Chimeric antigen receptor, human induced pluripotent stem cells, myeloid precursor cell-like macrophages and preparation methods thereof

Abstract

The present invention provides a chimeric antigen receptor, human induced pluripotent stem cells, myeloid precursor cell-like macrophages and preparation methods thereof, relating to the field of biomedical technology. The chimeric antigen receptor constructed by the present invention can enable hiPSCs expressing the chimeric antigen receptor to 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 recovery, as well as low immunogenicity and migration and infiltration into solid tumors. They shorten the cycle of their preparation and clinical treatment, reduce secondary risks such as host-versus-graft disease, and provide a continuous and reliable cell quantity guarantee for the clinical treatment of tumors. This makes it a truly "off-the-shelf" cell product. The myeloid precursor cell-like macrophages can be stimulated to mature in the presence of exogenous factors and the tumor microenvironment, and then have stronger target activation ability, M1 polarization level and antigen presentation ability, with more promising clinical translation 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] hiPSCs (human induced pluripotent stem cells) have the potential to differentiate into all cell types in the human body. Myeloid cells derived from iPSCs, including macrophages (iMACs) and dendritic cells (iDCs), hold significant clinical significance for cancer immune cell therapy and vaccine development. However, currently, iPSC-derived myeloid cells must be differentiated on-demand based on the needs of on-demand cancer treatment. This is time-consuming and labor-intensive, and poses challenges in effective cryopreservation and maintaining high resuscitation activity after cryopreservation. This significantly prolongs treatment cycles and may even miss the optimal therapeutic window. Therefore, high viability and efficient proliferation of immune cells after cryopreservation are crucial to achieving the "off-the-shelf" strategy of readily available immune cells, thereby seizing therapeutic opportunities. Currently, there are no studies or patents demonstrating the differentiation of iPSCs into myeloid progenitor cells with rapid proliferation and high resuscitation activity after cryopreservation. For example, the myeloid cells derived from patents for iPSC differentiation into macrophages (e.g., authorization publication numbers CN109082411B, CN109266618B, CN116731967A, CN115433715A) and iPSC differentiation into DCs (e.g., CN106852385A, CN110238265A, CN105272293A, US20110286965A1, WO2013142677A1, EP2612336A1) are all terminally differentiated cells, which cannot be effectively cryopreserved or rapidly proliferate after differentiation. Therefore, developing readily available myeloid immune cells with high growth potential and high vitality after resuscitation will be key to promoting the timely and effective treatment of corresponding diseases with iPSC-derived myeloid immune cells and shortening treatment cycles.

[0003] Furthermore, iPSC-derived chimeric antigen receptor (CAR) macrophages (CAR-iMACs) currently used in cell therapy face challenges such as poor targeted activation, low M1 polarization, susceptibility to M2 polarization in the tumor microenvironment, insufficient antigen presentation capacity, and low cytokine secretion, which severely hinder their clinical translation.

[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 human induced pluripotent stem cells.

[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:

[0013] In a first aspect, the present invention provides a chimeric antigen receptor comprising, from N-terminus to C-terminus, an extracellular domain, a transmembrane domain, and an intracellular signaling domain;

[0014] 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;

[0015] The transmembrane domain is the transmembrane domain of CD8α protein, and the nucleic acid sequence is shown in SEQ ID NO.5;

[0016] 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.

[0017] 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;

[0018] The nucleic acid sequence of the single-chain variable region targeting EGFRvIII is shown in SEQ ID NO.2;

[0019] The nucleic acid sequence of the single-chain variable region targeting GPC3 is shown in SEQ ID NO.3.

[0020] 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;

[0021] The linker nucleic acid sequence is shown in SEQ ID NO.8.

[0022] In a second aspect, the present invention provides a nucleic acid encoding the chimeric antigen receptor.

[0023] In a third aspect, the present invention provides a vector carrying the above-mentioned nucleic acid.

[0024] 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.

[0025] In a fifth aspect, the present invention provides a method for preparing myeloid precursor cell-like macrophages, comprising the following steps:

[0026] The human induced pluripotent stem cells are suspended and cultured in MI medium containing a 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 cultured in ME-2 medium to harvest myeloid progenitor cell-like macrophages;

[0027] The MI medium is APEL II medium containing 30-80 ng / mL BMP4 and 30-50 ng / mL Activin A;

[0028] The HS medium is 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;

[0029] The ME-1 medium is 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;

[0030] 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.

