invention provides a method for preparing myeloid precursor cell-like macrophages, comprising the following steps: The human induced pluripotent stem cells are suspended and cultured in MI medium containing ROCK inhibitor for 2-4 days to form embryoid bodies, and then cultured in MI medium for 1-2 days, HS medium for 5-7 days, and ME-1 medium for 3-4 days, and then continue to be cultured in ME-2 medium and harvest myeloid precursor cell-like macrophages; The MI medium is an APEL II medium containing 30-80 ng / mL BMP4 and 30-50 ng / mL Activin A, wherein the concentration of BMP4 can be, for example, but not limited to 30 ng / mL, 50 ng / mL or 80 ng / mL; the concentration of Activin A can be, for example, but not limited to 30 ng / mL, 40 ng / mL or 50 ng / mL; The HS medium is APEL containing 30-80 ng / mL BMP4, 50-100 ng / mL VEGF, 100-150 ng / mL SCF, 20-50 ng / mL IL-3, 25-50 ng / mL IL-6, 25-50 ng / mL IL-11 and 25-50 ng / mL Flt-3L. II culture medium, wherein the concentration of BMP4 may be, for example, but not limited to 30 ng / mL, 50 ng / mL or 80 ng / mL; the concentration of VEGF may be, for example, but not limited to 50 ng / mL, 80 ng / mL or 100 ng / mL; the concentration of SCF may be, for example, but not limited to 100 ng / mL, 120 ng / mL or 150 ng / mL; the concentration of IL-3 may be, for example, but not limited to 20 ng / mL, 40 ng / mL or 50 ng / mL; the concentration of IL-6 may be, for example, but not limited to 25 ng / mL, 40 ng / mL or 50 ng / mL; the concentration of IL-11 may be, for example, but not limited to 25 ng / mL, 40 ng / mL or 50 ng / mL; the concentration of Flt-3L may be, for example, but not limited to 25 ng / mL, 40 ng / mL or 50 ng / mL; The ME-1 medium is an APEL II medium containing 10-20 ng / mL bFGF, 50-100 ng / mL VEGF, 50-100 ng / mL SCF, 10-20 ng / mL IGF1, 25-50 ng / mL IL-3, 30-50 ng / mL TPO, 25-50 ng / mL M-CSF and 25-50 ng / mL GM-CSF, wherein: The concentration of bFGF may be, for example, but not limited to 10 ng / mL, 15 ng / mL or 20 ng / mL; the concentration of VEGF may be, for example, but not limited to 50 ng / mL, 80 ng / mL or 100 ng / mL; the concentration of SCF may be, for example, but not limited to 50 ng / mL, 80 ng / mL or 100 ng / mL; the concentration of IGF1 may be, for example, but not limited to 10 ng / mL, 15 ng / mL or 20 ng / mL; the concentration of IL-3 may be, for example, but not limited to 25 ng / mL, 40 ng / mL or 50 ng / mL; the concentration of TPO may be, for example, but not limited to 30 ng / mL, 40 ng / mL or 50 ng / mL; the concentration of M-CSF may be, for example, but not limited to 25 ng / mL, 40 ng / mL or 50 ng / mL; the concentration of GM-CSF may be, for example, but not limited to 25 ng / mL, 40 ng / mL or 50 ng / mL; The ME-2 medium is X-VIVO containing 20-40 ng / mL bFGF, 50-100 ng / mL VEGF, 50-80 ng / mL SCF, 10-20 ng / mL IGF1, 25-50 ng / mL IL-3, 25-50 ng / mL M-CSF and 25-50 ng / mL GM-CSF. TM -15 medium or StemSpan™ H3000 medium, wherein the concentration of bFGF can be, for example, but not limited to 20 ng / mL, 30 ng / mL or 40 ng / mL; the concentration of VEGF can be, for example, but not limited to 50 ng / mL, 80 ng / mL or 100 ng / mL; the concentration of SCF can be, for example, but not limited to 50 ng / mL, 60 ng / mL or 80 ng / mL; the concentration of IGF1 can be, for example, but not limited to 10 ng / mL, 15 ng / mL or 20 ng / mL; the concentration of IL-3 can be, for example, but not limited to 25 ng / mL, 40 ng / mL or 50 ng / mL; the concentration of M-CSF can be, for example, but not limited to 25 ng / mL, 40 ng / mL or 50 ng / mL; the concentration of GM-CSF can be, for example, but not limited to 25 ng / mL, 40 ng / mL or 50 ng / mL.

