Cell line with ADCC effect and application

Abstract

The invention relates to a cell line with ADCC effect and application, and the cell line overexpresses CD16 protein and lacks CD3 protein. According to the invention, CD3 is knocked out, so that the Jurkat cell does not express a TCR complex, a downstream signal channel cannot be activated after a CD3 antibody is added, and only the activation of Fc [gamma] RIII in an ADCC effect can be realized. According to the invention, CD3 is knocked out through a CRISPR-Cas9 gene editing technology, Fc [gamma] RIIIa is over-expressed, an ADCC Fc [gamma] RIIIa (158V) Reporter CD3KO cell line is developed, interference of a TCR signal on ADCC is eliminated, and an NK cell mediated ADCC effect is enhanced.

Description

Technical Field

[0001] The present invention relates to the technical field of immune cell therapy, and in particular to a cell line with ADCC effect and its application. Background Art

[0002] ADCC (Antibody-Dependent Cellular Cytotoxicity) is an immune effector mechanism that activates a cytotoxic immune response by bridging effector cells with target cells through antibodies. It is a key effector in the anti-tumor mechanism of therapeutic antibody drugs and also helps eliminate virus-infected cells.

[0003] CD16 (Cluster of Differentiation 16, FcγRIII) is primarily expressed on the surface of natural killer (NK) cells. When antibodies bind to antigens on the surface of target cells, CD16 specifically binds to the Fc region of IgG antibodies. CD16 plays a key role in ADCC. When CD16 on NK cells binds to the Fc region of antibodies, the NK cells are activated, releasing perforin and granzymes, inducing apoptosis in target cells. CD16a (FcγRIIIa) is the primary effector receptor for ADCC and has two common polymorphic sites: 158V and 158F. The FcγRIIIa 158V allele is associated with high-affinity binding to the IgG Fc region, enhancing NK cell-mediated ADCC activity.

[0004] CD3 is part of the T cell receptor (TCR) and is composed of four distinct polypeptide chains: CD3γ (CD3gamma; CD3G), CD3δ (CD3delta; CD3D), CD3ε (CD3epsilon; CD3E), and CD3ζ (CD3zeta; CD247). These polypeptide chains interact to form a stable complex that exists on the T cell surface along with the TCR. They participate in antigen recognition signaling, mediating T cell activation and function. They are all expressed on the T cell surface and are central to T cell-mediated immune responses.

[0005] Currently, studies have shown that knocking out CD3 can easily lead to T cell dysfunction, reduce T cell-mediated immune regulation and cytokine secretion, and have no direct promoting or inhibiting effect on ADCC, but may indirectly affect the synergistic effect of NK cells and other immune cells, thereby regulating the ADCC effect.

[0006] Therefore, studying the mechanism and function of CD3 and CD16 in affecting ADCC and exploring their potential cross-links with NK cells can provide direction for the study of the ADCC production process and offer new methods for the design of new antibodies and clinical trial verification. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a cell line with ADCC effect and its application. The present invention knocks out CD3 to reduce the influence of TCR signal on ADCC pathway and overexpresses CD16, thereby enhancing the ADCC effect.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a cell line having ADCC effect, wherein the cell line overexpresses CD16 protein and lacks CD3 protein.

[0010] In this study, CD3 is deleted, preventing cells from expressing the TCR complex. Adding CD3 antibodies prevents downstream signaling pathways from being activated, leaving only FcγRIII activation through ADCC. Using CRISPR-Cas9 gene editing technology to knock out CD3 and overexpress FcγRIIIa, an ADCC FcγRIIIa(158V)Reporter CD3 KO cell line was developed, eliminating the interference of TCR signaling on ADCC and enhancing NK cell-mediated ADCC.

[0011] Preferably, the CD16 protein comprises an F158V mutation.

[0012] In the present invention, the above mutation of FcγRIIIa 158V polymorphism can significantly enhance ADCC activity, which is a key factor in improving the efficacy of immunotherapy.

