Application of protein A magnetic beads coated with recombinant protein as model for specifically evaluating CAR-NK (Chimeric Antigen Receptor-Natural Killer) efficiency

Abstract

The invention provides application of protein A magnetic beads coated with recombinant protein as a model for specifically evaluating CAR-NK efficiency, and belongs to the technical field of evaluation models. The invention provides application of protein A magnetic beads coated with recombinant protein in preparation of a model for specifically evaluating the efficiency of CAR molecules on CAR-NK cells, almost all CAR-NK cells can be adsorbed on the surfaces of the protein A magnetic beads coated with the recombinant protein, CAR-NK can be specifically activated, and a downstream channel of the CAR-NK can be remarkably activated; it is proved that the protein A magnetic bead coated recombinant protein not only can specifically evaluate the unique effect of CAR molecules on CAR-NK, but also can obtain pure CAR-NK cells for subsequent western blot and other experiments due to the fact that the magnetic beads are easily, rapidly and conveniently separated.

Description

Technical Field

[0001] The present invention belongs to the technical field of evaluation models, and in particular relates to the use of protein A magnetic beads coated with recombinant proteins as a model for specifically evaluating the efficacy of CAR-NK. Background Art

[0002] NK cells retain their killing effect on most tumors. CAR-NK refers to loading chimeric antigen receptors on NK cells. CAR-NK can kill target cells in a CAR molecule-dependent and -independent manner. In theory, this can better overcome target loss and tumor heterogeneity. However, it is precisely because of this characteristic that general target cells cannot specifically evaluate the unique effect of CAR molecules on CAR-NK. For example, although some studies have confirmed that the killing effect of CAR-NK with γ chain as the intracellular segment is better than that of ζ chain, some studies have also reached the opposite conclusion. For example, if NK cell-sensitive cell lines are used as target cells, such as Raji cells, it is impossible to distinguish the effects of CAR molecule-independent killing and the interaction between potential activating receptors and CAR molecules on CAR-NK killing of target cells. Therefore, choosing a suitable model is crucial for specifically evaluating the unique effect of CAR molecules on CAR-NK.

[0003] In order to avoid interference caused by sensitive cell lines, previous studies have used P815 (mouse mastocytoma cells) coupled to antibodies against NK cell activation receptors to study the single or synergistic effects of NK cell activation receptors. Although this model largely avoids potential interference, P815 expresses ICAM-1, which can bind to the NK cell activation receptor LFA-1 and affect the results. In addition, in co-culture experiments, the cell model used for stimulation is difficult to be quickly and completely separated. Due to the mixing of other cells or the long separation time, the use of experiments such as Western blotting to explore the CAR-NK downstream pathway is disturbed. Therefore, a model that can specifically activate CAR-NK and can be quickly and completely cleared is of great significance for evaluating the efficacy of CAR-NK. Summary of the invention

[0004] The present invention provides the use of protein A magnetic beads coated with recombinant proteins as a model for specifically evaluating the efficacy of CAR-NK. The protein A magnetic beads coated with recombinant proteins can specifically evaluate the unique effect of CAR molecules on CAR-NK. At the same time, since the magnetic beads can be quickly and conveniently separated, pure CAR-NK cells can be obtained for subsequent experiments such as protein blotting.

[0005] The present invention provides the use of protein A magnetic beads coated with recombinant proteins in preparing a model for specifically evaluating the efficacy of CAR molecules on CAR-NK cells.

[0006] In a specific embodiment of the present invention, the recombinant protein includes a target protein for cellular immunotherapy.

[0007] In a specific embodiment of the present invention, the target protein comprises an Fc tag.

[0008] In a specific embodiment of the present invention, the recombinant protein includes human B3H7-Fc recombinant protein.

[0009] In a specific embodiment of the present invention, the method for preparing the protein A magnetic beads coated with the recombinant protein comprises the following steps: washing the protein A magnetic beads and mixing them with the recombinant protein, removing the supernatant using a magnetic rack and then washing them, and then resuspending them using NK cell culture medium to obtain a suspension of protein A magnetic beads coated with the recombinant protein.

[0010] In a specific embodiment of the present invention, the ratio of the recombinant protein to the protein A magnetic beads is 2 μg / 100 μL.

[0011] The present invention also provides a method for evaluating the efficacy of CAR molecules on CAR-NK cells, comprising the following steps: respectively co-culturing NK cells and CAR-NK cells with protein A magnetic beads coated with recombinant proteins, detecting NK cell degranulation by flow cytometry, and evaluating the efficacy of CAR molecules on CAR-NK cells according to the flow cytometry results;

[0012] The method for preparing protein A magnetic beads coated with recombinant protein comprises the following steps: washing the protein A magnetic beads and mixing them with the recombinant protein, removing the supernatant with a magnetic stand and then washing, and then resuspending with NK cell culture medium to obtain a protein A magnetic bead suspension coated with the recombinant protein.

