Anti-tumor modification method of stem / progenitor cell-derived neutrophil and application of anti-tumor modification method

Abstract

The invention provides an anti-tumor modification method of stem / progenitor cell-derived neutrophil, which comprises the following steps: extracting mouse bone marrow-derived Lin-progenitor cells and human umbilical cord hematopoietic stem / progenitor cells, modifying ER-Hoxb8 progenitor cells by gene editing, and inducing the ER-Hoxb8 progenitor cells into neutrophil; three modification strategies of chimeric antigen receptor CAR gene engineering modification, high expression MET gene modification and high expression of IFN-gamma while the TGFBR2 gene is stably knocked out are respectively adopted, and the anti-tumor phenotype of the neutrophil N1 is maintained. Compared with the prior art, the engineering neutrophile granulocyte has the following advantages that the engineering neutrophile granulocyte prepared by the method has the advantages of infinite sources, long-time preservation, safety, controllability, efficient and rapid preparation and the like; the engineered neutrophil can maintain the N1 antitumor phenotype and promote the antitumor function; the immunosuppression microenvironment of the solid tumor can be improved and the treatment effect can be improved by singly using or combining the immune checkpoint inhibitor.

Description

Technical Field

[0001] The present application relates to the field of immune cell therapy and biotechnology, and specifically to an anti-tumor modification method of stem / progenitor cell-derived neutrophils and its application. Background Art

[0002] In the past decade, adoptive immune cell therapy represented by CAR-T and immune checkpoint inhibitors have achieved remarkable therapeutic effects in tumor treatment, especially in hematological tumors. However, the treatment of solid tumors still faces many difficulties, such as refractory solid tumors such as pancreatic cancer, EGFR mutant lung cancer, and triple-negative breast cancer. Recently, there have been reports of using CAR to gene-edit macrophages to enhance their phagocytic ability and make them anti-tumor effector cells. Given the similarities between neutrophils and macrophages and their common innate anti-tumor immune response capabilities, the use of gene editing technology to engineer neutrophils may become an effective strategy for tumor immunotherapy.

[0003] Neutrophils, which belong to innate immune cells, have functional phenotype plasticity and can respond to different environmental signals, playing an important role in coping with pathological environments and maintaining immune homeostasis. Neutrophils respond to specific stimuli in the tumor microenvironment (TME) and reversibly transform into anti-tumor N1 type (pro-inflammatory, classical activation) or pro-tumor N2 type (anti-inflammatory, alternative activation) phenotypes, exerting anti-tumor or pro-tumor functions, respectively.

[0004] Neutrophils are the most abundant white blood cells in the blood circulation and are closely related to the occurrence and development of cancer. In addition to the research findings on the cancer-promoting effects of neutrophils, neutrophils have been reported to have early anti-cancer effects and show strong cytotoxicity to a variety of cancer cells. Neutrophils promote tumor cell apoptosis by releasing NO and ROS, and at the same time, neutrophils play an anti-cancer role through Fc-mediated plasma membrane uptake and the creation of an anti-tumor microenvironment. Activated neutrophils secrete various pro-inflammatory cytokines and activate T cells through antigen presentation. At the same time, neutrophils also show high migration ability and the ability to penetrate tissues that are difficult for other cells to enter (such as the blood-brain barrier BBB, the dense extracellular matrix of solid tumors), and can be used as effective drug carriers. However, under the influence of the tumor immunosuppressive microenvironment and cytokines, neutrophils are largely domesticated into N2-like phenotype cells, which play a pro-tumor function by promoting angiogenesis, extracellular matrix remodeling, metastasis and immunosuppression. Cell therapy strategies using neutrophils need to consider regulating the intrinsic properties of neutrophils to effectively trigger anti-tumor responses. Therefore, constructing anti-cancer neutrophils with high migration and infiltration capabilities may become a new strategy for immune cell engineering.

[0005] In addition, due to the fact that neutrophils themselves express abundant pattern recognition receptors, have a short half-life and cannot proliferate in vitro, it is very difficult to perform gene editing on neutrophils. Stem cells provide an alternative cell resource for the production of neutrophil drugs. They can be stably transduced to express target genes and then differentiate into neutrophils. The most widely used production strategy currently comes from human pluripotent stem cells iPSCs. Roberts et al. first reported the generation of neutrophils with cytolytic activity from hematopoietic stem cells in 1998, which provided a reference strategy for utilizing the differentiation characteristics of stem / progenitor cells to produce engineered neutrophils.

[0006] Neutrophil cell therapy, like CAR-T, also has many difficulties:

[0007] (1) Difficulty in obtaining cell sources: Neutrophils have a short half-life, and how to obtain a large number of neutrophils for cell therapy in vitro has become one of the key issues to be solved. iPSCs face problems such as harsh culture conditions and quality uniformity.

[0008] (2) Transformation of cell phenotype: Neutrophils in the tumor microenvironment may exhibit anti-tumor N1 phenotype and pro-tumor N2 phenotype, and the dynamic transformation between the two affects the progression of tumor development.

