CN114410588B

CN114410588B - Alpha 1 beta 1 integrin-dependent enhanced CAR macrophage and preparation method and application thereof

Info

Publication number: CN114410588B
Application number: CN202210112070.0A
Authority: CN
Inventors: 宁蓬勃; 王忠良; 杜付玉; 龚春梅; 张伟洁
Original assignee: Xidian University
Current assignee: Xidian University
Priority date: 2022-01-29
Filing date: 2022-01-29
Publication date: 2022-11-04
Anticipated expiration: 2042-01-29
Also published as: CN114410588A; WO2023142635A1

Abstract

The invention discloses alpha 1 beta 1 integrin-dependent enhanced CAR macrophages and a preparation method and application thereof, and belongs to the field of immunology and tumor therapeutics. The CAR macrophage comprises an alpha 1 beta 1 integrin transmembrane region, an intracellular signal regulating motif, an Fc gamma RI intracellular signal transduction motif and an extracellular antigen binding region, and the nucleotide sequences are respectively shown in SEQ ID NO 1-4. The invention also constructs CAR macrophages based on the alpha 1 beta 1 integrin mediated Fc gamma R I intracellular signaling domain, and experiments prove that the CAR macrophages activate macrophage activation signals and promote macrophage phagocytosis ability to synergistically enhance the anti-tumor effect. According to the invention, by designing the specific element suitable for the macrophage signal transduction function, a CAR macrophage technical platform in the true sense is constructed, and the CAR macrophage specific therapeutic method is widely suitable for application and extension design of various CAR macrophages for treating solid tumors in the future.

Description

Alpha 1 beta 1 integrin-dependent enhanced CAR macrophage and preparation method and application thereof

Technical Field

The invention relates to the technical field of immunology and tumor therapeutics, in particular to alpha 1 beta 1 integrin-dependent enhanced CAR macrophage and a preparation method and application thereof.

Background

Immunotherapy plays an important role in the field of tumor therapy and continues to make important progress. In recent years, CAR-T cell therapy has been approved by the FDA in the united states and the drug administration in our country as a clinical drug for the treatment of specific hematological cancers because of its excellent therapeutic effect in the field of hematological tumors. However, CAR-T cell therapy suffers from the problems of T cells being difficult to infiltrate solid tumors, and T cells experiencing immune failure in the tumor microenvironment. To date, CAR-T cell therapy has progressed slowly in the treatment of malignant solid tumors. The current search for more types of immune cells for the development of adoptive cell therapy to enable the development of specific effective cellular immunotherapy has been a hot spot and trend in the development of tumor immunotherapy. Macrophages have tumor chemotaxis in nature and can deeply infiltrate into solid tumors, wherein the infiltration degree of the macrophages in part of the tumors reaches 50%, and the infiltration degree is gradually increased along with the increase of the malignancy degree of the tumors. Therefore, the development of a CAR-Macrophage (CAR-M) tumor cell therapy technology based on the construction of a chimeric antibody by macrophages is expected to overcome the development of solid tumors in the future.

The activation of T cells requires the combination of surface TCR-CD3 complex and MHC molecule complex to provide a first signal for the activation of T cells, however, the expression level of MHC molecules in the tumor microenvironment is low, and the first activation signal for stimulating T cells cannot be effectively provided, so that the immune response and the killing effect of T cells are inhibited. Therefore, researchers have creatively designed CAR-T cell technology to replace the extracellular domain of the TCR with a scFv segment containing extracellular specific recognition of tumor antigens, independent of the role of the MHC molecule. Arthur Weiss et al found that the chimeric antigen receptor contained the T cell internal signal domain CD3 zeta that activated T cell signaling. CAR-T therefore selects CD3 ζ as a motif for transmitting intracellular activation signals.

CAR-M tumor cell therapy, as a novel tumor immunotherapy technology, focuses on another important immune cell-macrophage in the body, developing a new field of cell therapy, however, CAR-M is currently built mostly along with CAR-T cell design units. For example, CAR-macrophage therapies reported by doctor Saar Gill and doctor Michael Klichinsky in Nature Biotechnology, from the university of Pennsylvania, employ the CD3 zeta signaling domain of classical CAR-T, which functions specifically for T cell signaling. If CAR-M could be used as a unique cellular immunotherapy mechanism, it is urgent to design specific elements really suitable for macrophage signal transduction.