[0043] The preparation method is simple and convenient. The prepared myeloid precursor cell-like macrophages have the characteristics and advantages of rapid proliferation and high activity after cryopreservation and resuscitation, as well as low immunogenicity and migration and infiltration into solid tumors. It shortens the preparation and clinical treatment cycle, reduces secondary risks such as host-versus-graft disease, and provides a continuous and reliable cell quantity guarantee for the clinical treatment of tumors.

[0044] In some optional embodiments, in the MI medium containing the ROCK inhibitor, the concentration of the ROCK inhibitor may be, for example, but not limited to, 8 nM, 9 nM or 10 nM; The ROCK inhibitor includes but is not limited to Y27632.

[0045] In a sixth aspect, the present invention provides a myeloid precursor cell-like macrophage prepared by the above-mentioned preparation method.

[0046] In a seventh aspect, the present invention provides a mature macrophage, which is obtained by incubating the myeloid progenitor-like macrophage in X-VIVO containing 20-50 ng / mL GM-CSF and 50-100 ng / mL M-CSF. TM-15 culture medium, wherein the concentration of M-CSF can be, for example, but not limited to 25 ng / mL, 40 ng / mL or 50 ng / mL; the concentration of GM-CSF can be, for example, but not limited to 25 ng / mL, 40 ng / mL or 50 ng / mL.

[0047] The myeloid precursor-like macrophages provided by the present invention can be stimulated to mature in the presence of exogenous factors and tumor microenvironment, and then have stronger targeted activation ability and M1 polarization level, as well as stronger antigen presentation ability, and have more promising clinical transformation prospects.

[0048] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0049] Example 1 Part I: Design of G-CAR expression sequence G-CAR and Truncated CAR were designed respectively, and their structures are shown in Figure 1 As shown, the G-CAR protein gene expression sequence in the present invention consists of an extracellular domain, a transmembrane domain and an intracellular signaling domain. The intracellular domain consists of a signal peptide, a single-chain variable region (scFv) that can target and recognize EGFRvIII and GPC3 antigens, and a CD8αhinge sequence. The transmembrane domain (TM) is a transmembrane domain protein sequence from the CD8α protein. The intracellular part is composed of the Box1 domain of GM-CSFRα and the GM-CSFRβ intracellular key activation domain connected in series through the Linker sequence. The GM-CSFRβ activation domain in the G-CAR cell is the 461-849 amino acid sequence (461-849aa). Truncated CAR without the intracellular domain is used as a negative control CAR for subsequent validation experiments.

[0050] The chimeric antigen receptors for EGFRvIII antigen are as follows: scFv (EGFRvIII)-G-CAR, the nucleic acid sequence of which is shown in SEQ ID NO.9; scFv (EGFRvIII) Truncated CAR, the nucleic acid sequence of which is shown in SEQ ID NO.10; The chimeric antigen receptors for GPC3 antigen are as follows: scFv (GPC3)-G-CAR, the nucleic acid sequence of which is shown in SEQ ID NO.11; scFv (GPC3) Truncated CAR, the nucleic acid sequence of which is shown in SEQ ID NO.12.

[0051] At the same time, the above CARs are connected to EGFP green fluorescent protein (nucleic acid sequence is shown in SEQ ID NO.14) through T2A element (nucleic acid sequence is shown in SEQ ID NO.13) to form a fusion expression sequence. The expression of EGFP can indicate the expression of CAR.

[0052] Part II: Construction of hiPSC differentiation system expressing CAR (1) Construction of a lentiviral overexpression system for CAR.

[0053] CAR was cloned into the lentiviral expression plasmid Lenti-EF1a-T2A-EGFP-PGK-Puromycin by molecular cloning, and scFv (EGFRvIII)-G-CAR, scFv (EGFRvIII) Truncated CAR, scFv (GPC3)-G-CAR, and scFv (GPC3) Truncated CAR lentiviral expression plasmids were constructed respectively. The plasmids were amplified by Escherichia coli.

[0054] (2) Construction of a hiPSC cell line that stably overexpresses CAR.