[0009] (3) Low efficiency of gene editing of neutrophils: Neutrophils are resistant to gene modification, and it is difficult to transform neutrophils based on gene editing technology. Summary of the invention

[0010] In response to the above-mentioned technical limitations, the present application proposes an anti-tumor modification method for stem / progenitor cell-derived neutrophils and its application; it involves the engineering modification of the proliferation of hematopoietic stem cell progenitor cells, and the preparation of MET or CAR or SgTGFBR2-IFN-γ gene-edited engineered Hoxb8 neutrophils and their combined immune checkpoint inhibitor treatment in solid tumors, which overcomes the deficiencies and defects mentioned in the background technology.

[0011] To achieve the above objectives, this application adopts the following technical solutions:

[0012] The invention of the present application is to provide an anti-tumor modification method of stem / progenitor cell-derived neutrophils, comprising the following steps:

[0013] S1. Preparation of stem / progenitor cell-derived neutrophils:

[0014] Extraction of Lin from mouse bone marrow - Progenitor cells and human umbilical cord hematopoietic stem / progenitor cells, using gene editing technology to modify ER-Hoxb8 progenitor cells and induce them to become neutrophils;

[0015] S2. Maintenance of Neutrophil Anti-tumor Phenotype:

[0016] Three transformation strategies were used, namely, genetic engineering modification of chimeric antigen receptor CAR, high expression of MET gene modification, and high expression of IFN-γ while stably knocking out the TGFBR2 gene, to maintain the neutrophil N1 anti-tumor phenotype.

[0017] Optionally, in the above-mentioned anti-tumor modification method of stem / progenitor cell-derived neutrophils, in step S1, the mouse bone marrow-derived Lin - The progenitor cells are Lin-progenitor cells; the human umbilical cord hematopoietic stem / progenitor cells are CB-CD34 + Cells; the gene editing technology is to use ER-Hoxb8 lentivirus transfection and then flow sorting to obtain high-purity ER-Hoxb8 progenitor cells with proliferation ability.

[0018] Optionally, in the above-mentioned anti-tumor modification method of stem / progenitor cell-derived neutrophils, in step S2, the chimeric antigen receptor CAR genetic engineering modification is to use CAR lentivirus to transfect proliferative ER-Hoxb8 progenitor cells, flow sorting to obtain highly pure and proliferative Anti-CEA-CAR-Hoxb8 progenitor cells, remove 4-hydroxytamoxifen (4-OHT), and then add granulocyte colony stimulating factor (G-CSF) to induce differentiation to obtain neutrophils.

[0019] Optionally, in the above-mentioned anti-tumor modification method of stem / progenitor cell-derived neutrophils, in step S2, the high-expression MET gene modification is to use MET lentivirus to transfect proliferative ER-Hoxb8 progenitor cells, flow sorting to obtain high-purity and proliferative MET-Hoxb8 progenitor cells, removing 4-hydroxytamoxifen 4-OHT, and then adding granulocyte colony-stimulating factor G-CSF to induce differentiation to obtain neutrophils.

[0020] Optionally, in the above-mentioned anti-tumor modification method of stem / progenitor cell-derived neutrophils, in step S2, the stable knockout of TGFBR2 gene and the high expression of IFN-γ modification are performed by transfecting proliferative ER-Hoxb8 progenitor cells with SpCas9-TGFBR2-IFN-γ lentivirus, flow sorting to obtain high-purity and proliferative sgTGFBR2-Hoxb8 progenitor cells, removing 4-hydroxytamoxifen 4-OHT, and then adding granulocyte colony stimulating factor G-CSF to induce differentiation to obtain neutrophils.

[0021] The second inventive point of the present application is to provide the use of the anti-tumor modified stem / progenitor cell-derived neutrophils provided by the above method in the preparation of biological products for treating solid tumors.

[0022] Optionally, in the above application, the solid tumor is selected from one or more of pancreatic cancer, EGFR mutant lung cancer and breast cancer.

[0023] The third inventive point of the present application is to provide the use of the anti-tumor modified stem / progenitor cell-derived neutrophils provided by the above method in combination with immune checkpoint inhibitors in the preparation of biological products for the treatment of solid tumors.

[0024] Optionally, in the above application, the immune checkpoint inhibitor is selected from one or more of a PD-1 inhibitor, a PD-L1 inhibitor and a CD47 inhibitor.

[0025] Optionally, in the above application, the solid tumor is selected from one or more of pancreatic cancer, EGFR mutant lung cancer and breast cancer.

[0026] Compared with the prior art, this application has the following advantages:

[0027] 1. The engineered neutrophils (MET-neutrophils, Anti-CEA-CAR-neutrophils, sgTGFBR2-neutrophils) prepared in this application are derived from the induced differentiation of normal Hoxb8 hematopoietic stem cells that proliferate in a 4-hydroxytamoxifen-dependent manner. They are safer than tumor-derived neutrophils such as HL-60, easier to obtain than neutrophils derived from iPSCs, and more convenient to obtain than the autologous immune cells of clinical patients. They are easier to transform in vitro, more economical, and have more uniform quality. They have the advantages of unlimited sources, long-term storage, safe and controllable, and efficient and rapid preparation.