Activation of macrophages must go through two stages: an sensitisation phase and an activation phase. Activation is driven by the coordinated expression of numerous genes, during which the involvement of multiple transmembrane proteins is required. Various protein molecules exist on the surface of macrophages, and are involved in the process of mediating macrophage activation, including α 1 β 1 integrin, fcR, CSF-1R, MR, CD36, CD163, and CD206, among others. α 1 β 1 integrin is normally expressed in macrophages and mediates the secretion of its cytokines through the interaction of the I-domain of the extracellular domain of the α 1 subunit with collagen molecules in the extracellular matrix (ECM). α 1 β 1 integrin plays an important role in the inflammatory response of macrophages, and plays an important role in mediating macrophage activation. In the immune system, α 1 β 1 is expressed on macrophages only after activation by antigens, superantigens or cytokines. Studies have shown that the expression of macrophage alpha 1/beta 1 integrin is specifically induced by IFN-gamma, thereby promoting macrophage activation and stimulating cytokine expression. Macrophages are an important component of immune infiltration of tumors. Tumor cells can secrete signaling molecules such as interleukins and chemokines to recruit macrophages, which allow macrophages to naturally move toward and infiltrate solid tumors. Unfortunately, after macrophages are recruited to tumor tissues, tumor cells are prevented from being attacked by macrophage immunity, and the tumor microenvironment mostly expresses low immune induction factors such as IFN-gamma and the like, so that effective activation of macrophage M1 type polarization and anti-cancer activation characteristics cannot be formed, and malignant progress of solid tumors is objectively promoted.

In recent years, although many researchers do research on constructing macrophages, the ability of macrophages to eliminate tumor cells such as innate immune phagocytosis is significantly inhibited due to inhibitory factors such as low expression of IFN-gamma in tumor microenvironment and the like, CAR-M is not successfully constructed in a real sense, and how to design a specific element with a CAR-M signal transduction function is more suitable for the anticancer potential mechanism of CAR-M, so that a real CAR-M technical system is formed, and the problem to be solved is urgently needed.

Disclosure of Invention

The invention aims to provide an alpha 1 beta 1 integrin dependent enhanced CAR macrophage and a preparation method and application thereof, and aims to solve the problems in the prior art, and the activated phagocytosis enhanced CAR-M is designed and constructed based on specific motifs of alpha 1 beta 1 integrin and Fc gamma RI, and after a tumor cell is killed in a targeting manner by CAR-M scFV, macrophage activation signals are activated, macrophage phagocytosis is promoted, and an anti-tumor effect is synergistically enhanced.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides an alpha 1 beta 1 integrin-dependent enhanced CAR macrophage comprising an intracellular alpha 1 beta 1 integrin transmembrane-mediated Fc gamma RI intracellular signaling motif, wherein the alpha 1 beta 1 integrin comprises an alpha 1 beta 1 integrin transmembrane region and an intracellular signaling regulatory motif.

Preferably, the nucleotide sequence of the transmembrane region of the alpha 1 beta 1 integrin is shown as SEQ ID NO. 1, and the nucleotide sequence of the intracellular signal regulatory motif of the alpha 1 beta 1 integrin is shown as SEQ ID NO. 2;

the nucleotide sequence of the Fc gamma RI intracellular signal transduction motif is shown as SEQ ID NO. 3.

Preferably, the CAR macrophage further comprises an extracellular antigen-binding region.

Preferably, the extracellular antigen-binding region is scFv, and the nucleotide sequence of the scFv is shown in SEQ ID NO. 4.

The invention also provides a method for preparing the alpha 1 beta 1 integrin-dependent enhanced CAR macrophage, which is characterized by comprising the step of introducing nucleotide sequences of a transmembrane region and an intracellular signal regulating motif of the alpha 1 beta 1 integrin, an Fc gamma RI intracellular signal transduction motif and an extracellular antigen binding region into a CAR macrophage cell.

The invention also provides a chimeric antigen receptor which comprises the nucleotide sequence of the alpha 1 beta 1 integrin transmembrane region, an intracellular signal regulating motif, an Fc gamma RI intracellular signal transduction motif and an extracellular antigen binding region.

The invention also provides a vector comprising the nucleotide sequence of the transmembrane region of α 1 β 1 integrin and an intracellular signal-modulating motif, an Fc γ rii intracellular signal transduction motif, an extracellular antigen-binding region, or the chimeric antigen receptor of claim 6.

The invention also provides a pharmaceutical composition comprising the CAR macrophage or the vector together with a pharmaceutically acceptable carrier.

The invention also provides application of the CAR macrophage in preparing a medicine for enhancing the innate immune phagocytic function of the macrophage.

The invention also provides application of the CAR macrophage in preparation of a solid tumor treatment drug.

Preferably, the solid tumor includes HER2 high expression solid tumors such as breast cancer, ovarian cancer, gastric cancer, lung cancer, colorectal cancer and the like.

The invention discloses the following technical effects:

the activated phagocytosis enhancement type CAR-M (Active CAR-M, ACTCAR-M) is designed and constructed based on specific motifs of alpha 1 beta 1 integrin and Fc gamma RI, after CAR-M scFV is combined with tumor cell surface antigen in a targeted mode, the activated phagocytosis enhancement type CAR-M activates the alpha 1 beta 1 integrin to form a co-stimulation molecule with the Fc gamma RI, the CAR-M gives an intracellular activation structure domain of the alpha 1 beta 1-Fc gamma RI adopted by the invention, the intracellular of the Fc gamma RI is directly constructed into the CAR cell to start a first signal of activation of a phagocytic function of macrophages, and the intracellular construction of the alpha 1 beta 1 integrin into the CAR cell to enhance the macrophage activity and play a role in activating the macrophages in a second step. The targeting killing of the tumor cells by the CAR-M scFV can be realized, the alpha 1 beta 1 integrin is stimulated to promote the activation of macrophages, and the macrophages can activate the scFv segment which can specifically recognize the tumor antigen extracellularly without Fc mediation to play the role of enhancing phagocytosis and other anti-tumor potentials, namely, the synergistic enhancement of the anti-tumor effect.