[0013] In one embodiment of the present invention, the co-culture is carried out in a cell culture plate, and the ratio of the number of NK cells or CAR-NK cells to the protein A magnetic bead suspension coated with the recombinant protein in each well is 5×10 5 _ 2×10 6 Cells: 100 μL.

[0014] In one embodiment of the present invention, the co-culture temperature is 37°C, and the culture time for activation marker or degranulation experiment is 4 hours. ， The culture time for exploring the downstream activation signaling pathway of CAR-NK was 0-30 minutes.

[0015] The present invention also provides the use of protein A magnetic beads coated with recombinant proteins in purifying CAR-NK cells.

[0016] Beneficial effects: The present invention found that after co-culturing the protein A magnetic beads coated with recombinant protein with CAR-NK cells, almost all CAR-NK cells were adsorbed on the surface of the magnetic beads, and compared with simple protein A magnetic beads, the protein A magnetic beads coated with recombinant protein can significantly increase CAR-NK degranulation, indicating that the protein A magnetic beads can specifically activate CAR-NK after being coated with recombinant protein; and the embodiment confirmed that the protein A magnetic beads coated with human B7H3 recombinant protein can significantly activate the ERK pathway of CAR-NK, confirming that the protein A magnetic beads coated with recombinant protein can specifically evaluate the unique effect of CAR molecules on CAR-NK, and at the same time, because the magnetic beads can be quickly and conveniently separated, pure CAR-NK cells can be obtained for subsequent experiments such as protein blotting. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of protein A magnetic beads coated with human B7H3 recombinant protein to stimulate NK cells;

[0018] Figure 2 This is a result diagram showing that protein A magnetic beads coated with human B7H3 recombinant protein specifically promote the expression of CAR-NK activation markers;

[0019] Figure 3 This is a graph showing the results of protein A magnetic beads coated with human B7H3 recombinant protein specifically promoting CAR-NK degranulation;

[0020] Figure 4 This is a graph showing the results of protein A magnetic beads coated with human B7H3 recombinant protein specifically activating the proximal signal Lck phosphorylation of CAR-NK;

[0021] Figure 5 This is a graph showing the phosphorylation results of the proximal signal Lyn that specifically activates CAR-NK by protein A magnetic beads coated with human B7H3 recombinant protein;

[0022] Figure 6 This is a graph showing the results of protein A magnetic beads coated with human B7H3 recombinant protein specifically activating the proximal signal Fyn phosphorylation of CAR-NK;

[0023] Figure 7 This is a graph showing the results of protein A magnetic beads coated with human B7H3 recombinant protein specifically activating the phosphorylation of the distal signal Erk of CAR-NK;

[0024] Figure 8 This is a graph showing the results of protein A magnetic beads coated with human B7H3 recombinant protein specifically activating the phosphorylation of the distal signal Akt of CAR-NK;

[0025] Fig. 9 This is a graph showing the results of protein A magnetic beads coated with human B7H3 recombinant protein specifically activating the phosphorylation of the distal signal PLC-γ1 of CAR-NK;

[0026] Fig.10 This is a graph showing the results of protein A magnetic beads coated with human B7H3 recombinant protein specifically activating the phosphorylation of the distal signal Vav1 of CAR-NK;

[0027] Fig.11 This is a graph showing the results of protein A magnetic beads coated with human B7H3 recombinant protein to specifically activate the distal signal NF-kB phosphorylation of CAR-NK. DETAILED DESCRIPTION

[0028] The present invention provides the use of protein A magnetic beads coated with recombinant proteins in preparing a model for specifically evaluating the efficacy of CAR molecules on CAR-NK cells.

[0029] The present invention does not specifically limit the source of the protein A magnetic beads. The protein A magnetic beads used in the examples were purchased from GenScript with a product number of Cat. No. L00273.

[0030] In one embodiment of the present invention, the recombinant protein includes a target protein for cellular immunotherapy, and the target protein includes an Fc tag, and the protein A magnetic beads can bind to the constant chain (Fc) of the antibody. In one embodiment of the present invention, the recombinant protein is a human B3H7-Fc recombinant protein purchased from Biolegend, catalog number 564604.