[0013] Preferably, the amino acid sequence of the CD16 protein includes the sequence shown in SEQ ID No. 1.

[0014] SEQ ID No. 1:

[0015] MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLR CHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK.

[0016] Preferably, the nucleic acid sequence encoding the CD16 protein includes the sequence shown in SEQ ID No. 2.

[0017] SEQ ID No. 2:

[0018] .

[0019] Preferably, the cell types of the cell line include Jurkat cells and / or HEK-293 cells.

[0020] The Jurkat (human T lymphocyte leukemia functional cell line) cells used in the present invention are a lymphocyte cell line commonly used in immunology and cell biology research, are derived from human T cell leukemia patients, and can express TCR complexes.

[0021] Preferably, the amino acid sequence of the CD3 protein includes the sequence shown in SEQ ID No.3.

[0022] SEQ ID No.3:

[0023] MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSK PEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI.

[0024] In a second aspect, the present invention provides a method for constructing a cell line having ADCC effect according to the first aspect, wherein the method comprises overexpressing CD16 protein in the cell and knocking out CD3 protein.

[0025] Preferably, the construction method specifically includes: connecting CD16 protein to vector 1 to form recombinant vector 1, connecting CD3sgRNA to vector 2 to form recombinant vector 2, and introducing the recombinant vector 1 and recombinant vector 2 into host cells to obtain the obtained product.

[0026] Preferably, the target sequence of the CD3 sgRNA is shown as SEQ ID No. 4.

[0027] SEQ ID No. 4: AGGGCATGTCAATATTACTG.

[0028] Preferably, the vector 1 comprises any one of lenti-EF1a-MCS-PGK-Puro, lenti-CMV-MCS-PGK-Puro, lenti-EF1a-MCS-PGK-Blasticidin, or lenti-CMV-MCS-PGK-Blasticidin, or a combination of at least two thereof.

[0029] Preferably, the vector 2 comprises PGMLV-Cas9 and / or Lenti-Cas9.

[0030] In a third aspect, the present invention provides a disease model, wherein the disease model comprises the cell line having ADCC effect according to the first aspect.

[0031] In a fourth aspect, the present invention provides a use of the cell line having ADCC effect according to any one of claims 1 to 5 or the disease model according to claim 9 in drug screening.

[0032] Compared with the prior art, the present invention has at least the following beneficial effects:

[0033] By knocking out CD3, the present invention prevents Jurkat cells from expressing the TCR complex. Adding CD3 antibodies prevents downstream signaling pathways from being activated, leaving only FcγRIII activation through ADCC. Using CRISPR-Cas9 gene editing technology to knock out CD3 and overexpress FcγRIIIa, an ADCC FcγRIIIa(158V)Reporter CD3 KO cell line was developed, eliminating the interference of TCR signaling on ADCC and enhancing the NK cell-mediated ADCC effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the complete map of the recombinant plasmid lenti-EF1a-H_FCGR3A(CD16A_158V)-PGK-puro.

[0035] Figure 2 The figure shows the flow cytometry validation results of ADCC FcγRIIIa(158V)Reporter CD3 KO Cell Line using Anti-CD3epsilonAntibody antibody.

[0036] Figure 3 This figure shows the flow cytometry validation results of the ADCC FcγRIIIa(158V)Reporter CD3 KO Cell Line using Anti-CD16 Antibody antibody.