The invention designs a specific element suitable for a macrophage signal transduction function based on an Fc gamma RI intracellular signal transduction domain mediated by alpha 1 beta 1 integrin transmembrane, designs and implements an alpha 1 beta 1 integrin dependent enhanced Active CAR-M (ACTCAR-M) cell technology platform, obviously improves CAR-M anti-tumor property, and is suitable for application and extension design of various types of CAR-M treatment solid tumors in the future.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 shows the construction and characterization of macrophage-specific activating ACT CAR-M of the present invention;

a: confocal microscopy of transmembrane expression of ACT CAR; b: qPCR for expression of ACT CAR; c: the HER2 protein binding capacity of ACTCAR-M is detected in a confocal manner, and the scale size is 30 mu M;

FIG. 2 shows the activity assay of ACT CAR-M of the present invention; a: CCK-8 detects the proliferation of ACTCAR-M; b: qPCR detection of p53 expression; c: qPCR detecting the expression of CCND-1; d: qPCR detection of Ki67 expression; * Indicates significant differences between the different groups, <0.001, <0.01, <0.05;

FIG. 3 shows the tumor phagocytosis assay of ACT CAR-M of the present invention; a: ACT CAR-M phagocytosis of HER2 ⁺ -4T1 tumor cells; b: ACT CAR-M phagocytosis of HER2 ⁺ -SKOV3 tumor cells;

FIG. 4 shows anti-HER2 ACTCAR-M vs. HER2 of the invention ⁺ Analysis of the role of the 4T1 HER2 signaling pathway; a: qPCR detecting the expression condition of AKT; b: qPCR detection of PI3K expression: c: qPCR detection of p53 expression; d: qPCR detection of CCND-1 expression: e: qPCR (quantitative polymerase chain reaction) is used for detecting the expression condition of Caspase-3; f: qPCR detection of Ki67 expression; * Denotes significant differences between the different groups, is P<0.001 and is P<0.01, is P<0.05；

FIG. 5 shows anti-HER2 ACTCAR-M vs. HER2 of the invention ⁺ -analysis of the role of the SKOV3 HER2 signaling pathway; a: qPCR detecting the expression condition of AKT; b: qPCR detection of PI3K expression: c: qPCR detection of p53 expression; d: qPCR detection of the expression of CCND-1; e: qPCR (quantitative polymerase chain reaction) is used for detecting the expression condition of Caspase-3; f: qPCR detection of Ki67 expression; * Indicates significant differences between the different groups, P<0.001, is P<0.01, is P<0.05；

FIG. 6 is an in vitro killing assay of anti-HER2 ACT CAR-M of the invention against tumor cells; a: CCK-8 detection of ACT CAR-MFor HER2 ⁺ -killing of SKOV3 cells; CCK-8 detection of ACTCAR-M on HER2 ⁺ -killing of 4T1 cells;

FIG. 7 shows the present invention mouse HER2 ⁺ -4T1 tumor model validation of anti-tumor outcome of ACT CAR-M; a: ACT CAR-M treatment of mouse HER2+ -4T1 tumor pattern; b: ACT CAR-M treatment mouse tumor growth curve; c: ACT CAR-M treatment mouse tumor survival curves; d: ACT CAR-M treatment of mouse body weight; * Indicates significant differences between the different groups, P<0.001 and is P<0.01, is P<0.05。

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but rather as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the documents are cited. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Example 1 preparation method and functional verification of α 1 β 1 integrin-dependent enhanced CAR macrophage

1. Construction and characterization of Activity-enhanced ACT CAR-M

In the early stage of the research, the in vitro and extracellular binding concept of CAR-T is referred to, the infection budding expression mechanism of enveloped virus is benefited, a specific CAR of macrophage and activity-enhanced ACT CAR-M are constructed by adopting a lentiviral vector, an activity-enhanced CAR-M platform is successfully established by taking HER2 as a model molecule, a transmembrane HER2 scFv extracellular segment is expressed on the surface of a cell membrane, and an ACT CAR-M stable cell line with high affinity HER2 binding activity is obtained by screening.