[0031] As a further technical solution, in the MI culture medium containing the ROCK inhibitor, the concentration of the ROCK inhibitor is 8-10 nM;

[0032] The ROCK inhibitors include Y27632.

[0033] In a sixth aspect, the present invention provides a myeloid precursor cell-like macrophage prepared by the above-mentioned preparation method.

[0034] In a seventh aspect, the present invention provides a mature macrophage, which is prepared 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.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] 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 recovery, as well as low immunogenicity and migration and infiltration into solid tumors. They shorten the cycle of their preparation and clinical treatment, reduce secondary risks such as host-versus-graft, and provide a continuous and reliable cell quantity guarantee for the clinical treatment of tumors. This makes it a truly "spot-type" cell product. In addition, the myeloid precursor-like macrophages can be stimulated to mature in the presence of exogenous factors and the tumor microenvironment, and then have stronger target activation ability and M1 polarization level, as well as stronger antigen presentation ability, and have more promising clinical transformation prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0038] Figure 1 Schematic diagram of the gene expression sequence structure of CAR;

[0039] Figure 2 G-CAR-iMACs differentiated from G-CAR-expressing hiPSCs exhibit phenotypic characteristics similar to myeloid progenitor cells;

[0040] Figure 3 Detection of the proliferation capacity of G-CAR-iMAC in myeloid precursor cell-like state;

[0041] Figure 4 To detect the activity of G-CAR-iMAC in myeloid precursor cell-like state after cryopreservation and resuscitation;

[0042] Figure 5 Detection of the induction of G-CAR-iMAC in myeloid precursor cell-like state into mature G-CAR-iMAC;

[0043] Figure 6 To detect the targeted anti-tumor ability of G-CAR-iMAC;

[0044] Figure 7 To detect the polarization level of G-CAR-iMAC targeting EGFRvIII;

[0045] Figure 8 To test the anti-inflammatory presentation ability of G-CAR-iMAC;

[0046] Figure 9 To verify the anti-tumor efficacy of G-CAR-iMAC. DETAILED DESCRIPTION

[0047] Below in conjunction with embodiment and example, embodiment of the present invention is described in detail, but those skilled in the art will appreciate that the following embodiment and example are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise all fall within the scope of protection of the present invention. Unspecified conditions are carried out according to the conditions of normal conditions or manufacturer's recommendations. Reagents used or instruments not specified by the manufacturer are conventional products that can be purchased commercially.

[0048] In a first aspect, the present invention provides a chimeric antigen receptor comprising, from N-terminus to C-terminus, an extracellular domain, a transmembrane domain, and an intracellular signaling domain;

[0049] The extracellular domain includes a signal peptide, a single-chain variable region targeting an antigen, and CD8 αhinge from the N-terminus to the C-terminus;

[0050] The nucleic acid sequence of the signal peptide is shown in SEQ ID NO.1.

[0051] The nucleic acid sequence of the CD8 αhinge is shown in SEQ ID NO.4.

[0052] The transmembrane domain is the transmembrane domain of CD8α protein, and the nucleic acid sequence is shown in SEQ ID NO.5.

[0053] The intracellular signaling domain includes, from the N-terminus to the C-terminus, the Box1 domain of GM-CSFRα and the key intracellular activation domain of GM-CSFRβ;

[0054] The nucleic acid sequence of the Box1 domain of GM-CSFRα is shown in SEQ ID NO.6.

[0055] The sequence of the GM-CSFRβ intracellular key activation domain (GM-CSFRβ 461-897 aa) is shown in SEQ ID NO.7.

[0056] Based on the concept of synthetic biology, this invention uses genetic engineering to synthesize a novel CAR (G-CAR) containing the key intracellular activation domains of GM-CSFRα and GM-CSFRβ. iPSCs expressing this novel G-CAR can differentiate into macrophage precursor cells with high proliferation capacity and high recovery activity after cryopreservation, and can be further induced to differentiate into functionally enhanced G-CAR macrophages. This strategy has achieved the following breakthroughs in the application of CAR macrophages in clinical treatment: 1. The ability to be frozen and the high activity after cryopreservation and thawing make iPSC-derived myeloid precursor-like macrophages have the advantage of being available for immediate use, shortening the cycle of macrophage preparation and clinical treatment; 2. Their high growth capacity 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 the presence of exogenous factors and the tumor microenvironment, and then have stronger target activation ability and M1 polarization level, as well as stronger antigen presentation ability, so the new generation of G-CAR macrophages has more promising clinical translation prospects; 4. The myeloid precursor-like macrophages of the present invention have the characteristics of low immunogenicity, which greatly enhances their versatility in clinical treatment applications and further gives them "off-the-shelf" characteristics. 5. The myeloid precursor-like macrophages of the present invention have smaller cell bodies and highly express tumor chemokine receptors, thus having stronger ability to migrate to tumors and infiltrate into solid tumors, with inherent advantages in treating solid tumors.

