293T multi-target target cells and their preparation method and application

Abstract

The present invention belongs to the field of cellular immune engineering technology, and in particular to a kind of 293T multi-target target cell and its preparation method and application.The target cell includes a nucleotide fragment a expressing antigen CEA, a nucleotide fragment b expressing antigen CD19, a nucleotide fragment c expressing antigen BCMA, and a nucleotide fragment d expressing antigen CD70. Its preparation method is to select a basic cell (293T cell), construct it by means of exogenous construction, and then detect its antigen expression by flow cytometry, and sort out target cells that simultaneously highly express antigens CEA, CD19, BCMA and CD70 as target cells. The target cell exogenously expresses multiple antigens that can be used for the detection of multiple targets, and the operation that needs to be repeated 4 times is condensed into an operation once, which reduces the workload of cell culture, greatly saves time and cost, and the background factor secretion amount is normal during detection, the factor detection result is stable, the signal-to-noise ratio is higher, and it is more conducive to the quality detection of subsequent products.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell immune engineering, and in particular relates to a 293T multi-target target cell and a preparation method and application thereof. Background Art

[0002] In existing cell-based in vitro efficacy evaluation methods, the target cells are usually single target cells corresponding to the indication. For example, in the evaluation of CAR-T cells for B-lineage acute lymphoblastic leukemia, the corresponding target cells are NALM6 cells that endogenously express CD19, and in the evaluation of CAR-T cells for multiple myeloma, the corresponding target cells are MM.1S cells that endogenously express BCMA. If the efficacy of CAR-T cells for multiple indications is to be evaluated, multiple tumor cells need to be cultured. This not only requires a large workload, but also greatly increases the error rate when culturing multiple cells at one time. In addition, in the experimental method for in vitro efficacy evaluation of cells, different indications will have a different negative control target cell. Currently, most negative single-target control target cells have the problem of high background factor secretion, which affects the overall in vitro efficacy evaluation and the quality control of the drug.

[0003] 293T cells are a cell line derived from 293 cells through genetic engineering. Transfected with the adenovirus E1A gene, they express the SV40 large T antigen and contain the SV40 origin of replication and promoter region. Many eukaryotic expression vectors, such as pcDNA3.1, contain the SV40 viral origin of replication, allowing replication in cell lines expressing the SV40 viral T antigen, thereby increasing the expression level of exogenous genes. Therefore, 293T cells are widely used in transient transfections to overexpress various target proteins. They are also easy to culture and grow rapidly. To facilitate experiments and achieve optimal overall in vitro efficacy evaluation, the present invention utilizes 293T cells as target cells. However, expressing multiple antigens on 293T cells can interfere with the expression of multiple antigens, and expressing multiple antigens can also have certain effects on the 293T cells themselves. To address these objectives and overcome the numerous uncertainties in the experimental process, the present invention ultimately constructs standardized 293T cells targeting multiple targets. Summary of the Invention

[0004] In view of this, the present invention provides a target cell, which can simultaneously express antigens CEA, antigen CD19, antigen BCMA and antigen CD70. Target cells expressing multiple antigens may have the expression of multiple antigens affecting each other, and the expression of multiple antigens may also have a certain impact on the target cell itself. The technical solution of the present invention overcomes many uncertain factors and finally constructs a target cell targeting multiple targets.

[0005] The target cells include a nucleotide fragment a expressing the antigen CEA, a nucleotide fragment b expressing the antigen CD19, a nucleotide fragment c expressing the antigen BCMA, and a nucleotide fragment d expressing the antigen CD70.

[0006] Furthermore, the target cell also includes a nucleotide fragment that expresses a marker protein.

[0007] Furthermore, the nucleotide fragment a and / or the nucleotide fragment b and / or the nucleotide fragment c and / or the nucleotide fragment d are respectively connected to different / the same expression vector; when connected to different expression vectors, the types of the expression vectors are the same or different.

[0008] Preferably, the expression vector may be a viral vector, or other applicable eukaryotic or prokaryotic expression vector.

[0009] Furthermore, the target cells are transformed by exogenous construction using 293T as the basic cells to obtain "293T-CEA-CD19-BCMA-CD70-Luc-GFP target cells" or "293T-CEA-CD19-BCMA-CD70 target cells".

[0010] The present invention further provides a method for preparing the aforementioned target cells, which comprises selecting a basic cell, constructing it by exogenous construction, and then detecting its antigen expression by flow cytometry to select target cells that simultaneously highly express four antigens (antigen CEA, antigen CD19, antigen BCMA and antigen CD70) as target cells.

[0011] The method for preparing target cells that highly express four antigens comprises: selecting appropriate infection coefficients for the nucleotide fragment a, the nucleotide fragment b, the nucleotide fragment c, and the nucleotide fragment d, respectively, and transfecting them into the target cell a; the infection coefficients of the nucleotide fragment a, the nucleotide fragment b, the nucleotide fragment c, and the nucleotide fragment d are all 8-12 MOI, preferably 10 MOI.

[0012] Furthermore, the transfection of the nucleotide fragments is performed in batches, and the transfection order can be any combination of orders. Preferably, the nucleotide fragment a, the nucleotide fragment b, the nucleotide fragment c, and the nucleotide fragment d are sequentially transfected into the target cell a.

[0013] Furthermore, the transfection method includes: connecting the nucleotide fragment a, the nucleotide fragment b, the nucleotide fragment c, and the nucleotide fragment d to different expression vectors for transfection. The expression vector can be a viral vector or other applicable eukaryotic or prokaryotic expression vector, preferably a viral vector. That is, the expression vector is selected from commercially available expression vectors or expression vectors disclosed in other patents of the applicant. The transfection method is conventional transfection known to those skilled in the art and is consistent with the transfection methods disclosed in other related patents of the applicant.