1.1 construction and packaging of lentiviruses

Based on a lentiviral vector, a chimeric antigen receptor HER2 scFv-GFP (sequence synthesis by bio-engineering (Shanghai) GmbH) is synthesized by connecting HER2 scFv, an alpha 1 beta 1 integrin transmembrane region, an alpha 1 beta 1 integrin intracellular signal regulatory motif, an Fc gamma R1 transmembrane signal transduction domain intracellularly (and an Fc gamma RI intracellular signal transduction motif) and GFP by using a linker, the macrophage is transfected by the chimeric antigen receptor HER2 scFv-GFP, a macrophage specific CAR is constructed, and the synthesized HER2 scFv-GFP-CAR lentiviral plasmid is co-transfected with three auxiliary plasmids of Rev, gag and VSV, so that the lentivirus is packaged into the complete HER2 scFv-GFP-CAR lentivirus.

The method comprises the following specific steps:

(1) Will be 4X 10 ⁵ HEK293T cells in logarithmic growth phase were plated uniformly in 6-well plates and were subjected to 5% CO at 37 ℃% ₂ And (3) statically culturing in a cell culture box until the cell confluence reaches 60% -70%, and adding 2.0 mu g of plasmids pCDH-Affibody-GFP, 0.67 mu g of Rev, 0.67 mu g of Gag and 0.67 mu g of VSV according to the proportion of 3Mixing in a centrifuge tube of 1.5mL with 400 μ L Opti-MEM, incubating at room temperature for 5min, adding 6.0 μ L TurboFect Transfection Reagent, gently blowing and beating the tip, and incubating at room temperature for 15-20min;

(2) Slowly dropwise adding the above incubated mixture to a 6-well plate inoculated with 293T cells, gently shaking the plate while dropwise adding, and incubating at 37 deg.C and 5% CO ₂ After 16-24h incubation, the transfection mixture was discarded from each well of the 6-well plate, 2-3mL of Advanced DMEM complete medium containing 10% Fetal Bovine Serum (FBS), 1 XCD (CD concentrated Gibico), 0.01mM cholesterol, 4.0mM L-glutamic acid (Glu), 0.01mM egg yolk lecithin was added, the cell culture plate was reset to 37 ℃ and 5% CO ₂ Culturing for 48h, and synthesizing into HER2 scFv-GFP-CAR lentivirus;

(3) To remove cell debris, the lentivirus-containing medium was centrifuged at 4000g for 10min at 4 ℃. The supernatant was filtered through a 0.22 μm filter and collected, centrifuged at 15000g for 2h at 4 ℃. Discarding the supernatant, resuspending virus particles with virus preservation solution, centrifuging for 5min at 10000g, and storing the supernatant at-80 deg.C;

(4) Subsequently, the titer of the packaged lentivirus was checked by adding 90 μ L of DMEM complete medium containing 6.0 μ g/mL polybrene to 9 sterile 1.5mL centrifuge tubes, adding 10 μ L of the obtained recombinant lentivirus particles HER2 scFv-GFP-CAR to the first centrifuge tube, pipetting, homogenizing, aspirating 10 μ L of the mixture to the second centrifuge tube, and so on until the last tube, with three replicates per group. Will be 5X 10 ⁴ Uniformly inoculating HEK293T cells into a 96-well plate, sequentially adding the diluted gradient lentivirus diluents when the cell confluence reaches 60-70%, and simultaneously setting the 293T cells without the virus diluents as negative control; after 24h incubation, fresh DMEM complete medium was replaced and after 48h the wells were observed under an inverted fluorescence microscope. Lentivirus titer was calculated as: viral titer (TU/mL) = number of mean green fluorescent cells x viral dilution per volume of virus inoculation solution (mL), unit TU/mL.

The sequences of the elements for constructing the chimeric antigen receptor are as follows:

HER2 scFv sequence (SEQ ID NO: 4):

atgaatttacaaccaattttctggattggactgatcagttcagtttgctgtgtgtttgctctagcttctcaggtac aactgcagcagtctggacctgaactgaagaagcctggagagacagtcaagatctcctgcaaggcctctgggtat cctttcacaaactatggaatgaactgggtgaagcaggctccaggacagggtttaaagtggatgggctggattaac acctccactggagagtcaacatttgctgatgacttcaagggacggtttgacttctctttggaaacctctgccaacact gcctatttgcagatcaacaacctcaaaagtgaagacatggctacatatttctgtgcaagatgggaggtttaccacgg ctacgttccttactggggccaagggaccacggtcaccgtttcctctggcggtggcggttctggtggcggtggctc cggcggtggcggttctgacatccagctgacccagtctcacaaattcctgtccacttcagtaggagacagggtcag catcacctgcaaggccagtcaggatgtgtataatgctgttgcctggtatcaacagaaaccaggacaatctcctaaa cttctgatttactcggcatcctcccggtacactggagtcccttctcgcttcactggcagtggctctgggccggatttc actttcaccatcagcagtgtgcaggctgaagacctggcagtttatttctgtcagcaacattttcgtactccattcacg。

linker sequence (SEQ ID NO: 5):

ggcggtggttccggcggtggatctggtggaggaactggaggaggttcaggaggtggt。

the α 1 β 1 integrin transmembrane region sequence (SEQ ID NO: 1):

ttatgggtcatcctgctgagtgcttttgccggattgttgctgttaatgctgctcattttagcactgtgg。

α 1 β 1 integrin intracellular signal-regulating motif (SEQ ID NO: 2):

aagatcggattcttcaagagacctctgaagaagaagatggagaag。

fc γ RI intracellular signaling motif (SEQ ID NO: 3):

aagatccatagactgcagagagagaagaagtacaacctggaggtgcctctggtgagcgagcagggcaa gaaggccaacagcttccagcaggtgaggagcgacggcgtgtacgaggaggtgaccgccaccgccagccaga ccacccctaaggaggcccctgacggccctaggagcagcgtgggcgactgtggccctgagcagcctgagcctc tgcctcctagcgacagcaccggcgcccagaccagccagagc。