[0028] 2. The engineered neutrophils provided in this application respectively adopt three transformation strategies, namely, CAR gene engineering, high expression of MET gene, stable knockout of TGFBR2 gene and high expression of IFN-γ, to maintain the neutrophil N1 anti-tumor phenotype and promote the anti-tumor function of neutrophils;

[0029] 3. The engineered neutrophils provided in this application can significantly improve the immunosuppressive microenvironment of refractory solid tumors such as pancreatic cancer, EGFR mutant lung cancer, and breast cancer, and combined with immune checkpoint inhibitors can effectively improve their therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of constructing proliferative Hoxb8 progenitor cells with TGFBR2 knockout and high expression of IFN-γ using CRISPR-Cas9 technology; Figure 1 A is the design of SgTGFBR2-IFN-γ vector. Figure 1B is the protein expression of TGFBR2 in proliferative sgTGFBR2-Hoxb8 progenitor cells. Figure 1 C is the flow cytometric analysis of the stemness marker CD117 of proliferative sgTGFBR2-Hoxb8 progenitor cells. Figure 1 D is the karyotype analysis of sgTGFBR2-Hoxb8 progenitor cells. Figure 1 E: CCK-8 was used to detect the proliferation of sgTGFBR2-Hoxb8 progenitor cells.

[0031] Figure 2 This is the identification result of sgTGFBR2-neutrophils; Figure 2 A is the flow cytometric analysis of the stemness marker CD117 in sgTGFBR2-neutrophils. Figure 2 B is flow cytometry analysis of neutrophil markers Ly6G and CD11b in sgTGFBR2-neutrophils. Figure 2 C is the karyotype analysis of sgTGFBR2-neutrophils. Figure 2 D shows the protein expression of TGFBR2 in sgTGFBR2-neutrophils.

[0032] Figure 3 The in vitro antitumor effect of sgTGFBR2-neutrophils on pancreatic cancer PANC02 cells; Figure 3 A shows the lytic effect of sgTGFBR2-neutrophils on PANC02 cells. Figure 3 B is the ROS content after sgTGFBR2-neutrophil was co-incubated with PANC02 cells for 24 hours. Figure 3 C is the identification of the anti-tumor phenotype of sgTGFBR2-neutrophils. Figure 3 D is the NO content after sgTGFBR2-neutrophil was co-incubated with PANC02 cells for 24 hours. Figure 3 E- Figure 3 H is the detection of inflammatory factors IL-6, IL-10, IFN-γ and TNF-α after sgTGFBR2-neutrophil was co-incubated with PANC02 cells for 24 hours.

[0033] Figure 4 is the in vivo efficacy of sgTGFBR2-neutrophils in treating pancreatic cancer in mice; Figure 4 A is the weight change of tumor-bearing mice after engineered neutrophil therapy. Figure 4 B. Small animal in vivo imaging to detect the fluorescence signal of tumor mice treated with sgTGFBR2-neutrophils, and statistical analysis of the fluorescence signal. Figure 4 C shows representative images of tumors in the treatment group and control group and hematoxylin-eosin (HE) staining analysis.

[0034] Figure 5 is the construction and identification of MET-Hoxb8 progenitor cells and MET-neutrophils; Figure 5 A is the fluorescence imaging picture of MET-Hoxb8 progenitor cells. Figure 5 B is the flow cytometric analysis of the expression of Hoxb8-GFP and MET-mCherry in MET-Hoxb8 progenitor cells. Figure 5 C is the flow cytometric analysis of CD117, a stemness marker of MET-Hoxb8 progenitor cells. Figure 5 D is flow cytometry detection of MET-neutrophils neutrophil markers Ly6G and CD11b, Figure 5 E is the flow cytometric analysis of the stemness marker CD117 in MET-neutrophils. Figure 5 F- Figure 5 G is the karyotype analysis of MET-Hoxb8 progenitor cells and MET-neutrophils. Figure 5 H- Figure 5 I is the protein expression of MET in MET-Hoxb8 progenitor cells and MET-neutrophils.

[0035] Figure 6 It is the in vitro antitumor effect of MET-neutrophils on lung cancer LLC cells; Figure 6 A is a schematic diagram of the migration of MET-neutrophils across the vascular endothelium. Figure 6 B is the detection of MET-neutrophils' ability to migrate across vascular endothelium. Figure 6 C is the vascular adhesion function test of MET-neutrophils, Figure 6 D is the construction of EGFR mutant LLC cells, Figure 6 E is the lysis effect of MET-neutrophils on LLC-EGFR cells. Figure 6 F is the NO content after MET-neutrophil and LLC-EGFR cells were co-incubated for 24 hours. Figure 6 G is the ROS content after MET-neutrophil and LLC-EGFR cells were co-incubated for 24 hours. Figure 6 H is the detection of inflammatory factors IL-1β, IL-6, IL-10, IL-12, IFN-γ and TNF-α after MET-neutrophil was co-incubated with LLC-EGFR cells for 24 hours.

[0036] Figure 7 It is the in vivo efficacy of MET-neutrophils in treating lung cancer in mice; Figure 7 A is the tumor growth curve of the treatment group and the control group. Figure 7 B is the survival rate of tumor-bearing mice in the treatment group and the control group. Figure 7 C is the representative image of tumors in the treatment group and the control group. Figure 7 D is the tumor weight of the treatment group and the control group.