[0055] The above-constructed CAR lentiviral expression plasmids and lentiviral packaging plasmids psPAX2 and pMD2.G were transfected into HEK293T cells at a mass ratio of 4:3:1. The cell supernatant was collected at 48, 60, and 72 hours of culture, and concentrated to obtain lentiviral particles containing the above different CAR expression genes. Subsequently, the lentiviral particles containing the above different CAR expression genes were infected into the wild-type hiPSC cell line using the cationic compound polybrane to construct a CAR-hiPSC cell line that stably overexpressed CAR. The lentiviral overexpression system can stably integrate the CAR expression gene into the genome of hiPSC. The expression ratio and fluorescence brightness of EGFP in hiPSC cells can characterize the expression efficiency of CAR in hiPSC.

[0056] (3) Construct a hiPSC differentiation system based on embryoid body (EB) formation.

[0057] Spin-EB-based hiPSC differentiation system 1) G-CAR-expressing hiPSCs and Truncated CAR-expressing hiPSCs were cultured in six-well plates coated with Matrigel (Corning, #354277) using serum-free medium (mTesR1, STEMCELL; or ncTarget medium, Zhongsheng Suyuan) until the confluence reached 60% to 80%, and then digested into single cells using TrypLE digestion solution (STEMCELL).

[0058] 2) Collect hiPSCs digested into single cells in the previous step and resuspend them in MI medium (APEL II medium + 30ng / mL BMP4 + 30ng / mL Activin A) containing ROCK inhibitor (Y27632). Transfer the resuspended cells to a low-adsorption round-bottom 96-well plate with a culture volume of 60ul per well and culture them in a 37°C, 5% CO2 cell culture incubator for 24 hours. This is day 0. After 24 hours of suspension culture, EBs can be formed.

[0059] 3) On day 1-2, replace the suspended EBs with MI medium (APEL II medium + 20ng / mL BMP4 + 30ng / mL Activin A) without ROCK inhibitor (Y27632). Continue culturing for 48 hours.

[0060] 4) On the 3rd to 7th day, change to HS medium (HS medium: APEL II medium + 30ng / mL BMP4 + 50ng / ml VEGF + 100ng / mL SCF + 20ng / mL IL-3 + 25ng / mL IL-6 + 25ng / mL IL-11 + 25ng / mLFlt-3L).

[0061] 5) On days 8-10, change to ME-1 medium (ME-1: APEL II medium + 10ng / ml bFGF + 50ng / ml VEGF + 50ng / mL SCF + 10ng / mL IGF1 + 25ng / mL IL-3 + 30ng / mL TPO + 50ng / mLM-CSF + 50ng / mL GM-CSF).

[0062] 6) On day 11, transfer EBs to a 6-well plate coated with Matrigel (Corning, #354277). Place 16 EBs in each well of the 6-well plate. At this time, change the culture medium to ME-2 medium (ME-2: X-VIVO TM-15 culture medium / StemSpan™ H3000 culture medium + 10ng / ml bFGF + 50ng / ml VEGF + 50ng / mL SCF + 10ng / mL IGF1 + 25ng / mL IL-3 + 25ng / mL M-CSF + 25ng / mL GM-CSF). EBs are cultured in this culture medium from now on. During this period, a large number of myeloid precursor cell-like G-CAR-iMAC (differentiated from G-CAR-hiPSC) and Truncated CAR-iMAC (differentiated from Truncated CAR-hiPSC) will be produced. At this stage, the culture supernatant is collected every two days, the cells differentiated from hiPSC in the supernatant are collected by centrifugation, resuspended with fresh ME-2 culture medium and transferred to a new 6-well plate.

[0063] (4) hiPSCs expressing G-CAR can be induced to differentiate into myeloid progenitor cell-like G-CAR-iMACs with naive characteristics.