GFP sequence (SEQ ID NO: 6):

gtgagtaagggcgaggagctgtttaccggcgtggtgcccatcctggtggagctggacggcgacgtgaa cggccacaagttcagcgtgagcggcgagggggagggcgacgccacctacggcaagctgaccctgaagttcat ttgcacaaccggcaagctgcccgtgccatggcctaccctggtgaccaccctgacatacggcgtccagtgttttag caggtatcccgaccacatgaagcagcacgacttctttaagagcgccatgcccgagggctacgtgcaggagagg accatcttcttcaaggacgacggtaactacaagaccagagccgaggtgaagttcgagggcgacaccctggtgaa ccggatcgagctgaagggcatcgacttcaaggaggacggtaacatcctgggccacaagctggagtataactata atagccacaacgtgtacatcatggccgacaagcagaagaacggcattaaggtgaacttcaagatcagacataac atcgaggacggatctgtgcagctggccgaccactaccagcagaacacccctatcggagatggaccagtgctgct gcctgataatcactatctgagcacacagtctgccctgagcaaagatcctaatgaaaagagagatcacatggtgctg ctggagtttgtgacagctgctggaataacactgggcatggatgagctgtataagtaa。

1.2 screening ACT CAR-M stably transfected cell lines

After packaging as a complete lentivirus and testing the titer thereof, macrophages were infected with HER2 scFv-GFP-CAR lentivirus and ACT CAR-M stable transgenic cell lines were obtained by drug selection, the specific steps were as follows:

(1) To determine the optimal concentration for drug screening, 5X 10 was taken ⁴ A number of untreated J774A.1 macrophages in log phase were seeded in 24-well plates and completely cultured in DMEM at 37 ℃ and 5% CO ₂ Cultured in a cell culture box. When the cell density was about 60 to 70%, the determination of the optimum lethal concentration was carried out by adding DMEM complete medium containing puromycin at final concentrations of 0.5. Mu.g/mL, 1. Mu.g/mL, 1.5. Mu.g/mL, 2. Mu.g/mL and 5. Mu.g/mL. Placing the 24-pore plate in a cell culture box for static culture, observing cell morphology and residual cell number once a day, and taking the final concentration of puromycin killing all macrophages on day 4 as the optimal drug screening concentration;

(2) Infecting J774A.1 macrophage with lentivirus, and collecting 2 × 10 ⁵ Each j774a.1 macrophage cell was inoculated into a 6-well plate and grown to a cell confluency of 60-70%, the ratio of each 9:1, adding HER2 scFv-GFP-CAR lentiviral particles into a culture medium for culturing J774A.1 macrophages, adding polybrene to enable the final concentration to be 6.0 mu g/mL, replacing a fresh DMEM complete culture medium after 24 hours, and completing the lentiviral infection of J774A.1 after 48 hours;

(3) And (3) screening the stable cell strain by using the optimal puromycin drug screening concentration, replacing a fresh DMEM culture medium containing puromycin once every 1 day to keep the puromycin concentration in the culture medium unchanged, and observing the cell growth condition and morphological change. After 3-4 times, the drug screening is completed, the cells after drug screening are trypsinized and inoculated in a 96-well plate according to the cell concentration of 0.5/100 mu L, the culture is continued by using a DMEM complete culture medium containing puromycin, the wells containing only single cells are selected for cell expansion culture, and the cells are passed to a new culture bottle or culture dish for amplification or frozen storage.

1.3 qPCR detection

Extracting total RNA of the stably transfected cell strain, detecting the expression condition of the fusion protein in the stably transfected cell strain by using a SuperReal PreMix Plus (SYBR Green) kit, and carrying out an experiment according to a kit instruction, wherein the qPCR experiment comprises the following steps:

(1) Normal lysis 2 XSuperReal Premix Plus, 50 XROX Reference Dye, template, primers and RNase-free ddH ₂ O, balancing all reagents at room temperature and thoroughly mixing;

(2) On ice, the cDNA template obtained by inversion is mixed with a forward primer, a reverse primer, mix, ROX and ddH according to a certain system ₂ O mix, add the mixture to an eight-tube using a pipette, 20 μ L system comprising: mu.L of 2 × SuperReal Premix Plus, 0.6. Mu.L of forward primer (10. Mu.M), 0.6. Mu.L of reverse primer (10. Mu.M), 0.1-2. Mu.L of cDNA template, 0.4. Mu.L of 50 × ROX Reference Dye, using RNase-free ddH ₂ Supplementing O to total volume of 20 μ L;