[0037] Figure 4 This figure shows the Luciferase detection results in the Anti-CD3 epsilon Antibody drug detection experiment in which ADCC FcγRIIIa(158V)Reporter CD3 KO Cell Line cells were co-cultured with H_CD3 HEK-293 cells. DETAILED DESCRIPTION

[0038] The technical solution of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0039] Example 1

[0040] This example constructs a recombinant vector

[0041] (1) Construction of CD16 overexpression vector

[0042] The vector lenti-EF1a-MCS-PGK-Puro (from Jiman Biotechnology (Shanghai) Co., Ltd.) was double-enzyme-digested using XbaI and BamHI. The nucleic acid sequence of CD16 protein H_FCGR3A (CD16A_158V) (SEQ ID No.2) was synthesized and double-enzyme-digested using XbaI and BamHI. The lenti-EF1a-MCS-PGK-Puro and H_FCGR3A (CD16A_158V) after enzyme digestion were connected and named lenti-EF1a-H_FCGR3A (CD16A_158V) -PGK-puro. Sequencing was performed using PEF-F and PGK-R primer sequences, and the sequencing was completed by Sangon Biotechnology (Shanghai) Co., Ltd. The sequencing results showed that the inserted gene sequence H_FCGR3A (CD16A_158V) was the same as the sequence shown in SEQ ID No.3. The structure map of the overexpression vector is as shown below. Figure 1 The primer sequences are shown in Table 1.

[0043] (2) Construction of CD3 protein knockout vector

[0044] The vector PGMLV-Cas9 (from Yoshiman Biotechnology (Shanghai) Co., Ltd.) was double-digested with BsmBI. The H_CD3 sgRNA oligo sequence (SEQ ID No. 4) was synthesized and ligated to the linear PGMLV-Cas9 vector after digestion using an annealing reaction. The resulting vector was named H_CD3 sgRNA-Puro (PGMLV-Cas9). Sequencing was performed using the Hu6-F primer by Sangon Biotech (Shanghai) Co., Ltd. The sequencing results showed that the inserted sequence H_CD3 sgRNA was identical to the sequence shown in SEQ ID No. 4. The primer sequences are shown in Table 1.

[0045] Table 1

[0046] name serial number Sequence 5'-3' PEF-F SEQ ID No.5 TCAAGCCTCAGACAGTGGTTC PGK-R SEQ ID No.6 GCCAGAGGCCACTTGTGTAG Hu6-F SEQ ID No.7 GAGGGCCTATTTCCCATGATT

[0047] Example 2

[0048] This example performs virus packaging

[0049] Lentiviral packaging cells were 293T anchorage-dependent epithelial cells, and the growth medium was DMEM (containing 10% FBS). Transfection was performed using lenti-EF1a-H_FCGR3A(CD16A_158V)-PGK-puro and H_CD3 sgRNA-Puro(PGMLV-Cas9) prepared in Example 1.

[0050] One day before transfection, subculture the grown cells into a 10 cm dish at an appropriate ratio. When the cells reach 80% growth, prepare for transfection. 2 hours before transfection, replace the cells with fresh DMEM (containing 10% FBS) at 12 mL / 10 cm dish. Take two sterile 1.5mL EP tubes, add 1mL of DMEM, 10μL of Lenti-HG Mix and 60μg of HGtransgene reagent respectively, then add 10μg of lenti-EF1a-H_FCGR3A(CD16A_158V)-PGK-puro and 10μg of H_CD3 sgRNA-Puro(PGMLV-Cas9) plasmids into the two EP tubes respectively, mix them separately, leave them at room temperature for 20min, and then evenly add them dropwise to the culture dish that had been changed in advance, and then culture them in a CO2 incubator. After 10h of transfection, evenly add 100×Enhancing buffer (120μL / dish) to promote transfection. After 20h of transfection, aspirate the cell culture medium and add 15mL of fresh cell culture medium to continue culture.

[0051] Virus collection: 48 hours after the fluid change, aspirate the cell supernatant into a 50 mL centrifuge tube, centrifuge at 4500 g for 5 min at 4°C, filter the supernatant with a 0.45 μm filter and transfer it to a new centrifuge tube. Finally, transfer the filtrate in batches to a 100 kD ultrafiltration tube and centrifuge at 3000 g for 10 min at 4°C. Discard the lower layer of liquid into a waste liquid cup filled with disinfectant. Adjust the centrifugation time again according to the required volume of the virus and centrifuge the ultrafiltration tube until the centrifugation is complete.