[0057] 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;

[0058] The nucleic acid sequence of the single-chain variable region targeting EGFRvIII is shown in SEQ ID NO.2.

[0059] The nucleic acid sequence of the single-chain variable region targeting GPC3 is shown in SEQ ID NO.3.

[0060] In some optional embodiments, the Box1 domain of GM-CSFRα and the key intracellular activation domain of GM-CSFRβ are connected via a Linker;

[0061] The linker nucleic acid sequence is shown in SEQ ID NO.8.

[0062] In a second aspect, the present invention provides a nucleic acid encoding the chimeric antigen receptor.

[0063] The nucleic acid is capable of expressing the chimeric antigen receptor of the present invention.

[0064] In a third aspect, the present invention provides a vector carrying the above-mentioned nucleic acid.

[0065] 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.

[0066] In some alternative embodiments, the vector comprises a lentiviral vector.

[0067] 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.

[0068] 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.

[0069] In a fifth aspect, the present invention provides a method for preparing myeloid precursor cell-like macrophages, comprising the following steps:

[0070] The human induced pluripotent stem cells are suspended and cultured in MI medium containing a 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 cultured in ME-2 medium to harvest myeloid progenitor cell-like macrophages;

[0071] 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;

[0072] 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 can be, for example, but not limited to 30 ng / mL, 50 ng / mL, or 80 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 100 ng / mL, 120 ng / mL, or 150 ng / mL; the concentration of IL-3 can be, for example, but not limited to 20 ng / mL, 40 ng / mL, or 50 ng / mL; the concentration of IL-6 can be, for example, but not limited to 25 ng / mL, 40 ng / mL, or 50 ng / mL; the concentration of IL-11 can be, for example, but not limited to 25 ng / mL, 40 ng / mL, or 50 ng / mL; the concentration of Flt-3L can be, for example, but not limited to 25 ng / mL, 40 ng / mL, or 50 ng / mL;

[0073] 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 can be, for example, but not limited to, 10 ng / mL, 15 ng / mL, or 20 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, 80 ng / mL, or 100 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 TPO can be, for example, but not limited to 30 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;

[0074] 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; and the concentration of GM-CSF can be, for example, but not limited to 25 ng / mL, 40 ng / mL or 50 ng / mL.

[0075] This 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 recovery, 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 diseases, and provides a continuous and reliable cell quantity guarantee for the clinical treatment of tumors.

[0076] In some optional embodiments, in the MI culture medium containing a ROCK inhibitor, the concentration of the ROCK inhibitor may be, for example, but not limited to, 8 nM, 9 nM or 10 nM;

[0077] The ROCK inhibitor includes but is not limited to Y27632.

[0078] In a sixth aspect, the present invention provides a myeloid precursor cell-like macrophage prepared by the above-mentioned preparation method.

[0079] In a seventh aspect, the present invention provides a mature macrophage, which is prepared 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.

[0080] The myeloid precursor-like macrophages provided by the present invention can be stimulated to mature in the presence of exogenous factors and the 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.

[0081] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.

[0082] Example 1

[0083] Part I: Design of G-CAR expression sequence

[0084] G-CAR and Truncated CAR were designed respectively, and their structures are as follows Figure 1As shown, the G-CAR protein gene expression sequence of 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 targets EGFRvIII and GPC3 antigens, and a CD8α hinge sequence. The transmembrane domain (TM) is derived from the transmembrane domain protein sequence of the CD8α protein. The intracellular portion is composed of the Box 1 domain of GM-CSFRα and the key intracellular activation domain of GM-CSFRβ connected in series via a linker sequence. The intracellular GM-CSFRβ activation domain of G-CAR is amino acids 461-849 (461-849aa). Truncated CAR, which does not contain the intracellular domain, served as a negative control CAR for subsequent validation experiments.

[0085] The chimeric antigen receptors for EGFRvIII antigen are as follows:

[0086] scFv (EGFRvIII)-G-CAR, the nucleic acid sequence of which is shown in SEQ ID NO.9;

[0087] scFv (EGFRvIII) Truncated CAR, the nucleic acid sequence of which is shown in SEQ ID NO. 10;

[0088] The chimeric antigen receptors for GPC3 antigen are as follows:

[0089] scFv (GPC3)-G-CAR, the nucleic acid sequence of which is shown in SEQ ID NO.11;

[0090] scFv (GPC3) Truncated CAR, the nucleic acid sequence of which is shown in SEQ ID NO.12.