[0031] In a specific embodiment of the present invention, the method for preparing the protein A magnetic beads coated with recombinant protein comprises the following steps: washing the protein A magnetic beads and mixing them with the recombinant protein, removing the supernatant with a magnetic stand and then washing, and then resuspending with NK cell culture medium to obtain a protein A magnetic bead suspension coated with recombinant protein. In a specific embodiment of the present invention, the ratio of the recombinant protein to the protein A magnetic beads is 2 μg / 100 μL. In the embodiment, during the specific operation, 400 μL of magnetic beads (GenScript, Cat. No. L00273) were taken in an EP tube, washed twice with PBS, resuspended with 400 μL PBS, 40 μL of human B7H3-Fc recombinant protein (Biolegend, 564604) was added, and the magnetic beads and recombinant protein B7H3 were rotated on a shaker for 40 minutes to fully mix the magnetic beads and the recombinant protein B7H3, and the supernatant was removed with a magnetic stand, washed twice with PBS, and resuspended in 400 μL NK cell culture medium for standby use.

[0032] The present invention also provides a method for evaluating the efficacy of CAR molecules on CAR-NK cells, comprising the following steps: respectively co-culturing NK cells and CAR-NK cells with protein A magnetic beads coated with recombinant proteins, detecting NK cell degranulation by flow cytometry, and evaluating the efficacy of CAR molecules on CAR-NK cells according to the flow cytometry results;

[0033] The method for preparing protein A magnetic beads coated with recombinant protein comprises the following steps: washing the protein A magnetic beads and mixing them with the recombinant protein, removing the supernatant with a magnetic stand and then washing, and then resuspending with NK cell culture medium to obtain a protein A magnetic bead suspension coated with the recombinant protein.

[0034] In one embodiment of the present invention, the co-culture is carried out in a cell culture plate, and the ratio of the number of NK cells or CAR-NK cells to the protein A magnetic bead suspension coated with the recombinant protein in each well is 5×10 5 _ 2×10 6 Cells: 100 μL. In a specific embodiment of the present invention, the co-culture temperature is 37° C., the culture time for activation marker or degranulation experiment is 4 hours, and the culture time for exploring the CAR-NK downstream activation signaling pathway is 0-30 minutes.

[0035] In a specific embodiment of the present invention, a method for flow cytometry detection of NK cell degranulation comprises: removing the supernatant using a magnetic rack, separating the magnetic beads from the cells, washing the cells twice with PBS, fixing them with 4% PFA, resuspending them in 100 μL PBS, adding 5 μL anti-human CD107a antibody (Biolegend, 328620), mixing evenly, staining at 4 degrees for half an hour, and detecting by flow cytometry; wherein CD107a is used as a marker for evaluating degranulation.

[0036] In a specific embodiment of the present invention, after the protein A magnetic beads coated with recombinant protein are removed, the method for purifying CAR-NK cells for subsequent experiments such as protein blotting includes: co-culturing the protein A magnetic beads coated with recombinant protein with CAR-NK for 0-30 minutes, removing the supernatant using a magnetic rack, separating the magnetic beads from the cells, washing the cells once with PBS, adding cell lysate and protease phosphatase inhibitors to the cell precipitate, preparing samples for protein blotting experiments, and performing protein blotting experiments through gel electrophoresis, membrane transfer, etc. Alternatively, the purified CAR-NK cells are directly used for bulk transcriptome or single cell sequencing.

[0037] In order to further illustrate the present invention, the use of the protein A magnetic beads coated with recombinant proteins provided by the present invention as a model for specifically evaluating the efficacy of CAR-NK cells is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1

[0039] 1. Protein A magnetic beads coated with recombinant protein: Take B7H3-Fc recombinant protein as an example for experimental verification. Take 400μL magnetic beads (GenScript, Cat.No.L00273) in an EP tube, wash twice with PBS, resuspend with 400μL PBS, add 40μL human B7H3-Fc recombinant protein (Biolegend, 564604), rotate on a shaker for 40 minutes to fully mix the magnetic beads and recombinant protein B7H3, remove the supernatant with a magnetic stand, wash twice with PBS, and resuspend in 400μL NK cell culture medium for later use.

[0040] 2. Protein A magnetic beads coated with recombinant protein and co-cultured with NK cells or CAR-NK cells: Take NK cells and CAR-NK cells with good growth status, collect the cells by centrifugation and resuspend them to 5×10 per ml 5 . 1 mL of NK cell and CAR-NK cell suspension was added to a 24-well plate, and 100 μL of protein A magnetic beads coated with recombinant protein B7H3 suspension was added to each well, and co-cultured at 37°C for 4 hours.

[0041] The co-culture model and actual picture of protein A magnetic beads coated with human B7H3 recombinant protein and CAR-NK cells are as follows Figure 1 As shown, it can be seen that almost all NK cells are adsorbed on the surface of the magnetic beads.