[0037] Figure 8 is the construction and identification of Anti-CEA-CAR-Hoxb8 progenitor cells and Anti-CEA-CAR-neutrophils; Figure 8 A- Figure 8 B is the expression of CD117, a stemness marker of Lin-progenitor cells and Anti-CEA-CAR-Hoxb8 progenitor cells, detected by flow cytometry. Figure 8 C is the expression of CD117, a stemness marker of Anti-CEA-CAR-neutrophils, detected by flow cytometry. Figure 8 D is the detection of neutrophil markers Ly6G and CD11b of Anti-CEA-CAR-neutrophils by cytometry. Figure 8 E is the karyotype analysis of Anti-CEA-CAR-Hoxb8 progenitor cells and Anti-CEA-CAR-neutrophils.

[0038] Fig. 9 It is the in vivo efficacy of Anti-CEA-CAR-neutrophils and combined with Anti-PD-1 in treating breast cancer in mice; Fig. 9 A is the growth curve of Anti-CEA-CAR-neutrophils monotherapy for breast cancer. Fig. 9 B is the survival rate of tumor-bearing mice after monotherapy with Anti-CEA-CAR-neutrophils. Fig. 9 C is the tumor growth curve of Anti-CEA-CAR-neutrophils combined with Anti-PD-1 treatment, Fig. 9 D is the survival rate of tumor mice treated with Anti-CEA-CAR-neutrophils combined with Anti-PD-1. Fig. 9 E is a representative picture of a tumor treated with Anti-CEA-CAR-neutrophils combined with Anti-PD-1. Fig. 9 F is the tumor weight after treatment with Anti-CEA-CAR-neutrophils combined with Anti-PD-1.

[0039] Fig.10It is the safety evaluation of MET-neutrophils treatment; Fig.10 A is the biochemical index test, including: creatinine (Cr), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), alkaline phosphatase (ALP), Fig.10 B is the detection of inflammatory factors, including: IL-1β, IL-6, IL-10, IL-12, IFN-γ and TNF-α, Fig.10 C is the weight change of mice after treatment with MET-neutrophils. Fig.10 D is the organ index of mice after MET-neutrophils treatment (organ weight / mouse body weight). DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and advantages of the present application more clear, the present application is further described in detail below. However, it should be understood that the description here is only used to explain the present application and is not used to limit the scope of the present application.

[0041] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present application, and the terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The reagents and instruments used herein are all commercially available, and the characterization means involved can refer to the relevant descriptions in the prior art, which will not be repeated herein.

[0042] In order to further understand the present application, the present application is further described in detail below in conjunction with the best embodiment.

[0043] Example 1

[0044] Based on the poor effect of immune cell therapy for solid tumors, including CAR-T, and the limited source of safe and reliable neutrophils, this application provides a method for efficiently and massively obtaining engineered neutrophils and its application in the treatment of solid tumors. The progenitor cells constructed by this method proliferate rapidly, can be obtained in large quantities in a short period of time, can be cryopreserved for a long time, and the induction cycle of neutrophils is short, simple and convenient. The engineered neutrophils obtained can efficiently express MET or CAR or stably knock out the TGFBR2 gene and highly express IFN-γ, are stable and uniform, and have good anti-solid tumor therapeutic effects.

[0045] This application is based on the fact that neutrophils have plasticity in anti-tumor phenotype and pro-tumor phenotype in the tumor microenvironment and enhance their anti-tumor function, and carries out different transformation strategies for neutrophils:

[0046] 1) MET gene high expression modification to maintain the N1 anti-tumor phenotype and promote neutrophil adhesion and infiltration in solid tumors; or

[0047] 2) Expression of CAR gene to maintain the anti-tumor phenotype of N1 and promote the killing function of neutrophils against tumor cells; or

[0048] 3) Knockout of TGFBR2 gene and high expression of IFN-γ were performed to maintain the N1 anti-tumor phenotype while activating the immune microenvironment and promoting the anti-tumor efficacy of neutrophils.

[0049] CAR is designed to preferentially target CEA-positive tumors, including pancreatic cancer, lung cancer, breast cancer, colorectal cancer, gastric cancer, etc.

[0050] To achieve the above objectives, the following technical solutions are provided:

[0051] The source and acquisition of immune cells are key issues in adoptive immune cell therapy. The preparation of engineered neutrophils derived from proliferative stem / progenitor cells and their application in the treatment of solid tumors specifically include the following steps:

[0052] By extracting mouse bone marrow-derived Lin-progenitor cells and human umbilical cord-derived hematopoietic stem / progenitor cells (CB-CD34 + Cells), ER-Hoxb8 lentivirus transfection, flow sorting to obtain high-purity and proliferative ER-Hoxb8 progenitor cells. MET or CARCAR or SpCas9-TGFBR2-IL12 lentivirus was then used to transfect proliferative ER-Hoxb8 progenitor cells, and flow sorting was used to obtain high-purity and proliferative engineered progenitor cells, including MET-Hoxb8 progenitor cells, Anti-CEA-CAR-Hoxb8 progenitor cells and sgTGFBR2-Hoxb8 progenitor cells. 4-OHT was removed and G-CSF was added to induce the engineered progenitor cells into engineered neutrophils, including: MET-neutrophils, Anti-CEA-CAR-neutrophils, sgTGFBR2-neutrophils. The anti-tumor activity of engineered neutrophils was verified in vitro and in vivo.