[0064] The myeloid precursor cell-like G-CAR-iMAC harvested in step (3) was further cultured in ME-2 medium for 10 days, and the cells were tested. The results were as follows: Figure 2 As shown. G-CAR-iMACs differentiated from hiPSCs expressing G-CAR targeting EGFRvIII induced by the differentiation platform of the present invention exhibited a myeloid precursor cell-like immature developmental phenotype compared to the control group cells (Truncated CAR-iMACs). After 10 days of in vitro culture, G-CAR-iMACs still maintained a myeloid precursor-like state ( Figure 2 A in Figure 1). CD14, a marker membrane protein of monocytes / macrophages, was detected by flow cytometry. The results showed that Truncated CAR-iMAC expressed high levels of CD14, while G-CAR-iMAC hardly expressed CD14, a marker membrane protein of monocytes / macrophages ( Figure 2 B in the figure). Flow cytometry was used to detect the marker membrane proteins of macrophages, CD80, CD86, CD206, and CD163. The results showed that the four membrane proteins were highly expressed in Truncated CAR-iMAC and weakly expressed in G-CAR-iMAC ( Figure 2 C in Figure 1). The above results indicate that G-CAR-iMAC differentiated from hiPSC expressing G-CAR is in a macrophage precursor-like state without any treatment.

[0065] (5) G-CAR-iMAC in the myeloid precursor cell-like state has the ability to proliferate rapidly.

[0066] 1×10^4 myeloid progenitor cell-like G-CAR-iMAC (differentiated from G-CAR-hiPSC) and Truncated CAR-iMAC (differentiated from Truncated CAR-hiPSC) were taken and incubated with 100ul X-VIVO containing 50ng / mL M-CSF and 50ng / mL GM-CSF, respectively. TM -15 culture medium was cultured in a well of a 96-well plate. 1ul of CCK8 reagent for detecting cell proliferation and toxicity was added to each well at 0 hours, 12 hours, 36 hours and 60 hours of culture, and the size and change of OD value were detected by microplate reader to judge the cell viability. At least 3 replica wells were set for each group of cell samples at each time point.

[0067] The results are as follows Figure 3 As shown. The CCK8 proliferation assay was used to detect the cell activity of hiPSCs expressing G-CAR targeting EGFRvIII and differentiated into G-CAR-iMAC in a myeloid precursor cell-like state for 0-60 hours. The results showed that G-CAR-iMAC in a myeloid precursor cell-like state had a stronger proliferation ability than the control group cells (Truncated CAR-iMAC).

[0068] (6) G-CAR-iMAC in the myeloid precursor cell-like state has high cell activity after cryopreservation and can continue to proliferate stably, indicating that G-CAR-iMAC has the characteristics of an off-the-shelf cell drug.

[0069] 1×10^6 myeloid progenitor cell-like G-CAR-iMAC (differentiated from G-CAR-hiPSC) and Truncated CAR-iMAC (differentiated from Truncated CAR-hiPSC) were taken, each was reselected with 1ml of commercial universal cell freezing solution and then frozen at -80℃. After freezing for 7 days, they were revived at 37℃, and 1×10^4 myeloid progenitor cell-like G-CAR-iMAC and Truncated CAR-iMAC were taken, and 100ul of X-VIVO containing 50ng / mL M-CSF and 50ng / mL GM-CSF were used respectively. TM -15 culture medium was cultured in a well of a 96-well plate. 1ul of CCK8 reagent for detecting cell proliferation and toxicity was added to each well at 0 hours, 24 hours, 48 ​​hours and 72 hours of culture, and the size and change of OD value were detected by microplate reader to judge the cell viability. At least 3 replica wells were set for each group of cell samples at each time point.

[0070] The results are as follows Figure 4As shown. The MTT experiment detected the cell activity of hiPSC expressing G-CAR targeting EGFRvIII and differentiated into G-CAR-iMAC in a myeloid precursor cell-like state for 0-70 hours. The results showed that G-CAR-iMAC in a myeloid precursor cell-like state had significantly higher activity than the control group cells (Truncated CAR-iMAC) after cryopreservation and recovery, and maintained continuous proliferation ability as the culture time increased. This result shows that G-CAR-iMAC in a myeloid precursor cell-like state differentiated from G-CAR hiPSC can be cryopreserved in large quantities, and then taken out when needed, shortening the treatment cycle based on G-CAR-iMAC. This invention makes G-CAR-iMAC a truly off-the-shelf drug.