[0052] Lentivirus titer determination: 293T cells in logarithmic growth phase were trypsinized and seeded at 8,000 cells / well in 96-well plates. Incubate at 37°C overnight. The next day, cells were infected at 40% confluency. The virus solution was serially diluted using cell culture medium supplemented with 10% FBS. Select the desired wells, aspirate 90 μL of the medium, and add 90 μL of the mixed lentivirus solution to each well. Incubate the wells overnight at 37°C in a cell culture incubator. On the third day, the lentivirus-containing medium was removed and replaced with 100 μL of complete culture medium. On the fifth day, viral titer was determined by RT-PCR. The RT-PCR system is shown in Table 2, the reaction conditions are shown in Table 3, and the primers used are shown in Table 4. The dilution method was as follows: Diluent #1: 10 μL of virus solution + 90 μL of virus dilution medium; Diluent #2: 10 μL of diluent #1 + 90 μL of virus dilution medium. The titer of lenti-EF1a-H_FCGR3A(CD16A_158V)-PGK-puro was 8.66×10 7TU / mL, the titer of H_CD3 sgRNA-Puro(PGMLV-Cas9) was 1.01×10 9 TU / mL.

[0053] Table 2

[0054] Components volume Ultrapure water 7.2μL 2×SYBR Mix 10 μL Upstream primer (10 μM) 0.4μL Downstream primer (10 μM) 0.4μL template 2μL Overall system 20 μL

[0055] Table 3

[0056]

[0057] Table 4

[0058] name serial number Sequence 5'-3' WPRE-F SEQ ID No.8 CGCTATGTGGATACGCTGCTTTA WPRE-R SEQ ID No.9 GCAACCAGGATTTATACAAGGAGGA GAPDH-F (internal reference) SEQ ID No.10 GTCTCCTCTGACTTCAACAGCG GAPDH-R (internal reference) SEQ ID No.11 ACCACCCTGTTGCTGTAGCCAA

[0059] Example 3

[0060] This example constructs a reporter gene cell line

[0061] The two virus solutions obtained in Example 2 were used to prepare two cell lines. One cell line used the two virus solutions to infect Jurkat cells at a virus MOI of 150, and the other cell line used the H_CD3 sgRNA-Puro (PGMLV-Cas9) virus solution to infect Jurkat cells at a virus MOI of 150.

[0062] On the first day, cells were cultured to the logarithmic growth phase and 4 × 10 4 The cells were placed in a 1.5 mL EP tube and centrifuged at 1000 rpm for 5 min. The supernatant was discarded and the cell pellet was kept for later use. The required volume of lentivirus was calculated according to MOI = 150. The virus solution was aspirated and added to another EP tube prepared in advance (containing 500 μL complete medium). After pipetting and mixing, it was added to 4 × 10 4 The cell pellet was gently pipetted into the EP tube to avoid bubbles. The new mixture was then transferred to a 24-well plate and centrifuged at 2100 rpm for 30 minutes. After centrifugation, the cell culture plate was placed in a 37°C, 5% CO2 incubator for overnight culture.

[0063] On the second day, 16 hours after infection, observe the cell status. If the cell status deteriorates significantly, aspirate the culture medium containing the lentiviral particles and replace it with a full volume of fresh culture medium. If the cell status is normal, replenish the medium to 1 mL of full culture medium and replace it with a full volume of fresh culture medium on the third day.

[0064] On the fourth day, continue to culture the cells and observe whether there are any abnormalities in the cell status.

[0065] On day 5, cells were screened for Puro resistance (Puro full lethal concentration 1.5 μg / mL, maintenance concentration 0.75 μg / mL) for two rounds of drug selection. Once cells were stable, they were maintained in RPMI 1640 complete medium supplemented with 10% FBS, 1% P.S., 3.5 μg / mL Blasticidin, and 0.75 μg / mL Puromycin. The prepared cells were designated ADCC FcγRIIIa(158V)Reporter CD3 KO Cell Line and H_CD3 HEK-293 Cell Line.