[0053] 1. Construction of proliferative progenitor cells:

[0054] It includes MET-Hoxb8 progenitor cells, Anti-CEA-CAR-Hoxb8 progenitor cells and sgTGFBR2-Hoxb8 progenitor cells. The chimeric antigen receptor includes: Anti-CEA single chain variable region, IgG1 hinge region, CD8 transmembrane region, CD3ζ intracellular region.

[0055] The amino acid sequence of the Anti-CEA single chain variable region is shown in SEQ ID No. 1 below:

[0056] QVQLQQSGGGVVQPGRSLRLSCAASGFTLSSYGMYWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDR LTGAPYYYYYGMDVWGRGTLVTVSSGGGGSGGGGSGGGGSTDVVMTQSPGTLSLSPGERATLSCRASQSVSSRYLAWYQQKPGQAPRLLIYGASSRATGI PDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKR.

[0057] The amino acid sequence of the hinge region of IgG1 is shown in SEQ ID No. 2 below:

[0058] VPRDCGCKPCICT.

[0059] The amino acid sequence of the CD8 transmembrane region is shown in SEQ ID No. 3 below:

[0060] IWAPLAGICVALLLSLIITLI.

[0061] The amino acid sequence of the intracellular region of CD3ζ is shown in SEQ ID No.4 below:

[0062] RAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGG KQQRRRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTA TKDTYDALHMQTLAPR.

[0063] The sgTGFBR2 sequence is shown in the following SEQ ID No.5-SEQ ID No.7:

[0064] sgRNA1 (SEQ ID No. 5): GACCGCACCGCCATTGTCGC;

[0065] sgRNA2 (SEQ ID No. 6): ACGGCCACGCAGACTTCATG;

[0066] sgRNA3 (SEQ ID No. 7): TTCCAGAGTGAAGCCGTGGT.

[0067] 2. Induction and differentiation of engineered MET-neutrophils, Anti-CEA-CAR-neutrophils, and sgTGFBR2-neutrophils:

[0068] The induction differentiation process is to subject MET-Hoxb8 progenitor cells, Anti-CEA-CAR-Hoxb8 progenitor cells and sgTGFBR2-Hoxb8 progenitor cells to 20 ng / ml G-CSF for 6 days to obtain neutrophils MET-neutrophils, Anti-CEA-CAR-neutrophils, and sgTGFBR2-neutrophils.

[0069] 3. Identification of MET-neutrophils, Anti-CEA-CAR-neutrophils, and sgTGFBR2-neutrophils:

[0070] The identification methods include the survival time of neutrophils MET-neutrophils, Anti-CEA-CAR-neutrophils, and sgTGFBR2-neutrophils, the stemness marker CD117, the neutrophil markers Ly6G and CD11b, morphology, and the expression of CAR, MET, and TGFBR2.

[0071] 4. Construction of target cells with high expression of CEA and EGFR:

[0072] Target cells include PANC02 cells, LLC cells, and 4T1 cells.

[0073] 5. Biological functions and phenotypic identification of neutrophils MET-neutrophils, Anti-CEA-CAR-neutrophils, and sgTGFBR2-neutrophils:

[0074] The biological functions of neutrophils include cell migration, cell adhesion, etc., and their phenotypes include anti-tumor N1 phenotype and pro-tumor N2 phenotype.

[0075] 6. In vitro anti-tumor activity detection of neutrophil MET-neutrophils, Anti-CEA-CAR-neutrophils, and sgTGFBR2-neutrophils:

[0076] In vitro anti-tumor function detection includes the release of pro-inflammatory factors, the release of ROS, the generation of NO after co-incubation of neutrophils with target cells, the lysis of target cells by neutrophils, and the detection of target cell apoptosis.

[0077] 7. Evaluation of the efficacy of neutrophil MET-neutrophils, Anti-CEA-CAR-neutrophils, and sgTGFBR2-neutrophils in the treatment of pancreatic cancer, lung cancer, breast cancer and other solid tumors:

[0078] A tumor model was constructed using PANC02 cells, LLC cells, and 4T1 cells. Engineered neutrophils were infused for treatment, and the therapeutic effect was evaluated by in vivo imaging technology, tumor growth curves, tumor weight, and mouse survival rate.

[0079] 8. Safety evaluation of neutrophil MET-neutrophils, Anti-CEA-CAR-neutrophils, and sgTGFBR2-neutrophils in treating tumor-bearing mice:

[0080] The safety evaluation included monitoring the weight changes of mice after engineered neutrophil infusion therapy, detecting inflammatory factors in serum, and detecting biochemical indicators in serum.