[0071] Part III: Further stimulation of myeloid progenitor cell-like G-CAR-iMAC to mature into G-CAR-iMAC (1) The collected myeloid progenitor cell-like G-CAR-iMAC was incubated in X-VIVO containing 50 ng / mL GM-CSF and 50 ng / ml M-CSF. TM -15 medium for 72 hours and then the cells were observed for their status, which showed that mature G-CAR-iMAC was gradually obtained ( Figure 5 A in ).

[0072] (2) Continue to culture G-CAR-iMAC in the myeloid precursor cell-like state until day 10.

[0073] (3) Flow cytometry analysis showed that after myeloid progenitor cell-like G-CAR-iMAC was stimulated with 50 ng / mL GM-CSF and 50 ng / ml M-CSF for different days, the expression level of CD11B, a marker protein of mature myeloid cells, increased with the increase of stimulation days ( Figure 5 B in the figure).

[0074] (4) Myeloid progenitor cell-like G1-CAR-iMACs stimulated with 50ng / mL GM-CSF and 50ng / ml M-CSF for different days were collected for transcriptome sequencing analysis. The results showed that the marker proteins of mature macrophages increased with the increase of stimulation days, indicating that myeloid progenitor cell-like G-CAR-iMACs were gradually stimulated into mature G-CAR-iMACs with macrophage characteristics ( Figure 5 C in Figure 2, control is Truncated CAR-iMAC control).

[0075] The results are as follows Figure 5As shown, myeloid precursor cell-like state G-CAR-iMAC differentiated from hiPSC expressing G-CAR targeting EGFRvIII can be matured into G-CAR-iMAC with true macrophage morphology and function by adding M-CSF and GM-CSF to stimulate it.

[0076] (5) G-CAR-iMAC has the ability to almost completely eliminate tumor cells.

[0077] The myeloid progenitor cell-like state G-CAR-iMAC differentiated from hiPSC expressing G-CAR was stimulated to mature in vitro with M-CSF and GM-CSF, and then co-cultured with tumor cells expressing luciferase at an effector-target ratio of 5 / 1 (E / T=5 / 1) for 48 hours, and then the activity of luciferase in the tumor was detected. Figure 6 shown.

[0078] The results showed that G-CAR-iMAC targeting EGFRvIII showed significant targeted killing ability against the glioblastoma cell line U87MG expressing EGFRvIII antigen both without pre-polarization and after IFN-gamma / LPS polarization. Compared with the control group Truncated CAR-iMAC, G-CAR-iMAC almost completely eliminated U87MG ( Figure 6 A in the figure, where the blank control represents neither tumor cells nor G-CAR-iMAC; the non-treated group represents U87MG tumor cells with only EGFRvIII antigen; EGFRvIII Truncated CAR-iMAC represents the use of this cell to kill U87MG cells expressing EGFRvIII antigen; EGFRvIII-G-CAR-iMAC represents the use of this cell to kill U87MG cells expressing EGFRvIII antigen; EGFRvIII-G-CAR-iMAC (IFN-gamma / LPS) represents the use of this cell to kill U87MG cells expressing EGFRvIII antigen after being treated with 100ug / ml IFN-gamma and LPS for 24 hours. G-CAR-iMAC targeting GPC3 showed significant targeted killing ability against HepG2 hepatocellular carcinoma cells expressing GPC3 antigen both without pre-polarization and after IFN-gamma / LPS polarization. Compared with the control group Truncated CAR-iMAC, G-CAR-iMAC almost completely eliminated HepG2 ( Figure 6In B, the blank control represents neither tumor cells nor G-CAR-iMAC; the non-treated group represents HepG2 tumor cells with only GPC3 antigen; GPC3 Truncated CAR-iMAC represents the use of the cells to kill HepG2 cells expressing GPC3 antigen; GPC3-G-CAR-iMAC represents the use of the cells to kill U87MG cells expressing EGFRvIII antigen; GPC3-G-CAR-iMAC (IFN-gamma / LPS) represents the use of the cells treated with 100ug / ml IFN-gamma and LPS for 24 hours to kill HepG2 cells expressing GPC3 antigen).

[0079] (6) G-CAR-iMAC can maintain a higher M1 polarization level during the anti-tumor process and has a stronger anti-tumor effect.

[0080] Mature G-CAR-iMAC and Truncated CAR-iMAC were co-cultured with U87MG cells expressing EGFRvIII at an effector-target ratio of 1 / 5 (E / T=1 / 1) for 0, 24, 72, and 120 hours, and the proportion of G-CAR-iMAC expressing macrophage polarization marker membrane proteins was detected by flow cytometry. Figure 7 shown.