(3) Covering a tube cover, blowing, beating and uniformly mixing, and centrifuging for 5-10s by using a microcentrifuge to ensure that all components are at the bottom of the tube;

(4) Placing the reaction system in an RT-qPCR instrument ABI 7300, setting instrument parameters, pre-denaturing at 95 ℃ for 15min, denaturing at 95 ℃ for 10s, annealing at 60 +/-1 ℃ for 20s, extending at 72 ℃ for 31s, and running a program for 40 cycles, deriving a Ct value after the program is finished, and utilizing 2 ^-ΔΔCt (Livak) method calculates the final result.

The qPCR results show: successful expression of ACT CAR-M fusion protein; confocal microscopy results successfully verified HER2 binding activity of HER2 scFv, as shown in figure 1. This provides a cell therapy model for later studies.

2. Activity assay of ACT CAR-M

(1) CCK cell proliferation assay

(1) 100. Mu.L of HER2 positive tumor target cell suspension was added to each well of a 96-well plate at a density of 1X 10 ⁵ Individual cells/mL, at 37 ℃ and 5% CO ₂ The incubator is kept still for culture;

(2) after HER2 positive tumor target cells adhere to the wall, a culture medium in a pore plate is sucked, the cells are washed by PBS for 2-3 times, 100 mu L of J774A.1 (UTD-M), empty-pCDH J774A.1 (Empty), HER2 scFv J774A.1 (CAR-like) and HER2 scFv CAR J774A.1 (ACTCAR-M) with different densities are respectively added into a 96 pore plate, effective target ratios are respectively set as 0:1, 0.25;

(3) after 24-48h of cell co-culture, the medium in the 96-well plate was aspirated, the cells were washed 2-3 times with PBS, 10. Mu.L of CCK8 assay solution was added to the 96-well plate, and the CO was 5% at 37 ℃ ₂ Culturing for 3-4h in the incubator;

(4) and (3) measuring the absorbance of each well at 450nm by using a microplate reader, wherein the activity of target cell cells is = (the absorbance of an effective target ratio action group-the absorbance of an effector cell control group)/(the absorbance of a target cell positive control group-the absorbance of blank wells).

(2) Meanwhile, total RNA of each group of cells is respectively extracted, and the ACTCAR-M activity is detected by adopting qPCR, wherein the detection method is the same as that of the qPCR.

Results As shown in FIG. 2, ACT CAR-M group cells proliferated significantly more than CAR-Like group (FIG. 2A). The qPCR detection result shows that the mRNA expression levels of proliferation factors CCND1 and Ki67 of ACT CAR-M group cells are remarkably stronger than those of CAR-Like group, and the p53 expression level is lower than that of CAR-Like group, and the results are shown in FIGS. 2B-D. The results of the study suggest that the basal activity of ACTCAR-M is significantly enhanced, which means that the intracellular transduction of α 1 β 1 integrin and FcyRI enhances the proliferative activity of ACTCAR-M.

3. In vitro phagocytic Activity assay of ACT CAR-M

ACT CAR-M constructed as described above was subjected to HER2 based on the effective-to-target ratio of 1:1, respectively ⁺ SKOV3 and HER2 ⁺ -4T1 tumor cells as a model, phagocytosis of tumor cells using ACT CAR-M, specifically operated as:

the cells were inoculated in 6-well plates at 4X 10 ⁵ UTD-M, empty, CAR-like, ACT CAR-M, HER2 positive tumor target cells, in 10% FBS DMEM high-sugar culture 37 ℃, 5% CO ₂ Performing static culture in an incubator, adding DiO dye with working concentration of 4 mu M into a macrophage group when cells grow to 70-80% of confluence degree, adding DiI dye with working concentration of 4 mu M into an HER2 positive tumor target cell group, and incubating for 4-6h; the supernatant was aspirated and photographed under a fluorescent microscope.

The results are shown in fig. 3A and 3B, compared with the CAR-like group, the tumor cell phagocytic capacity of the ACT CAR-M group is significantly improved, which means that the intracellular transduction signals of α 1 β 1 integrin and fcyr enhance the phagocytic capacity of the ACT CAR-M, and suggest that the activity of the ACT CAR-M is enhanced, thereby laying a theoretical foundation for later-stage studies.