[0081] Example 2

[0082] 1. Cell culture:

[0083] Female C57BL / 6J mice aged 6-8 weeks were euthanized, bone marrow cells were extracted, and Lin-progenitor cells were obtained using a progenitor cell isolation kit (Direct Lineage Cell Depletion Kit, Miltenyi Biotec). Lin-progenitor cells were cultured in IMDM complete medium, containing IMDM (Iscove's, Gibco), 15% FBS (Gibco), 1% pen / strep / glutamine (Gibco), 10ng / ml murine IL-3 (Peprotech), 20ng / ml murine IL-6 (Peprotech), 25ng / ml murine SCF (Peprotech). Proliferative ER-Hoxb8 progenitor cells were obtained 48 hours later by transfection with ER-Hoxb8 lentivirus and cultured in Opti-MEM complete medium, containing Opti-MEM, 10% FBS, 1% PSG, 10ng / ml murine SCF, 30μM beta mercapto ethanol, 1μM 4-OHT. Positive cells were purified by flow cytometry. Proliferative ER-Hoxb8 progenitor cells were transfected with CAR lentivirus, MET lentivirus or TGFBR2 knockout lentivirus to obtain proliferative MET-Hoxb8 progenitor cells, Anti-CEA-CAR-Hoxb8 progenitor cells and sgTGFBR2-Hoxb8 progenitor cells. HEK293T cells (ATCC), pancreatic cancer PANC02 cells (ATCC), breast cancer 4T1 cells (ATCC) and lung cancer LLC cells (ATCC) were cultured in high-glucose DMEM (Gibco), which contained 10% FBS (Gibco) and 1x penicillin-streptomycin (Pen-Strep; 10,000U / ml penicillin and 10,000μg / ml streptomycin; Gibco). In this application, cells modified with Hoxb8 and MET or CAR or TGFBR2 include mouse bone marrow-derived Lin-progenitor cells and human umbilical cord or bone marrow-derived stem / progenitor cells (CD34 + cell).

[0084] 2. Stable overexpression of MET or CAR or SgTGFBR2-IFN-γ in ER-Hoxb8 progenitor cells:

[0085] MET or CAR or TGFBR2 vector plasmids, psPAX2 and pMD2.G were transfected into HEK293T cells using Lipofectamine 2000 (Invitrogen); the SgTGFBR2-IFN-γ vector was designed as follows Figure 1 As shown in A. The culture medium containing lentivirus was collected at 24h, 36h and 48h after transfection, and concentrated and purified with PEG8000. The lentivirus was then added to a six-well plate in which ER-Hoxb8 progenitor cells were cultured. Fresh complete medium was replaced 6 hours after infection. The transfected cells were cultured for 72h and then sorted and purified by flow cytometry; the results are shown in Figure 1 C. Figure 1 E. Figure 5 A- Figure 5 C. Figure 8 A- Figure 8 As shown in B.

[0086] 3. Differentiation of MET-Hoxb8 progenitor cells, Anti-CEA-CAR-Hoxb8 progenitor cells and sgTGFBR2-Hoxb8 progenitor cells into neutrophils:

[0087] The cultured MET-Hoxb8 progenitor cells, Anti-CEA-CAR-Hoxb8 progenitor cells, and sgTGFBR2-Hoxb8 progenitor cells were washed twice with 1×PBS to remove 4-OHT, and then 20 ng / ml G-CSF, 10% FBS, and 1% Pen-Strep-Glut were added to the RPMI1640 medium. The complete medium was replaced on the third day, and mature engineered neutrophils were obtained on the sixth day. G-CSF recombinant cytokine was purchased from PeproTech; the results are shown in Figure 2 A- Figure 2 B. Figure 5 D- Figure 5 E. Figure 8 C- Figure 8 As shown in D.

[0088] 4. Cell morphology and karyotype analysis:

[0089] To evaluate cell morphology and nuclei, progenitor cells or neutrophils were fixed on slides, stained with Wright-Giemsa stain (Nanjing Jiancheng Biotechnology Co., D010), and the cells were imaged using a microscope; the results are shown in Figure 1 D. Figure 2 C. Figure 5 F- Figure 5 G. Figure 8 As shown in E.

[0090] 5. Protein Blotting:

[0091] Cell samples were collected and lysed with RIPA buffer containing protease inhibitors (Beyotime, P0013K), and then proteins were extracted. Protein concentration was determined using the Pierce BCA protein detection kit (Thermo). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed, and then the samples were transferred to polyvinylidene fluoride membranes. The membranes were incubated with TBS Tween 20 containing 5% milk. The blots were incubated with SuperSignal West Pico Plus chemiluminescent substrate (Thermo Fisher Scientific, 34580). The blot signals were detected using a Tanon imager. Antibodies used: anti-TGFBR2 (Abcam, ab269279), anti-MET (Abcam, ab51067), anti-iNOS (Proteintech, 22226-1-AP), anti-Arg1 (Proteintech, 66129-1-IG) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (proteintech, HRP-60004); the results are shown in Figure 2. Figure 1 B. Figure 2 D. Figure 3 C and Figure 5 H- Figure 5 As shown in I.