[0066] After infection according to the experimental process, the cells were in normal condition and screened for two rounds. Microscopic observation showed that no large-scale cell death occurred during the screening process.

[0067] Example 4

[0068] This embodiment performs flow verification

[0069] Wait for the cell growth rate to stabilize. On the day of the experiment, digest the ADCC FcγRIIIa (158V) Reporter CD3 KOCell Line and take 100 μL of cell suspension (adjust the concentration to 2×10 after counting the cells with PBS). 6 cells / mL), add 10ug / mL of surface antibody Anti-CD3 epsilon Antibody, incubate at 4℃ in the dark for 1h. Add 2mL PBS to rinse and repeat this step. Add fluorescent labeled secondary antibody and incubate at 4℃ in the dark for 30min. Centrifuge at 1000rpm for 5min, remove the supernatant, and resuspend in 300μL PBS. Immediately test on the instrument to verify the expression. Figure 2 Flow cytometry results showed that compared with uninfected Jurkat cells, the CD3 gene was successfully knocked out in ADCCFcγRIIIa(158V)Reporter CD3 KO Cell Line cells. Figure 3 Flow cytometry results showed that CD16 was still expressed.

[0070] Example 5

[0071] This example conducts drug validation tests

[0072] The cell number of ADCC FcγRIIIa(158V)Reporter CD3 KO Cell Line was 1×10 5 cells / well, H_CD3 HEK-293Cell Line cell volume is 1.5×10 4This experiment used Anti-CD3 epsilon Antibody (150 kDa; hereinafter referred to as: Anti-CD3) as an agonist and Human IgG1 Isotype Control (hereinafter referred to as Human IgG1) as a control.

[0073] The first well concentration (Conc.01) of the Anti-CD3 epsilon Antibody and Human IgG1 Isotype Control Antibody was 30 μg / mL. A 4-fold serial dilution was performed. Wells 1 through 9 were arranged in the second row (B2-B10) and third row (C2-C10) of the cell plate, respectively. Wells B11 and C11 served as the 0 concentration control. 100 μL of PBS was added to the surrounding wells to prevent evaporation.

[0074] 20 h before the experiment, H_CD3 HEK-293Cell Line cells were removed from the culture flask, digested and centrifuged to collect the cell pellet, and resuspended in an appropriate amount of complete medium. Cell viability was detected and counted, and the cell concentration was adjusted to 1.5×10 5 cells / mL. Using a pipette, add 100 μL of cells / well to the center 10 wells. Add 100 μL of PBS to the surrounding wells. Cover the plate and incubate overnight in an incubator.

[0075] 2 h before the experiment, ADCC FcγRIIIa(158V)Reporter CD3 KO Cell Line cells were removed from the culture flask, digested and centrifuged to collect the cell pellet, and resuspended in Assay Buffer. Cell viability was detected and counted, and the cell concentration was adjusted to 2×10 6 cells / mL.

[0076] Prepare drug dilutions in a sterile 96-well V-bottom plate. The following steps use Anti-CD3 as an example: Use one row for each antibody to be tested (e.g., B2-B10), with an Anti-CD3 concentration of 2.595 mg / mL and a Human IgG1 concentration of 2 mg / mL. Add Assay Buffer to the 96-well V-bottom plate. For example, add 71.64 μL of Assay Buffer to well B2 and 55 μL of Assay Buffer to wells B3-B10. Pipette different volumes of the test sample stock solution and add them to the first well of the serial dilution series, e.g., add 1.7 μL of Anti-CD3 to well B2. Mix thoroughly.