4. anti-tumor mechanism analysis of anti-HER2 ACT CAR-M acting on HER2 target of tumor cell

After different groups of macrophages are cultured by the CCK cell proliferation test in the 2 and ACT CAR-M activity detection, HER2 signal channel downstream related molecules (AKT and PI 3K) are adopted, and tumor related active molecules p53, caspase-3, CCND-1 and Ki67 are detected simultaneously, so that the tumor killing of ACT CAR-M is further proved. The method comprises the following specific steps:

(1) Adding 4X 10 of the mixture into each hole of a 6-hole plate ⁵ HER2 positive tumor target cells, incubated at 37 ℃ and 5% CO ₂ The incubator is kept still for culture;

(2) After HER2 positive tumor target cells adhere to the wall, a culture medium in a pore plate is sucked, the cells are washed for 2-3 times by PBS, J774A.1 (UTD-M), empty-pCDH J774A.1 (Empty), HER2 scFv J774A.1 (CAR-like) and HER2 scFv CAR J774A.1 (ACTCAR-M) with different densities are respectively added into a 6 pore plate, the effective target ratios are set to be 0:1, 0.25;

(3) After 24-48h of cell co-culture, the media in the 6-well plate was aspirated, the cells were washed 2-3 times with PBS, 1mL of trizol was added to each well of the 6-well plate, RNA was extracted, and experiments were performed using the super PreMix Plus (SYBR Green) kit, according to the kit instructions.

(4) Normal lysis 2 XSuperReal Premix Plus, 50 XROX Reference Dye, template, primers and RNase-free ddH ₂ O, balancing all reagents at room temperature and thoroughly mixing;

(5) The cDNA template obtained by inversion is mixed with a forward primer, a reverse primer, mix, ROX and ddH in a certain system on ice ₂ O mix, add the mixture to an eight-tube using a pipette, 20 μ L system comprising: mu.L of 2 × SuperReal Premix Plus, 0.6. Mu.L of forward primer (10. Mu.M), 0.6. Mu.L of reverse primer (10. Mu.M), 0.1-2. Mu.L of cDNA template, 0.4. Mu.L of 50 × ROX Reference Dye, using RNase-free ddH ₂ Supplementing O to total volume of 20 μ L;

(6) Covering a tube cover, blowing, beating and uniformly mixing, and centrifuging for 5-10s by using a microcentrifuge to ensure that all components are at the bottom of the tube;

(7) Placing the reaction system in an RT-qPCR instrument ABI 7300, setting instrument parameters, pre-denaturing at 95 ℃ for 15min, denaturing at 95 ℃ for 10s, annealing at 60 +/-1 ℃ for 20s, extending at 72 ℃ for 31s, and running a program for 40 cycles, deriving a Ct value after the program is finished, and utilizing 2- ^ΔΔCt The (Livak) method calculates the final result.

The results are shown in FIG. 4 and FIG. 5, compared with CAR-like group, AKT and PI3K expression of ACTCAR-M group are significantly reduced, which shows that ACTCAR-M blocks HER2 signal conduction, thus inducing p53 and Caspase-3 expression to be significantly up-regulated, and finally leading to tumor killing inhibition, which is shown in that Ki67 and CCND-1 expression are significantly down-regulated, and the ACTCAR-M cell blocking is suggestedHER2 ⁺ Tumor cell HER2 signal pathway conduction, HER2 inhibition ⁺ Tumor cell cycle progression results in the occurrence of tumor apoptotic events. The method lays a foundation for the research of the in-vitro tumor killing effect of ACT CAR-M, and also provides reliable theoretical support for the design of activity-enhanced CAR-M for later-stage tumor regulation.

6. In vitro killing detection of anti-HER2 ACT CAR-M on multiple tumor cell lines

In the early phase of the study, the in vitro killing ability of anti-HER2 ACT CAR-M was tested at the cellular level using the CCK-8 technique (as described above in "CCK cell proliferation assay for Activity assay of 2, ACTCAR-M"), using HER2+ -4T1 cells and HER2 ⁺ SKOV3 cells, and the effectiveness of anti-HER2 ACT CAR-M in killing HER2 targets is determined, and the effective target ratio is determined.

As shown in FIG. 6, ACT CAR-M group vs CAR-like group for HER2 ⁺ The tumor cells have obvious killing effect, which shows that the tumor inhibition effect of ACTCAR-M is better than that of CAR-like, and signals of alpha 1 beta 1 integrin and Fc gamma RI which are transduced in cells enhance the tumor killing capability of ACTCAR-M.

7. Validation of Effect of ACT CAR-M on in vivo treatment of tumors

Verification of tumor targeting ability of ACT CAR-M in vivo at the early stage of this study, using mouse HER2 ⁺ Verification of the antitumor Effect of ACTCAR-M, HER2, by the 4T1 tumor model ⁺ -the establishment of the 4T1 tumor model comprises the steps of:

(1) Mouse breast cancer cell line HER2 ⁺ -4T1 utilization of 1640 medium (hyclone) at 37 ℃ C. And 5% CO ₂ Culturing in a cell culture box;

(2) After being purchased, 4-6 weeks old magnetic BALB/c mice need to be fed for more than one week in a constant-temperature sterile ventilated mouse feeding room, and then are subjected to mouse experiments;