[0042] 3. Flow cytometry detection of NK cell degranulation: Use a magnetic rack to remove the supernatant and separate the magnetic beads from the cells. The cells were washed twice with PBS, fixed with 4% PFA, resuspended in 100 μL PBS, and 5 μL anti-human CD107a antibody (Biolegend, 328620) was added. The cells were mixed evenly and stained at 4 degrees for half an hour, and then detected by flow cytometry.

[0043] 3.1 In order to explore whether protein A magnetic beads coated with human B7H3 recombinant protein can activate CAR-NK, the embodiment of the present invention constructed a B7H3-targeted CAR-NK and a negative control (Mock) of the same vector on NK-92MI cells. This CAR-NK used the CAR molecule (NKG2D(TM)-2B4-FCER1G) with the best efficacy verified by the system, named CAR4. For the convenience of the experiment, the CAR molecule carried green fluorescent protein GFP.

[0044] The specific construction process is as follows: Gene sequence codon optimization was performed using GenScript codon optimization software (https: / / www.genscript.com.cn / tools / gensmart-codon-optimization). After optimization, the gene sequence was sent to Anshengda for gene synthesis. The geneIDs involved were: CD8α (GenBank: NM_001145873.1), anti-B7H3scFv (US20100143245A1), NKG2D (GenBank: AF461811.1), 2B4 (GenBank: AF107761.2), FCER1G (GenBank: NM_004106.2). Max DNA Polymerase (Takara, Code No. R045Q) PCR was used to obtain the relevant target gene fragment, which was then double-digested with NheI / XhoI and connected to the plasmid vector pIRES-EGFP-puro (Addgene, #45567). After the CAR molecular screening plasmid was constructed, the plasmid was extracted using a midiprep kit (ZymoPURE II Plasmid Midiprep Kit Cat #D4201) for later use.

[0045] Take NK-92MI cells with good growth status, collect cells by centrifugation, resuspend cells with celetrix special electroporation solution 103buffer (partA: partB = 1:1), add CAR molecular screening plasmid to electroporate cells. Electroporation conditions: 100μL electroporation tube, 10M cells, CAR molecular screening plasmid 8μg, voltage cell line 430V, electroporation solution 103buffer, electroporator model Celetrix Cell Electroporator Model CTX-1500A LE+. The electroporation operation was carried out according to the instructions of the celetrix electroporator. After electroporation, the cells were transferred to 1 well of a 6-well plate for culture, and 3ml of NK-92MI cell special culture medium (Procell, CAT#CM-0533) was added to each well for culture. After electroporation, the cells were transferred to a 6-well plate, grown in a 37°C incubator for 3 days, and then cultured in separate wells. The cells in a single well of the 6-well plate were filled with about 80%, and 2.0μg / ml puromycin was added for selection. After two weeks, the flow cytometry was run to identify the positive rate.

[0046] After co-culture of protein A magnetic beads coated with human B7H3 recombinant protein and CAR-NK cells, the magnetic beads were removed by a magnetic rack, fixed with tissue fixative and stained with fluorescent group-labeled CD69 antibody (Biolegend, 310909), and the expression of CD69 was detected by flow cytometry.

[0047] The results are as follows Figure 2As shown, compared with protein A magnetic beads, protein A magnetic beads coated with human B7H3 recombinant protein can effectively promote CAR-NK degranulation, while the degranulation of Mock used as a control did not increase, indicating that protein A magnetic beads coated with human B7H3 recombinant protein can specifically activate CAR-NK.

[0048] 3.2 Degranulation refers to the process in which NK cells release perforin and granzyme in the cytoplasm after receiving activation signals. Degranulation more directly marks the activation of NK cells and is also the main means for NK cells to kill target cells. Currently, the most commonly used marker for evaluating degranulation is CD107a.

[0049] After co-culture of Mock or CAR-NK with protein A magnetic beads coated with human B7H3 recombinant protein, the beads were removed by magnetic rack, fixed with tissue fixative and stained with CD107a antibody labeled with fluorescent group (Biolegend, 328620), and degranulation was observed by flow cytometry.

[0050] The results are as follows Figure 3 As shown, compared with protein A magnetic beads, protein A magnetic beads coated with human B7H3 recombinant protein can significantly increase CAR-NK degranulation, proving that protein A magnetic beads coated with human B7H3 recombinant protein can specifically activate CAR-NK.