[0091] At the same time, the above CARs are linked to EGFP green fluorescent protein (nucleic acid sequence shown in SEQ ID NO. 14) via a T2A element (nucleic acid sequence shown in SEQ ID NO. 13) to form a fusion expression sequence. The expression of EGFP can indicate the expression of the CAR.

[0092] Part II: Construction of hiPSC differentiation system expressing CAR

[0093] (1) Construct a lentiviral overexpression system for CAR.

[0094] CARs were cloned into the lentiviral expression plasmid Lenti-EF1a-T2A-EGFP-PGK-Puromycin through 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. Plasmids were amplified in Escherichia coli.

[0095] (2) Construct a hiPSC cell line that stably overexpresses CAR.

[0096] The constructed CAR lentiviral expression plasmid and the lentiviral packaging plasmids psPAX2 and pMD2.G were transfected into HEK293T cells at a mass ratio of 4:3:1. Cell supernatants were harvested at 48, 60, and 72 hours of culture and concentrated to produce lentiviral particles containing the various CAR expression genes. Subsequently, the lentiviral particles containing the various CAR expression genes were infected into wild-type hiPSC cell lines using the cationic compound polybrane to construct CAR-hiPSC cell lines stably overexpressing CAR. This lentiviral overexpression system allows for the stable integration of CAR expression genes into the hiPSC genome. The expression ratio and fluorescence brightness of EGFP in hiPSC cells indicate the efficiency of CAR expression in hiPSCs.

[0097] (3) Construct a hiPSC differentiation system based on embryoid body (EB) formation.

[0098] Spin-EB-based hiPSC differentiation system

[0099] 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).

[0100] 2) Collect the hiPSCs digested to single cells in the previous step and resuspend them in MI medium (APEL II medium + 30 ng / mL BMP4 + 30 ng / mL Activin A) containing the ROCK inhibitor (Y27632). Transfer 60 μl of culture volume per well to a low-adhesion round-bottom 96-well plate and incubate in a 37°C, 5% CO2 incubator for 24 hours. This is day 0. After 24 hours of suspension culture, EBs will form.

[0101] 3) On day 1-2, replace the suspended EBs with MI medium (APEL II medium + 20 ng / mL BMP4 + 30 ng / mL Activin A) without the ROCK inhibitor (Y27632). Continue culturing for 48 hours.

[0102] 4) On days 3-7, 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 / mL Flt-3L).

[0103] 5) On days 8-10, change to ME-1 medium (ME-1: APEL II medium + 10 ng / ml bFGF + 50 ng / ml VEGF + 50 ng / mL SCF + 10 ng / mL IGF1 + 25 ng / mL IL-3 + 30 ng / mL TPO + 50 ng / mL M-CSF + 50 ng / mL GM-CSF).

[0104] 6) On day 11, EBs were transferred to a 6-well plate coated with Matrigel (Corning, #354277). 16 EBs were placed in each well of the 6-well plate. At this time, the culture medium was changed to ME-2 medium (ME-2: X-VIVO TM-15 Medium / StemSpan™ H3000 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 medium continuously. During this period, a large number of myeloid progenitor cell-like G-CAR-iMACs (differentiated from G-CAR-hiPSCs) and Truncated CAR-iMACs (differentiated from Truncated CAR-hiPSCs) will be generated. During this stage, the supernatant is collected every two days, and the hiPSC-derived cells in the supernatant are collected by centrifugation. They are resuspended in fresh ME-2 Medium and transferred to a new 6-well plate.

[0105] (4) hiPSCs expressing G-CAR can be induced to differentiate into myeloid progenitor cell-like G-CAR-iMACs with naive characteristics.

[0106] The myeloid precursor cell-like G-CAR-iMAC harvested in step (3) was 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 in 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 barely 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 these four membrane proteins were highly expressed in Truncated CAR-iMAC and weakly expressed in G-CAR-iMAC ( Figure 2 C). The above results indicate that G-CAR-iMACs differentiated from G-CAR-expressing hiPSCs are in a macrophage precursor-like state without any treatment.

[0107] (5) G-CAR-iMAC in the myeloid precursor cell-like state has the ability to proliferate rapidly.