(3) HER2 ⁺ -4T1 cells in expanded culture, and after the mice have been fed for about one week, the HER2 cells are digested ⁺ -4T1 cells, washed 2-3 times with PBS and resuspended in PBS at a dilution density of 10 ⁷ Each cell/mL, 100 μ L of cell resuspension was injected subcutaneously above the right limb of the mouse;

(4) UTD-M, empty, CAR-like and ACT CAR-M cells were each assayed using DMEM high-sugar culture at 37 ℃ and 5% CO ₂ Culturing in a cell culture box;

(5) To HER2 ⁺ After nodulation of 4T1 cells, parallel experiments with 4 mice were set up in each group, 100. Mu.L PBS or cell resuspension was injected by tail vein, and 3X 10 cells of UTD-M, empty, CAR-like and ACT CAR-M fractions were injected ⁶ Cells, injected at 5d after the first injection, for a second injection, at a dose of 3X 10 per group ⁶ One cell, injected twice in total.

(6) The length and width of the mouse tumor are measured by an electronic vernier caliper, the weight of the mouse is measured by an electronic balance, the volume of the mouse tumor is = (length is 2) and width is 2), the measurement is carried out every two days, and finally, the survival curve of the mouse is calculated.

The results are shown in FIG. 7, and compared with the CAR-like group, ACTCAR-M group tumors were significantly inhibited, had prolonged survival, and had significant therapeutic effects. In addition, ACT CAR-M group showed no significant changes in body weight relative to CAR-like group, suggesting better safety.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention are intended to fall within the scope of the present invention defined by the claims.

Sequence listing

<110> university of west' an electronic technology

<120> alpha 1 beta 1 integrin dependent enhanced CAR macrophage, preparation method and application thereof

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accgccaccg ccagccagac cacccctaag gaggcccctg acggccctag gagcagcgtg 180

ggcgactgtg gccctgagca gcctgagcct ctgcctccta gcgacagcac cggcgcccag 240

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ctagcttctc aggtacaact gcagcagtct ggacctgaac tgaagaagcc tggagagaca 120

gtcaagatct cctgcaaggc ctctgggtat cctttcacaa actatggaat gaactgggtg 180

aagcaggctc caggacaggg tttaaagtgg atgggctgga ttaacacctc cactggagag 240

tcaacatttg ctgatgactt caagggacgg tttgacttct ctttggaaac ctctgccaac 300

actgcctatt tgcagatcaa caacctcaaa agtgaagaca tggctacata tttctgtgca 360

agatgggagg tttaccacgg ctacgttcct tactggggcc aagggaccac ggtcaccgtt 420

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cctaaacttc tgatttactc ggcatcctcc cggtacactg gagtcccttc tcgcttcact 660

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ctgagcacac agtctgccct gagcaaagat cctaatgaaa agagagatca catggtgctg 660

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Claims

1. Use of an α 1 β 1 integrin-dependent enhanced CAR macrophage in the preparation of a medicament for treating a solid tumor, wherein the CAR macrophage comprises an intracellular α 1 β 1 integrin transmembrane-mediated fcyri intracellular signaling motif, and the α 1 β 1 integrin comprises an α 1 β 1 integrin transmembrane region and an intracellular signaling regulatory motif;

the nucleotide sequence of the transmembrane region of the alpha 1 beta 1 integrin is shown as SEQ ID NO. 1, and the nucleotide sequence of the intracellular signal regulating motif of the alpha 1 beta 1 integrin is shown as SEQ ID NO. 2;

the nucleotide sequence of the Fc gamma RI intracellular signal transduction motif is shown as SEQ ID NO. 3;

the CAR macrophage further comprises an extracellular antigen binding region, wherein the extracellular antigen binding region is scFv, and the nucleotide sequence of the scFv is shown as SEQ ID NO. 4; the solid tumors are ovarian cancer and breast cancer.

2. A method of making the α 1 β 1 integrin-dependent enhanced CAR macrophage of claim 1, comprising the step of introducing a nucleotide sequence of the α 1 β 1 integrin transmembrane region and intracellular signaling motif, fcyri intracellular signaling motif, extracellular antigen binding region into a CAR macrophage;

the CAR macrophage further comprises an extracellular antigen binding region, wherein the extracellular antigen binding region is scFv, and the nucleotide sequence of the scFv is shown as SEQ ID NO. 4.

3. A chimeric antigen receptor comprising the α 1 β 1 integrin transmembrane region of claim 2 and an intracellular signaling motif, an fcyri intracellular signaling motif, an extracellular antigen-binding region;

the nucleotide sequence of the Fc gamma RI intracellular signal transduction motif is shown in SEQ ID NO. 3;

4. A vector comprising the nucleotide sequence of the transmembrane region of α 1 β 1 integrin and the intracellular signaling motif, fc γ RI intracellular signaling motif, and extracellular antigen binding region of claim 2.

5. A pharmaceutical composition comprising the CAR macrophage of claim 1 or the vector of claim 4 together with a pharmaceutically acceptable carrier.

6. Use of the CAR macrophage of claim 1 in the preparation of a medicament for enhancing innate immune phagocytic function of the macrophage.