Application of engineering modification of macrophages by using SPARC protein to activate immune function

Abstract

The invention relates to application of engineering modification of macrophages by using SPARC protein to activate an immune function, and belongs to the technical field of biological medicines. The SPARC protein is overexpressed in the macrophage or the endogenous expression of the SPARC protein is enhanced through a genetic engineering means, so that the polarization of the macrophage to a pro-inflammatory M1 type is remarkably promoted, specifically, the secretion amount of TNF-alpha is increased, the positive rate of HLA-C surface expression is increased, and an M2 type transformation marker CD206 is effectively inhibited. According to the present invention, the SPARC expression element is innovatively integrated into the chimeric antigen receptor macrophage (CAR-Macrophage) system; in-vivo experiments prove that the engineered cell can improve the tumor volume inhibition rate, and the action mechanism of the engineered cell relates to SHARPIN-mediated NF-kappa B pathway continuous activation) and PSMB10-driven antigen processing capacity improvement. The invention provides a novel cell drug development scheme with targeting specificity and immune activation efficacy for solid tumor immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and specifically to the application of engineering macrophages with SPARC protein to activate immune functions. Background Art

[0002] In existing cell engineering research and application technologies, cell functions are mainly directionally modified through transgenic technologies. Among them, CAR cell immunotherapy (Chimeric Antigen Receptor Cell immunotherapy), that is, chimeric antigen receptor cell immunotherapy is currently the most advanced and has the best clinical effect in the modified therapies, especially in the application effect for tumor treatment. However, tumors can hijack immune cells, changing immune cells from immune activation type to immune suppression type, such as T cell exhaustion, macrophage M2 polarization, etc., which is a challenge for the existing technology applications. Therefore, it is very important to maintain the "immune characteristics" of immune cells and their "opposing identity" to tumors during treatment. Summary of the Invention

[0003] In order to solve the problems of the existing technology, the present invention provides the application of engineering macrophages with SPARC protein to activate immune functions.

[0004] To solve the above technical problems, the present invention is achieved through the following technical solutions: In a first aspect, the application of engineering macrophages with SPARC protein to activate immune functions, overexpressing SPARC protein in macrophages or increasing the expression element of endogenous SPARC protein, so as to promote the polarization of macrophages into the M1 type, and the characteristics of the M1 type polarization are manifested as the up-regulation of the levels of tumor necrosis factor (TNF), major histocompatibility antigen complex class I C (HLA-C), TASL protein, proteasome subunit beta type 10 (PSMB10), and SHARPIN protein.

[0005] In the present application, specifically:

[0006] SPARC protein engineering method

[0007] Overexpression means: Transfecting macrophages through an exogenous plasmid vector (such as pcDNA3.1-SPARC), or using a lentiviral vector to carry the SPARC gene for stable expression.

[0008] Endogenous regulation: Using the CRISPR / dCas9 activation system to target the promoter region of the SPARC gene (such as the region -500 bp upstream of the transcription start site of NCBI GeneID: 6678), or inserting an endogenous enhancer element (such as the SV40 enhancer).

[0009] Detection of M1 polarization markers.

[0010] The upregulation of TNF, HLA-C, TASL, PSMB10, and SHARPIN was verified as follows:

[0011] Protein level: Detected by Western blot or flow cytometry (e.g., TNF antibody: Abcam ab66579);

[0012] Functional verification: The phagocytosis rate of macrophages against tumor cells increased by ≥ 40%;

[0013] (Co-culture experiment with Calcein-AM labeled tumor cells).

[0014] In a specific embodiment of the first aspect, the engineering modification of SPARC protein can maintain the M1 phenotype characteristics of macrophages, manifested as the continuous expression of tumor necrosis factor (TNF) and major histocompatibility complex class I C (HLA-C).

[0015] Application of engineering macrophages with SPARC protein to activate immune function M1 phenotype maintenance mechanism

[0016] Continuous TNF secretion: Detected by ELISA, the concentration of TNF-α in the culture supernatant was ≥ 500 pg / mL (for more than 72 hours);

[0017] Surface expression of HLA-C: Detected by flow cytometry, the proportion of HLA-C positive cells was ≥ 80% (anti-HLA-C antibody: BioLegend 306602).

[0018] Application of engineering macrophages with SPARC protein to activate immune function Opposing identity verification

[0019] After co-culturing macrophages with tumor cells, the expression levels of M2 markers (CD206, IL-10) decreased to less than 30% of the untreated group.

[0020] In the second aspect, a preparation method of chimeric antigen receptor macrophages (CAR-Macrophage) integrates a SPARC protein expression element into a CAR expression vector, and the element is selected from at least one of an exogenous SPARC overexpression vector or an endogenous SPARC expression enhancing regulatory sequence, so that the obtained CAR-Macrophage simultaneously has the ability to recognize target antigens and enhanced M1 polarization stability.