[0108] 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 Cells were cultured in a 96-well plate using a 15-well culture medium. At 0, 12, 36, and 60 hours of culture, 1 μl of CCK8 reagent (to monitor cell proliferation and toxicity) was added to each well. Cell viability was assessed by measuring OD values using a microplate reader. At least three replicate wells were set up for each cell sample at each time point.

[0109] The results are as follows Figure 3 The CCK8 proliferation assay was used to detect the cell viability of hiPSCs expressing G-CAR targeting EGFRvIII and differentiating them into myeloid progenitor cell-like G-CAR-iMAC cells 0-60 hours later. The results showed that myeloid progenitor cell-like G-CAR-iMAC cells had a stronger proliferation capacity than the control group (Truncated CAR-iMAC cells).

[0110] (6) G-CAR-iMAC in a myeloid precursor cell-like state has high cell activity after cryopreservation and can continue to proliferate steadily, indicating that G-CAR-iMAC has the characteristics of a spot cell drug.

[0111] 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 each reselected with 1 ml of commercial universal cell freezing medium and then frozen at -80°C. After 7 days of freezing, they were revived at 37°C and 1×10^4 myeloid progenitor cell-like G-CAR-iMAC and Truncated CAR-iMAC were each taken and incubated with 100 μl of X-VIVO containing 50 ng / mL M-CSF and 50 ng / mL GM-CSF, respectively. TM Cells were cultured in a 96-well plate using a 15-well culture medium. At 0, 24, 48, and 72 hours of culture, 1 μl of CCK8 reagent (to monitor cell proliferation and toxicity) was added to each well. Cell viability was assessed by measuring OD values using a microplate reader. At least three replicate wells were set up for each cell sample at each time point.

[0112] The results are as follows Figure 4As shown. The MTT assay was used to detect cell activity 0-70 hours after hiPSC expressing G-CAR targeting EGFRvIII differentiated into G-CAR-iMAC in a myeloid precursor cell-like state. 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 maintained sustained proliferation capacity as the culture time increased. This result indicates 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.

[0113] Part III: Further stimulation of myeloid progenitor cell-like G-CAR-iMAC to mature into G-CAR-iMAC

[0114] (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 After 72 hours of continuous culture in -15 medium, the cells were observed to show that mature G-CAR-iMAC was gradually obtained ( Figure 5 A in ).

[0115] (2) G-CAR-iMAC in the myeloid precursor cell-like state was cultured until day 10.

[0116] (3) Flow cytometry analysis showed that after myeloid precursor 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 ).

[0117] (4) Myeloid progenitor cell-like G1-CAR-iMACs stimulated with 50 ng / mL GM-CSF and 50 ng / 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).

[0118] The results are as follows Figure 5As shown, G-CAR-iMACs in a myeloid precursor cell-like state differentiated from hiPSCs expressing G-CAR targeting EGFRvIII can be matured into G-CAR-iMACs with true macrophage morphology and function by adding M-CSF and GM-CSF to stimulate them.

[0119] (5) G-CAR-iMAC has the ability to almost completely eliminate tumor cells.

[0120] The myeloid progenitor cell-like state G-CAR-iMAC differentiated from hiPSC expressing G-CAR was matured in vitro by M-CSF and GM-CSF respectively, 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 luciferase activity in the tumor was detected. Figure 6 shown.

[0121] The results showed that G-CAR-iMAC targeting EGFRvIII showed significant targeted killing ability against 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 Figure 2, where the blank control represents neither tumor cells nor G-CAR-iMAC; the untreated group represents U87MG tumor cells expressing only EGFRvIII antigen; the EGFRvIII Truncated CAR-iMAC represents the use of these cells to kill U87MG cells expressing EGFRvIII antigen; the EGFRvIII-G-CAR-iMAC represents the use of these cells to kill U87MG cells expressing EGFRvIII antigen; and the EGFRvIII-G-CAR-iMAC (IFN-gamma / LPS) represents the use of these cells treated with 100 μg / ml IFN-gamma and LPS for 24 hours to kill U87MG cells expressing EGFRvIII antigen. G-CAR-iMAC targeting GPC3 exhibited significant targeted killing ability against HepG2 hepatocellular carcinoma cells expressing GPC3 antigen both without prepolarization and after IFN-gamma / LPS polarization. Compared with the control Truncated CAR-iMAC, G-CAR-iMAC almost completely eliminated HepG2 ( Figure 6In Figure B, the blank control represents the absence of both tumor cells and G-CAR-iMAC; the untreated group represents HepG2 tumor cells expressing only GPC3 antigen; GPC3 Truncated CAR-iMAC represents the use of these cells to kill HepG2 cells expressing GPC3 antigen; GPC3-G-CAR-iMAC represents the use of these cells to kill U87MG cells expressing EGFRvIII antigen; GPC3-G-CAR-iMAC (IFN-gamma / LPS) represents the use of these cells treated with 100 μg / ml IFN-gamma and LPS for 24 hours to kill HepG2 cells expressing GPC3 antigen).