[0077] Pipette 18.33 μL from the first gradient dilution well, B2, and add it to the second gradient dilution well, B3, and mix thoroughly. Repeat this process until the ninth gradient dilution well (B10). Remove the cell plate cultured overnight in step 1, discard 100 μL of culture medium from each well, and add 50 μL / well of ADCC FcγRIIIa (158V) Reporter CD3 KO Cell Line cells. Add 50 μL of the previously prepared antibody gradient dilution solution to each well. Cover the plate and incubate in a 37°C CO2 incubator for 6 hours. Detect the results using a Luciferase Assay Kit.

[0078] like Figure 4 As shown, combined with flow cytometry results, it was confirmed that CD3 knockout prevented the CD3 antibody from binding to the TCR complex. This phenomenon suggests that the T cell activation mechanism has been altered, thereby affecting its response to the antibody. However, the ADCC effect observed in the experiment was still significant, especially in the modified FcγRIIIa(158V)Reporter CD3 KO cell line, which showed that it could still activate the ADCC pathway. The core objective of this experiment was to explore the potential of enhancing ADCC by eliminating TCR-mediated signaling through CD3 knockout and simultaneously overexpressing CD16 (i.e., FcγRIII). Using CD16 as the primary receptor effectively focused the antibody-mediated cellular effect on the interaction between the Fc region and FcγRIII, independent of the TCR signaling pathway. The EC50 value was 0.01183 μg / mL, indicating that ADCC was still significant even in the absence of CD3. This result confirms that enhancing CD16 expression on the NK cell surface can effectively promote ADCC and can be subsequently applied to the detection of ADCC activity of TCE-based CD3 bispecific antibodies.

[0079] In summary, the present invention reduces the influence of TCR signal on ADCC pathway by knocking out CD3 and overexpressing CD16, thereby enhancing ADCC effect.

[0080] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A cell line with ADCC effect, characterized in that: The cell line overexpresses CD16 protein and lacks CD3 protein.

2. The cell line with ADCC effect according to claim 1, characterized in that The CD16 protein includes an F158V mutation; Preferably, the amino acid sequence of the CD16 protein includes the sequence shown in SEQ ID No.

1.

3. The cell line with ADCC effect according to claim 1 or 2, characterized in that The nucleic acid sequence encoding the CD16 protein includes the sequence shown in SEQ ID No.

2.

4. The cell line having ADCC effect according to any one of claims 1 to 3, characterized in that The cell types of the cell line include Jurkat cells and / or HEK-293 cells.

5. The cell line having ADCC effect according to any one of claims 1 to 4, characterized in that The amino acid sequence of the CD3 protein includes the sequence shown in SEQ ID No.

3.

6. A method for constructing a cell line having ADCC effect according to any one of claims 1 to 5, characterized in that: The construction method comprises the steps of overexpressing CD16 protein in cells and knocking out CD3 protein.

7. The method for constructing a cell line with ADCC effect according to claim 6, characterized in that: The construction method specifically includes: connecting CD16 protein with vector 1 to form recombinant vector 1, connecting CD3 sgRNA with vector 2 to form recombinant vector 2, and introducing the recombinant vector 1 and recombinant vector 2 into a host cell to obtain the result; Preferably, the target sequence of the CD3 sgRNA is shown as SEQ ID No.

4.

8. The method for constructing a cell line with ADCC effect according to claim 7, characterized in that: The vector 1 comprises any one of lenti-EF1a-MCS-PGK-Puro, lenti-CMV-MCS-PGK-Puro, lenti-EF1a-MCS-PGK-Blasticidin or lenti-CMV-MCS-PGK-Blasticidin, or a combination of at least two thereof; Preferably, the vector 2 comprises PGMLV-Cas9 and / or Lenti-Cas9.

9. A disease model, characterized in that The disease model comprises the cell line having ADCC effect according to any one of claims 1 to 5.

10. Use of the cell line having ADCC effect according to any one of claims 1 to 5 or the disease model according to claim 9 in drug screening.

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