[0021] CAR-SPARC dual-element integration strategy

[0022] Vector design: Insert the SPARC expression cassette (such as CMV-SPARC-IRES-Puro) into the CAR expression vector (such as pLVX-EF1α-CAR);

[0023] Endogenous enhancement: Insert the SPARC mRNA stability element (such as the ARE deletion mutant) into the 3'UTR region of the CAR gene.

[0024] Application function co-verification of using SPARC protein to engineer macrophages to activate immune function

[0025] CAR targeting efficiency: The binding rate to HER2+ tumor cells is ≥90% (flow cytometry detection of anti-HER2-CAR expression);

[0026] M1 stability: Under the conditions of simulating the tumor microenvironment (IL-4 / IL-13 stimulation), the maintenance rate of M1 markers is ≥70%.

[0027] In a specific manner of the second aspect, the SPARC protein expression element is delivered to macrophages through a genetic engineering vector.

[0028] Vector delivery system

[0029] Viral vector: The transduction efficiency of lentivirus (such as VSV-G pseudotype) is ≥60% (GFP reporter gene detection);

[0030] Non-viral vector: Electroporation transfection of plasmid (parameters: voltage 250V, pulse duration 10ms).

[0031] In the third aspect, an application of using SPARC protein to engineer macrophages in the preparation of anti-tumor drugs, by maintaining M1 polarization and preventing M2 conversion, enhancing the phagocytosis and antigen presentation function of macrophages against tumor cells.

[0032] Quantitative indicators of anti-tumor effect

[0033] Phagocytic function: The phagocytosis index against A549 lung cancer cells is ≥2.5 (fluorescence microscopy counting);

[0034] Antigen presentation: The efficiency of macrophages presenting tumor antigens to T cells is increased by 3 times (CFSE-labeled T cell proliferation experiment).

[0035] In a specific implementation manner of the third aspect, the activity of the pro-inflammatory signaling pathway is maintained by upregulating the SHARPIN protein level.

[0036] SHARPIN mechanism of action

[0037] By inhibiting the dissociation of the LUBAC complex, maintain the linear ubiquitination of NEMO protein (verified by immunoprecipitation);

[0038] NF-κB pathway activation: the nuclear translocation ratio of p65 ≥ 80% (detected by immunofluorescence).

[0039] The beneficial effects of the present invention are as follows:

[0040] 1. The engineering transformation of SPARC protein significantly enhances the M1 polarization ability and phenotypic stability of macrophages. Experimental data show that overexpression of SPARC increases the secretion of TNF-α in macrophages by 4.3-fold (650 pg / mL vs 150 pg / mL), and the positive rate of HLA-C surface expression reaches 82%, which is 5.5-fold higher than that of the control group. This polarization effect is significantly persistent: in the tumor microenvironment simulated by IL-4 / IL-13, engineered macrophages maintain high expression of TNF (ΔCt = 3.2) for more than 72 hours, while effectively inhibiting the expression of M2 marker CD206 to less than 10%. More importantly, SPARC promotes the continuous activation of the NF-κB pathway (a 4-fold increase in nuclear p65 protein) by upregulating SHARPIN protein by 2.8-fold and stabilizing the assembly of the LUBAC complex (a 2.3-fold increase in the binding rate), thereby breaking the regulation of macrophage function by the tumor immunosuppressive microenvironment;

[0041] 2. Innovatively integrate the SPARC expression element into the CAR-Macrophage system to achieve a dual synergistic effect of targeted killing and innate immune activation. In the vector design, the co-expression of SPARC and anti-HER2-CAR enables macrophages to achieve a binding rate to HER2+ tumor cells exceeding 95%, and still maintain a 70% M1 phenotype under the stimulation of IL-13. Animal experiments show that the tumor inhibition rate of this bifunctional cell therapy group is 27 percentage points higher than that of traditional CAR-Macrophage (72% vs 45%). Its mechanism of action is reflected in: on the one hand, the antigen processing ability is enhanced by upregulating PSMB10 by 2.5-fold, and the T cell proliferation efficiency is increased by 3.5-fold; on the other hand, the secretion level of pro-inflammatory factors is maintained, and the phagocytosis index of A549 lung cancer cells reaches 2.5 in the co-culture experiment, which is 67% higher than that of the unmodified group. This technological breakthrough provides a new cell drug development strategy with both specificity and persistence for solid tumor immunotherapy. Brief Description of the Drawings

[0042] Figure 1 It is a schematic diagram of the modified macrophage pattern of the present invention.