[0122] (6) G-CAR-iMAC can maintain a higher M1 polarization level during the anti-tumor process and has stronger anti-tumor efficacy.

[0123] 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.

[0124] By detecting the proportion 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 proportion 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.

[0125] (7) G-CAR-iMAC has stronger antigen presentation ability.

[0126] 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:

[0127] Jurkat group: experimental group with only Jurkat cells;

[0128] U87MG EGFRvIII-NY+Jurkat: U87MG expressing NY-ESO-1 EGFRvIII experimental group in which cells were co-cultured with Jurkat cells;

[0129] 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;

[0130] Truncated CAR-iMAC+U87MG EGFRvIII :Truncated CAR-iMAC and U87MG EGFRvIII Co-culture experimental group;

[0131] G-CAR-iMAC+Jurkat: experimental group in which G-CAR-iMAC targeting EGFRvIII was co-cultured with Jurkat;

[0132] U87MG EGFRvIII +Truncated CAR-iMAC+Jurkat: U87MG EGFRvIII The experimental group in which three cells were co-cultured: Truncated CAR-iMAC and Jurkat cells;

[0133] U87MG EGFRvIII +G-CAR-iMAC+Jurkat: U87MG EGFRvIII , G-CAR-iMAC targeting EGFRvIII, and Jurkat cell co-cultured experimental group;

[0134] 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;

[0135] U87MG EGFRvIII -NY+G-CAR-iMAC+Jurkat: U87MG expressing NY-ESO-1 EGFRvIII The experimental group co-cultured three cells: cells, G-CAR-iMAC targeting EGFRvIII, and Jurkat cells.

[0136] The above experimental groups were cultured for 24 hours, and then the expression ratio of T cell activation marker protein CD69 was detected. Figure 8The 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 the positive control cells (U87MG EGFRvIII This result indicates that G-CAR-iMAC has enhanced antigen presentation function.

[0137] (8) G-CAR-iMAC has significantly enhanced tumor therapeutic efficacy.

[0138] Immunodeficient mice were subcutaneously inoculated with 1×10^5 HepG2 cells expressing the GPC3 antigen. Tumors formed after one week. The tumors were then inoculated with PBS, followed by 5×10^5 GPC3-targeting Truncated CAR-iMACs and mature GPC3-G-CAR-iMACs. Tumors were harvested 60 days after treatment, and tumor volumes were measured and recorded.

[0139] The results are as follows Figure 9 The results showed that after 80 days of treatment with G-CAR-iMAC targeting GPC3, the progression of tumor malignancy in HepG2 tumor-bearing mice was significantly alleviated. Four of the five tumor-bearing mice achieved almost complete remission ( Figure 9 The statistical results of tumor volume after tumor treatment are as follows. Figure 9 As shown in Figure 3 B. This result indicates that G-CAR-iMAC has ideal tumor therapeutic efficacy.

[0140] 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 above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above 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 The nucleic acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.9 or SEQ ID NO.

11.

2. A nucleic acid, characterized in that The nucleic acid encodes the chimeric antigen receptor of claim 1.

3. A carrier, characterized in that The vector carries the nucleic acid according to claim 2.

4. A human induced pluripotent stem cell, characterized in that The human induced pluripotent stem cells carry the nucleic acid of claim 2, or contain the vector of claim 3, or express the chimeric antigen receptor of claim 1.

5. 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 4 are suspended and cultured in MI medium containing a 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 cultured in ME-2 medium to harvest myeloid progenitor cell-like macrophages. The MI medium is APEL II medium containing 30-80 ng / mL BMP4 and 30-50 ng / mL Activin A; The HS medium is 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 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.

6. The preparation method according to claim 5, characterized in that In the MI culture medium containing the ROCK inhibitor, the concentration of the ROCK inhibitor is 8-10 nM; The ROCK inhibitors include Y27632.

7. A myeloid precursor cell-like macrophage, characterized in that: The preparation method according to claim 5 or 6 is used for preparation.

8. A mature macrophage, characterized in that: The myeloid precursor cell-like macrophages according to claim 7 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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