[0081] By detecting the percentage of positive cells for the M1 polarization membrane protein CD80, it was shown that G-CAR-iMAC could maintain a higher level and longer duration of M1 polarization in the anti-tumor process compared with Truncated CAR-iMAC ( Figure 7 A and B in Figure 1). By detecting the percentage of positive cells for the M2 polarization membrane protein CD206, it was shown that G-CAR-iMAC can maintain a lower level of M1 polarization than TruncatedCAR-iMAC during the anti-tumor process ( Figure 7 This assay result indicated that G-CAR-iMAC could maintain a higher level of anti-tumor activity.

[0082] (7) G-CAR-iMAC has stronger antigen presentation ability.

[0083] Studies have shown that NY-ESO-1 can activate T cells expressing IG4-TCR. Cells expressing HLA:0201 can activate T cells by presenting NY-ESO-1. Based on this, the following experimental groups were designed: Jurkat group: experimental group with only Jurkat cells; U87MG EGFRvIII -NY+Jurkat: U87MG expressing NY-ESO-1EGFRvIII Experimental group in which cells were co-cultured with Jurkat cells; U87MG EGFRvIII -HLA-NY+Jurkat: U87MG expressing HLA:0201 and NY-ESO-1 EGFRvIII Experimental group in which cells were co-cultured with Jurkat cells; Truncated CAR-iMAC+U87MG EGFRvIII : Truncated CAR-iMAC and U87MG EGFRvIII Co-culture experimental groups; G-CAR-iMAC+Jurkat: experimental group in which G-CAR-iMAC targeting EGFRvIII was co-cultured with Jurkat; U87MG EGFRvIII +Truncated CAR-iMAC+Jurkat: U87MG EGFRvIII The experimental group in which three types of cells were co-cultured: Truncated CAR-iMAC and Jurkat; U87MG EGFRvIII +G-CAR-iMAC+Jurkat: U87MG EGFRvIII , G-CAR-iMAC targeting EGFRvIII, and Jurkat cells co-cultured together; U87MG EGFRvIII -NY+Truncated CAR-iMAC+Jurkat: U87MG expressing NY-ESO-1 EGFRvIII The experimental group in which cells, Truncated CAR-iMAC and Jurkat cells were co-cultured; U87MG EGFRvIII -NY+G-CAR-iMAC+Jurkat: U87MG expressing NY-ESO-1 EGFRvIII The experimental group co-cultured three types of cells: cells, G-CAR-iMAC targeting EGFRvIII, and Jurkat cells.

[0084] The above experimental groups were cultured for 24 hours, and then the expression ratio of T cell activation marker protein CD69 was detected. Figure 8 The results showed that the control cells Truncated CAR-iMAC in U87MG EGFRvIII In the presence of -NY, Jurkat cells could not be activated significantly, while G-CAR-iMAC in U87MG EGFRvIII-NY can significantly activate Jurkat cells in the presence of NY, and its activation level is significantly higher than that of positive control cells (U87MG EGFRvIII -HLA-NY) on the activation level of Jurkat cells. This result shows that G-CAR-iMAC has enhanced antigen presentation function.

[0085] (8) G-CAR-iMAC has significantly enhanced tumor therapeutic efficacy.

[0086] 1×10^5 HepG2 cells expressing GPC3 antigen were subcutaneously inoculated into immunodeficient mice. Tumors formed after one week. Then PBS, 5×10^5 Truncated CAR-iMAC targeting GPC3 and mature GPC3-G-CAR-iMAC were inoculated at the tumor site for treatment. Tumors were collected from tumor-bearing mice 60 days after treatment, and the tumor volumes were measured and recorded in groups.