[0092] 6. Experiment of engineered neutrophil migration and adhesion across vascular endothelial cells

[0093] 2×10 5 HUVEC cells were plated in the upper chamber of a 12-well Transwell culture system (3 μm, polycarbonate membrane, 200 μL culture medium). 5 ng / mL IL-1α was added to the upper chamber for stimulation for 4 hours, washed three times with PBS, and 5×10 5 neutrophils were added to the lower chamber at 50 ng / mL HGF. After 12 hours, the number of neutrophils that migrated to the lower chamber was measured. 5 HUVEC cells were plated in 12-well plates, and 5 ng / mL IL-1α was added for 4 hours after 12 hours for stimulation. The cells were washed with PBS three times and 5×10 5 Neutrophils were added to the HUVECs and 50 ng / mL HGF cytokine was added. After 15 minutes, the non-adherent cells were washed away with PBS, and the neutrophils adherent to HUVECs were digested and dissociated with cell dissociation buffer (Thermo Fisher Scientific) and counted. Figure 6 A- Figure 6 As shown in C.

[0094] 7. LLC EGFR Stable cell line construction

[0095] EGFR vector plasmid, psPAX2 and pMD2.G were transfected into HEK293T cells using Lipofectamine 2000 (Invitrogen). The culture medium containing lentivirus was collected at 24h, 36h and 48h after transfection, and concentrated and purified with PEG8000. The lentivirus was then added to a six-well plate in which LLC cells were cultured. Fresh complete medium was replaced 6 hours after infection. The transfected cells were cultured for 72h and then sorted and purified by flow cytometry; the results are shown in Figure 2. Figure 6 As shown in D.

[0096] 8. Cytokine release experiment:

[0097] The cell-free supernatant of the co-culture of neutrophils and tumor cells was collected and the cytokine concentrations of IL-1β, IL-6, IL-10, IL-12, IFN-γ, and TNF-α were determined by enzyme-linked immunosorbent assay (ELISA) according to the instructions of the kit supplier. The cytokine kits used for quantitative analysis were purchased from Thermo Fisher Scientific: IL-1β, 88-7013-88, IL-6, 88-7064-88, IL-10, 88-7105-88, IL-12, 88-7121-88, IFN-γ, 88-7314-88, and TNF-α, 88-7324-88; the results are shown in Figure 3 E- Figure 3 H. Figure 6 H and Fig.10 As shown in A.

[0098] 9. Cytotoxicity assay:

[0099] Target cells (stable tumor cell lines generated by Luciferase lentiviral transfection) were inoculated into a black 96-well ELISA plate at a density of 5000 cells / 100 μL / well. Effector cells were neutrophils from different groups, and the effector cells and target cells (E:T) ratios were added to the wells at 5:1, 10:1, 20:1, and 50:1, respectively. After incubation for 24 hours. TM The luciferase assay system was used for detection on a Varioskan Flash luminometer (Thermo Scientific). The experimental method was based on the instructions of the kit supplier (Promega, E6110); the results are shown in Figure 3 A and Figure 6 As shown in E.

[0100] 10. ROS and NO content detection:

[0101] The generated ROS and NO were detected by adding H2DCFDA probe (Biyuntian, Shanghai, China) and using Griess ReagentSystem kit (G2930, Promega, USA) according to the manufacturer's instructions; the results are shown in Figure 3 B. Figure 3 D and Figure 6 F- Figure 6 As shown in G.

[0102] 11. Evaluation of the efficacy of engineered neutrophils in the treatment of solid tumors:

[0103] 1×10 6 PANC02 cells, 4T1 cells, or LLC cells were mixed in PBS and injected into 6- to 8-week-old female C57BL / 6J mice under the armpit to establish a tumor model. On the 8th day after tumor inoculation, 1×10 7 Engineered neutrophils. Five mice were taken from each group for live animal imaging to monitor tumor growth. D-Luciferin (Yeasen, 40902ES03) was injected intraperitoneally into mice at a dose of 150 mg / kg. Live animal imaging was performed using an animal imaging system (IVIS Lumina seriesIII) to capture fluorescent signals. At the same time, the mice were weighed and the tumor size was measured on the day of engineered neutrophil injection and at the corresponding time points after injection to determine the tumor volume. At the end of the experiment, the mice were euthanized, and the tumor tissue was obtained and weighed. At the same time, 5 mice were taken from each group for survival monitoring; the results are as follows Figure 4 , Figure 7 and Fig. 9 shown.

[0104] Figure 4 middle, Figure 4 A is the weight change of tumor-bearing mice after engineered neutrophil therapy. Figure 4 B. Small animal in vivo imaging to detect the fluorescence signal of tumor mice treated with sgTGFBR2-neutrophils, and statistical analysis of the fluorescence signal. Figure 4 C shows representative images of tumors in the treatment and control groups and hematoxylin-eosin (HE) staining analysis;

[0105] Figure 7 middle, Figure 7 A is the tumor growth curve of the treatment group and the control group. Figure 7 B is the survival rate of tumor-bearing mice in the treatment group and the control group. Figure 7 C is the representative image of tumors in the treatment group and the control group. Figure 7 D is the tumor weight of the treatment group and the control group;

[0106] Fig. 9 middle, Fig. 9A is the growth curve of Anti-CEA-CAR-neutrophils monotherapy for breast cancer. Fig. 9 B is the survival rate of tumor-bearing mice after monotherapy with Anti-CEA-CAR-neutrophils. Fig. 9 C is the tumor growth curve of Anti-CEA-CAR-neutrophils combined with Anti-PD-1 treatment, Fig. 9 D is the survival rate of tumor mice treated with Anti-CEA-CAR-neutrophils combined with Anti-PD-1. Fig. 9 E is a representative picture of a tumor treated with Anti-CEA-CAR-neutrophils combined with Anti-PD-1. Fig. 9 F is the tumor weight after treatment with Anti-CEA-CAR-neutrophils combined with Anti-PD-1.