[0051] 3.3NK cell activation usually involves a series of protein phosphorylation modifications. These phosphorylated proteins can be divided into two categories. One is the phosphorylation of proximal signaling proteins, and the common proteins are SRC family (Lck, Lyn, Fyn). The other is the phosphorylation of distal signaling proteins, and the common proteins are Erk, Akt, PLC-γ1, Vav1, and NF-kB. Since the magnetic beads can be easily and completely removed by the magnetic stand, the next step is to explore whether protein A magnetic beads coated with human B7H3 recombinant protein can specifically activate the downstream pathways of NK cells. After co-culture with Mock or CAR-NK for 0, 2, 5, 10, and 30 minutes, the magnetic beads were removed, the proteins were extracted, and gel electrophoresis was performed using 4-20% gradient gel and blocked with 5% skim milk powder. Lck (immunoway, YT2546) and phosphorylated Lck antibodies (Abcam, ab318960), Lyn (Abcam, ab318191) and phosphorylated Lyn antibody (Abcam, ab300118), Fyn (Abcam, ab125016) and phosphorylated Fyn antibody (Abcam, ab188319), ERK (CST, 4695) and phosphorylated ERK antibody (CST, 4370), Akt (Abcam, ab185633) and phosphorylated Akt antibody (Abcam, ab192623), PLC-γ1 (CST, 5690) and phosphorylated PLC-γ1 antibody (CST, 2821), Vav1 (immunoway, YT4862) and phosphorylated Vav1 antibody (Abcam, ab76225), NF-kB (CST, 8242) and phosphorylated NF-kB antibody (CST, 3033) were used as primary antibodies, and Actin was used as an internal control.

[0052] The results showed that due to the use of NK-92MI cells, the proximal signaling proteins of NK cells in the Mock group were significantly phosphorylated, including Lck ( Figure 4 )、Lyn( Figure 5 )、Fyn( Figure 6 ), after stimulation by protein A magnetic beads coated with human B7H3 recombinant protein, the proximal signaling proteins of CAR-NK were significantly phosphorylated, but there was no significant increase compared with the Mock group. Compared with the NK cells in the Mock group, protein A magnetic beads coated with human B7H3 recombinant protein can significantly activate the phosphorylation of distal signaling proteins of CAR-NK, including Erk ( Figure 7 )、Akt( Figure 8 )、PLC-γ1( Fig. 9 )、Vav1( Fig.10 )、NF-kB( Fig.11 ). It proves that protein A magnetic beads coated with human B7H3 recombinant protein can specifically activate the downstream signaling pathway of CAR-NK.

[0053] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of protein A magnetic beads coated with recombinant proteins in the preparation of a model for specific evaluation of the efficacy of CAR molecules on CAR-NK cells.

2. The use according to claim 1, characterized in that: The recombinant protein includes a target protein for cellular immunotherapy.

3. The application according to claim 2, characterized in that: The target protein includes an Fc tag.

4. The use according to claim 3, characterized in that: The recombinant protein includes human B3H7-Fc recombinant protein.

5. The use according to claim 1, characterized in that: The method for preparing protein A magnetic beads coated with recombinant protein comprises the following steps: washing the protein A magnetic beads and mixing them with the recombinant protein, removing the supernatant with a magnetic stand and then washing, and then resuspending with NK cell culture medium to obtain a protein A magnetic bead suspension coated with the recombinant protein.

6. The use according to claim 4, characterized in that: The ratio of the recombinant protein to the protein A magnetic beads was 2 μg / 100 μL.

7. A method for evaluating the efficacy of CAR molecules on CAR-NK cells, characterized in that: The following steps are involved: NK cells and CAR-NK cells were co-cultured with protein A magnetic beads coated with recombinant proteins, and NK cell degranulation was detected by flow cytometry. The efficacy of CAR molecules on CAR-NK cells was evaluated based on the flow cytometry results. The method for preparing protein A magnetic beads coated with recombinant protein comprises the following steps: washing the protein A magnetic beads and mixing them with the recombinant protein, removing the supernatant with a magnetic stand and then washing, and then resuspending with NK cell culture medium to obtain a protein A magnetic bead suspension coated with the recombinant protein.

8. The method according to claim 7, characterized in that: The co-culture was performed in a 24-well cell culture plate, and the ratio of the number of NK cells or CAR-NK cells to the protein A magnetic bead suspension coated with the recombinant protein in each well was 5×10 5 ~2×10 6 Cells: 100 μL.

9. The method according to claim 8, characterized in that: The co-culture temperature is 37° C., the culture time for activation marker or degranulation experiments is 4 hours, and the culture time for exploring the CAR-NK downstream activation signaling pathway is 0-30 minutes.

10. Application of protein A magnetic beads coated with recombinant protein in purifying CAR-NK cells.

Images