[0087] The results are as follows Fig. 9 The results showed that the malignant progression of tumors in mice inoculated with HepG2 tumor cells was significantly alleviated after 80 days of treatment with G-CAR-iMAC targeting GPC3. Four of the five tumor-bearing mice achieved almost complete remission ( Fig. 9 The statistical results of tumor volume after tumor treatment are shown in Figure A. Fig. 9 As shown in B. This result shows that G-CAR-iMAC has ideal tumor therapeutic efficacy.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A chimeric antigen receptor, characterized in that From N-terminus to C-terminus, it includes the extracellular domain, transmembrane domain and intracellular signaling domain; The extracellular domain includes a signal peptide, a single-chain variable region targeting an antigen, and a CD8α hinge from the N-terminus to the C-terminus; the nucleic acid sequence of the signal peptide is shown in SEQ ID NO.1; the nucleic acid sequence of the CD8α hinge is shown in SEQ ID NO.4; The transmembrane domain is the transmembrane domain of CD8α protein, and the nucleic acid sequence is shown in SEQ ID NO.5; The intracellular signaling domain includes the Box1 domain of GM-CSFRα and the GM-CSFRβ intracellular key activation domain from the N-terminus to the C-terminus; the nucleic acid sequence of the Box1 domain of GM-CSFRα is shown in SEQ ID NO.6; the sequence of the GM-CSFRβ intracellular key activation domain is shown in SEQ ID NO.

7.

2. The chimeric antigen receptor according to claim 1, characterized in that The single-chain variable region targeting the antigen includes a single-chain variable region targeting EGFRvIII or a single-chain variable region targeting GPC3; The nucleic acid sequence of the single-chain variable region targeting EGFRvIII is shown in SEQ ID NO.2; The nucleic acid sequence of the single-chain variable region targeting GPC3 is shown in SEQ ID NO.

3.

3. The chimeric antigen receptor according to claim 1, characterized in that The Box1 domain of GM-CSFRα and the key intracellular activation domain of GM-CSFRβ are connected by a Linker; The linker nucleic acid sequence is shown in SEQ ID NO.

8.

4. A nucleic acid, characterized in that The nucleic acid encodes the chimeric antigen receptor according to any one of claims 1 to 3.

5. A carrier, characterized in that The vector carries the nucleic acid of claim 4.

6. A human induced pluripotent stem cell, characterized in that: The human induced pluripotent stem cells carry the nucleic acid of claim 4, or contain the vector of claim 5, or express the chimeric antigen receptor of any one of claims 1-3.

7. A method for preparing myeloid precursor cell-like macrophages, characterized in that: The following steps are involved: The human induced pluripotent stem cells according to claim 6 are suspended and cultured in MI medium containing ROCK inhibitor for 2-4 days to form embryoid bodies, and then cultured in MI medium for 1-2 days, HS medium for 5-7 days, and ME-1 medium for 3-4 days, and then continue to be cultured in ME-2 medium and harvest myeloid precursor cell-like macrophages; The MI medium is an APEL II medium containing 30-80 ng / mL BMP4 and 30-50 ng / mL Activin A; The HS medium is an APEL II medium containing 30-80 ng / mL BMP4, 50-100 ng / mL VEGF, 100-150 ng / mL SCF, 20-50 ng / mL IL-3, 25-50 ng / mL IL-6, 25-50 ng / mL IL-11 and 25-50 ng / mL Flt-3L; The ME-1 medium is an APEL II medium containing 10-20 ng / mL bFGF, 50-100 ng / mL VEGF, 50-100 ng / mL SCF, 10-20 ng / mL IGF1, 25-50 ng / mL IL-3, 30-50 ng / mL TPO, 25-50 ng / mL M-CSF and 25-50 ng / mL GM-CSF; The ME-2 medium is X-VIVO containing 20-40 ng / mL bFGF, 50-100 ng / mL VEGF, 50-80 ng / mL SCF, 10-20 ng / mL IGF1, 25-50 ng / mL IL-3, 25-50 ng / mL M-CSF and 25-50 ng / mL GM-CSF. TM -15 medium or StemSpan™ H3000 medium.

8. The preparation method according to claim 7, characterized in that: In the MI medium containing ROCK inhibitor, the concentration of ROCK inhibitor is 8-10 nM; The ROCK inhibitors include Y27632.

9. A myeloid precursor cell-like macrophage, characterized in that: The preparation is obtained by the preparation method described in claim 7 or 8.

10. A mature macrophage, characterized in that: The myeloid precursor cell-like macrophages of claim 9 are cultured in X-VIVO containing 20-50 ng / mL GM-CSF and 50-100 ng / mL M-CSF. TM -15 medium.

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