[0043] Figure 2 It is a schematic diagram showing the positive correlation between the high content of SPARC-positive macrophages and good survival in the analysis results of public data such as TCGA of the present invention for clinical patient samples.

[0044] Figure 3The results of protein mass spectrometry sequencing of the present invention show that overexpression of SPARC in macrophages can significantly promote the polarization of macrophages into pro-inflammatory M1 type, specifically manifested as an increase in the secretion of TNF-α and the positive rate of surface expression of HLA-C.

[0045] Figure 4 The in-vivo experiment of the animal model of the present invention shows that adoptive transfer of SPARC-positive macrophages can significantly inhibit tumor size in combination with platinum drugs. Detailed implementation manners

[0046] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0047] Please refer to Figures 1 to 4 as shown in:

[0048] As shown in the attached Figure 1 the application of engineering macrophages with SPARC protein to activate immune function. Overexpress the SPARC protein in macrophages or increase the endogenous SPARC protein expression element to promote the polarization of macrophages into M1 type. The characteristics of the M1 type polarization are manifested as up-regulation of the levels of tumor necrosis factor (TNF), major histocompatibility complex class I C (HLA-C), TASL protein, proteasome subunit beta type 10 (PSMB10), and SHARPIN protein, as shown in the attached Figure 2 as shown.

[0049] Overexpress the SPARC protein in macrophages by genetic engineering means or enhance its endogenous expression to induce and maintain M1 type polarization.

[0050] Specific implementation steps:

[0051] The application of engineering macrophages with SPARC protein to activate immune function. Construction of SPARC overexpression vector:

[0052] Clone the human SPARC cDNA (GenBank: NM_003118.3) into the lentiviral vector pLVX-IRES-Puro (Clontech) to construct pLVX-SPARC.

[0053] After transfection of the human monocytic cell line THP-1 (ATCC TIB-202), induce differentiation into macrophages with 100 ng / mL PMA.

[0054] Application of engineering macrophages with SPARC protein to activate immune function: Endogenous SPARC enhancement strategy

[0055] Design a CRISPR / dCas9 activation system: The sgRNA targets the promoter region of the SPARC gene (chr5:151,636,402 - 151,636,423, GRCh38) and is co-transfected with the dCas9-VPR fusion protein.

[0056] Application of engineering macrophages with SPARC protein to activate immune function: Detection of M1 markers

[0057] Application of engineering macrophages with SPARC protein to activate immune function: TNF-α secretion: The culture supernatant was detected using an ELISA kit (R&D Systems DTA00D). The concentration in the SPARC overexpression group reached 650 ± 120 pg / mL (vs 150 ± 30 pg / mL in the control group).

[0058] Application of engineering macrophages with SPARC protein to activate immune function: HLA-C surface expression: Flow cytometry was used with an anti-HLA-C antibody (BioLegend 306602), and the positive rate increased from 15% to 82%.

[0059] Engineering of the SPARC protein can maintain the M1 phenotypic characteristics of macrophages, manifested as the continuous expression of tumor necrosis factor (TNF) and major histocompatibility complex class I C (HLA-C).

[0060] Maintain the M1 phenotype of macrophages through continuous SPARC expression to prevent tumor microenvironment-induced M2 conversion.

[0061] Specific implementation steps:

[0062] Application of engineering macrophages with SPARC protein to activate immune function: Long-term culture verification:

[0063] Co-culture SPARC-engineered macrophages with SKOV3 ovarian cancer cells for 7 days, and supplement 10 ng / mL IL-4 every 48 hours to simulate the tumor microenvironment.

[0064] Application of engineering macrophages with SPARC protein to activate immune function: Detection of phenotypic stability:

[0065] Application of engineering macrophages with SPARC protein to activate immune function: M1 maintenance: qPCR showed that the TNF mRNA level was maintained at a high expression level (ΔCt = 3.2, vs ΔCt = 8.1 in the untreated group).

[0066] Application of engineering macrophages with SPARC protein to activate immune function. M2 inhibition: The proportion of CD206+ cells ≤ 10% (vs 65% of unmodified macrophages).

[0067] A method for preparing a chimeric antigen receptor macrophage (CAR-Macrophage), integrating a SPARC protein expression element into a CAR expression vector, where the element is selected from at least one of an exogenous SPARC overexpression vector or an endogenous SPARC expression enhancing regulatory sequence, so that the obtained CAR-Macrophage simultaneously has the ability of targeted antigen recognition and enhanced M1-type polarization stability.

[0068] Integrating a SPARC expression element in CAR-Macrophage to achieve the synergy of targeted killing and immune activation.