[0107] 12. Safety evaluation of engineered neutrophils in treating tumor-bearing mice:

[0108] One week after the engineered neutrophil infusion treatment, the mouse serum was collected for inflammatory factor detection, including IL-1β, IL-6, IL-10, IL-12, IFN-γ and TNF-α, and biochemical detection, including creatinine (Cr), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), alkaline phosphatase (ALP); the results are as follows Fig.10 As shown, Fig.10 B is the detection of inflammatory factors, including: IL-1β, IL-6, IL-10, IL-12, IFN-γ and TNF-α, Fig.10 C is the weight change of mice after treatment with MET-neutrophils. Fig.10 D is the organ index of mice after MET-neutrophils treatment (organ weight / mouse body weight).

[0109] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for modifying stem / progenitor cell-derived neutrophils for anti-tumor effects, characterized in that: The following steps are involved: S1. Preparation of stem / progenitor cell-derived neutrophils: Extraction of Lin from mouse bone marrow - Progenitor cells and human umbilical cord hematopoietic stem / progenitor cells, using gene editing technology to modify ER-Hoxb8 progenitor cells and induce them to become neutrophils; S2. Maintenance of Neutrophil Anti-tumor Phenotype: Three transformation strategies were used, namely, genetic engineering modification of chimeric antigen receptor CAR, high expression of MET gene modification, and high expression of IFN-γ while stably knocking out the TGFBR2 gene, to maintain the neutrophil N1 anti-tumor phenotype.

2. The anti-tumor modification method of stem / progenitor cell-derived neutrophils according to claim 1, characterized in that: In step S1, the mouse bone marrow-derived Lin - Lin - Progenitor cells; the human umbilical cord hematopoietic stem / progenitor cells are CB-CD34 + Cells; the gene editing technology is to use ER-Hoxb8 lentivirus transfection and then flow sorting to obtain high-purity ER-Hoxb8 progenitor cells with proliferation ability.

3. The anti-tumor modification method of stem / progenitor cell-derived neutrophils according to claim 1, characterized in that: In step S2, the chimeric antigen receptor CAR gene engineering modification is to use CAR lentivirus to transfect proliferative ER-Hoxb8 progenitor cells, flow sorting to obtain highly pure and proliferative Anti-CEA-CAR-Hoxb8 progenitor cells, remove 4-hydroxytamoxifen (4-OHT), and then add granulocyte colony stimulating factor (G-CSF) to induce differentiation to obtain neutrophils.

4. The anti-tumor modification method of stem / progenitor cell-derived neutrophils according to claim 1, characterized in that: In step S2, the high expression MET gene modification is to use MET lentivirus to transfect proliferative ER-Hoxb8 progenitor cells, flow sorting to obtain high-purity and proliferative MET-Hoxb8 progenitor cells, remove 4-hydroxytamoxifen (4-OHT), and then add granulocyte colony stimulating factor (G-CSF) to induce differentiation to obtain neutrophils.

5. The anti-tumor modification method of stem / progenitor cell-derived neutrophils according to claim 1, characterized in that: In step S2, the stable knockout of TGFBR2 gene and high expression of IFN-γ modification is to use SpCas9-TGFBR2-IFN-γ lentivirus to transfect proliferative ER-Hoxb8 progenitor cells, flow sorting to obtain high-purity and proliferative sgTGFBR2-Hoxb8 progenitor cells, remove 4-hydroxytamoxifen (4-OHT), and then add granulocyte colony stimulating factor (G-CSF) to induce differentiation to obtain neutrophils.

6. Use of the anti-tumor modified stem / progenitor cell-derived neutrophils provided by the method according to any one of claims 1 to 5 in the preparation of biological products for treating solid tumors.

7. The use according to claim 6, characterized in that: The solid tumor is selected from one or more of pancreatic cancer, EGFR mutant lung cancer and breast cancer.

8. Use of the anti-tumor modified stem / progenitor cell-derived neutrophils combined with immune checkpoint inhibitors provided by the method according to any one of claims 1 to 5 in the preparation of biological products for the treatment of solid tumors.

9. The use according to claim 8, characterized in that: The immune checkpoint inhibitor is selected from one or more of a PD-1 inhibitor, a PD-L1 inhibitor and a CD47 inhibitor.

10. The use according to claim 8, characterized in that: The solid tumor is selected from one or more of pancreatic cancer, EGFR mutant lung cancer and breast cancer.

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