[0069] Specific implementation steps:

[0070] Bifunctional vector design:

[0071] Construct an anti-HER2-CAR vector (the scFv sequence is derived from trastuzumab) and a SPARC co-expression system:

[0072] Vector structure: EF1α-CAR-T2A-SPARC.

[0073] Transfect primary macrophages by electroporation (Neon system, 1400V, 20ms, 2 pulses).

[0074] Function verification of the application of engineering macrophages with SPARC protein to activate immune function:

[0075] Targeting ability of the application of engineering macrophages with SPARC protein to activate immune function: The binding rate to HER2+ BT474 breast cancer cells > 95% (flow cytometry for CAR expression detection).

[0076] An application of engineering macrophages with SPARC protein in the preparation of anti-tumor drugs, enhancing the phagocytosis and antigen presentation functions of macrophages against tumor cells by maintaining M1-type polarization and preventing M2-type transformation.

[0077] Use SPARC-engineered macrophages for the preparation of drugs for solid tumor treatment.

[0078] Specific implementation steps:

[0079] In vitro anti-tumor experiment of the application of engineering macrophages with SPARC protein to activate immune function:

[0080] Application of engineering macrophages with SPARC protein to activate immune function - Phagocytosis function: Calcein-AM-labeled MDA-MB-231 breast cancer cells were co-cultured with macrophages for 4 hours, and the phagocytosis index (proportion of phagocytic cells) increased from 15% to 58%.

[0081] Application of engineering macrophages with SPARC protein to activate immune function - Antigen presentation: Macrophages presented tumor antigens to autologous T cells, and the T cell proliferation rate (CFSE dilution method) increased by 3.5 times.

[0082] Application of engineering macrophages with SPARC protein to activate immune function - In vivo model verification:

[0083] SPARC-CAR-M was injected into A549 lung cancer xenografts (volume 100 mm 3 ) in NOD / SCID mice, and the tumor volume inhibition rate in the treatment group reached 72% (vs 45% for CAR-M alone).

[0084] Maintain the activity of the pro-inflammatory signaling pathway by upregulating the SHARPIN protein level.

[0085] Enhance the activity of the pro-inflammatory signaling pathway through SHARPIN regulation.

[0086] Specific implementation steps:

[0087] Application of engineering macrophages with SPARC protein to activate immune function - Mechanism verification experiment:

[0088] Analysis of the LUBAC complex in the application of engineering macrophages with SPARC protein to activate immune function: Co-immunoprecipitation (anti-SHARPIN antibody, Abcam ab140985) showed that the binding of SHARPIN to HOIP increased by 2.3 times in the SPARC overexpression group.

[0089] Activation of the NF-κB pathway in the application of engineering macrophages with SPARC protein to activate immune function: The nuclear p65 protein level increased by 4 times (nuclear protein extraction kit, Thermo 78833).

[0090] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made in these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Application of engineering macrophages with SPARC protein to activate immune function, characterized in that: Overexpressing the SPARC protein or increasing the expression element of endogenous SPARC protein in macrophages to promote the polarization of macrophages into the M1 type, and the characteristics of the M1 type polarization are manifested as the upregulation of the levels of tumor necrosis factor (TNF), major histocompatibility complex class I C (HLA-C), TASL protein, proteasome subunit beta type 10 (PSMB10), and SHARPIN protein.

2. Use of engineered macrophages with SPARC protein to activate immune function according to claim 1, characterized in that: The engineering transformation of the SPARC protein can maintain the M1 type phenotypic characteristics of macrophages, manifested as the continuous expression of tumor necrosis factor (TNF) and major histocompatibility complex class I C (HLA-C).

3. A method for preparing chimeric antigen receptor macrophages (CAR-Macrophage), characterized in that: Integrating the SPARC protein expression element into the CAR expression vector, and the element is selected from at least one of an exogenous SPARC overexpression vector or an endogenous SPARC expression enhancing regulatory sequence, so that the obtained CAR-Macrophage simultaneously has the ability of targeted antigen recognition and enhanced M1 type polarization stability.

4. The preparation method of a chimeric antigen receptor macrophage (CAR-Macrophage) according to claim 3, characterized in that: The SPARC protein expression element is delivered to macrophages through a gene engineering vector.

5. Use of SPARC protein-engineered macrophages in the preparation of anti-tumor drugs, characterized in that: Enhance the phagocytosis and antigen presentation functions of macrophages on tumor cells by maintaining M1 type polarization and preventing M2 type transformation.

6. Use of engineered macrophages using the SPARC protein in the preparation of anti-tumor drugs according to claim 5, characterized in that: Maintain the activity of the pro-inflammatory signaling pathway through the upregulation of the SHARPIN protein level.

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