[0014] Furthermore, the preparation method further comprises: transfecting the target cell a with a nucleotide sequence of a marker protein, preferably, the marker protein is Luc-GFP.

[0015] Furthermore, the preparation method further includes: after transfection is completed and culture is performed, the target cells are labeled with corresponding antibodies, and then a cell population that expresses positive antigens CEA, CD19, BCMA, and CD70 is sorted out.

[0016] Preferably, the sorting method is to detect the antigen expression by flow cytometry, generally using a flow cytometry instrument for sorting. After sorting, the cells are expanded and cultured and a library is established.

[0017] Furthermore, the culture after transfection is conventional target cell culture, using conventional culture medium and for conventional time.

[0018] Preferably, the target cell a is a 293T cell. 293T cells are a cell line derived from 293 cells through genetic engineering. They are transfected with the adenovirus E1A gene and can express the SV40 large T antigen. They contain the SV40 replication origin and promoter region. Many eukaryotic expression vectors, such as pcDNA3.1, contain the SV40 viral replication origin, allowing replication in cell lines expressing the SV40 viral T antigen, thereby increasing the expression level of exogenous genes. Therefore, 293T cells are widely used in transient transfection to overexpress various target proteins. They are also easy to culture and grow rapidly.

[0019] The present invention also aims to provide an application of the target cells that simultaneously highly express four antigens as described above. The target cells can be used as a reagent for evaluating the efficacy of cells in vitro, and the target cells can also be used in the preparation of a reagent or device for evaluating the efficacy of cells in vitro.

[0020] Furthermore, the in vitro cell efficacy evaluation reagent or device is a reagent or device for testing the killing effect of CAR-T cells or a reagent or device for testing the secretion amount of CAR-T cell factors, and the CAR-T cells target at least one of CEA, CD19, BCMA, and CD70.

[0021] Preferably, the target cells can be used as positive control target cells for in vitro efficacy evaluation of one or more of the four indications: B-lineage acute lymphoblastic leukemia, multiple myeloma, CEA-positive malignant tumors, and renal cell carcinoma.

[0022] The beneficial effects of the present invention are

[0023] The target cells provided by the present invention that simultaneously express four antigens, namely, CEA, CD19, BCMA, and CD70, exogenously express multiple targets, condensing an operation that needs to be repeated four times into one operation, reducing the workload of cell culture and greatly saving time and cost.

[0024] The 293T target cells provided by the present invention simultaneously express four antigens, namely CEA, CD19, BCMA, and CD70. During detection, the background factor secretion amount is normal, the factor detection results are stable, and the signal-to-noise ratio is higher, which is more conducive to the quality detection of subsequent products.

[0025] The target cells provided by the present invention simultaneously express four antigens, namely, CEA, CD19, BCMA, and CD70. The four antigens expressed on 293T cells do not affect each other, can all be expressed normally, and can also achieve the quality control effect of the corresponding targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a flow cytometric plot of CEA antigen expression in multi-target cells.

[0027] Figure 2 This is a flow cytometric plot of CD19 antigen expression on multi-target target cells.

[0028] Figure 3 This is a flow cytometric plot of BCMA antigen expression in multi-target target cells.

[0029] Figure 4 This is a flow cytometric plot of CD70 antigen expression on multi-target target cells.

[0030] Figure 5 This is a flow cytometry graph of GFP expression in multi-target cells.

[0031] Figure 6 Comparison of cell killing results between CEA target and conventional target cells for multi-target target cells.

[0032] Figure 7 Comparison of cytokine secretion experimental results of CEA target cells and conventional target cells in multi-target target cells.

[0033] Figure 8 Comparison of cell killing results between CEA target of multi-target cells and single-target cells.

[0034] Figure 9The results of the cytokine secretion experiments of CEA target cells with multiple targets are compared with those of single target cells.

[0035] Figure 10 This is the cell killing result of CD19 target on multiple target cells.

[0036] Figure 11 The results of cytokine secretion of CD19 target in multi-target target cells.

[0037] Figure 12 This is the cell killing result of BCMA target in multiple target cells.

[0038] Figure 13 These are the cytokine secretion results of the BCMA target in multi-target cells.

[0039] Figure 14 This is the cell killing result of CD70 target on multiple target cells.

[0040] Figure 15 These are the cytokine secretion results of the BCMA target in multi-target cells. DETAILED DESCRIPTION

[0041] The examples are provided to better illustrate the present invention, but are not intended to limit the present invention to the examples. Therefore, non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0042] In the embodiment of the present invention, the sources of the antigen sequences expressed on the target cells (including nucleotide fragments expressing the antigens CEA, CD19, BCMA, and CD70) are shown in Table 1 below:

[0043] Table 1 Antigens used in the examples and their sources

[0044] antigen Genebank sequences CEA NM_004363.6 CD19 NM_001770.6 BCMA NM_001192.3 CD70 NM_001252.5

[0045] In the embodiment of the present invention, the expression vector and transfection method are selected from another patent of the applicant 201710301492.1, a humanized monoclonal antibody targeting the human CD19 antigen.

[0046] In an embodiment of the present invention, the feasibility of standardizing cells as positive target cells for in vitro efficacy evaluation of each target was verified by detecting cell killing by Luciferase method and detecting cytokine (IFN-γ) secretion by ELISA method, and the results of efficacy evaluation of target cells and single-target exogenous constructed cells were compared to evaluate their consistency. Luciferase method for detecting cell killing is to transfect target cells with the reporter enzyme luciferase (Luc) gene of eukaryotic organisms by gene transfection technology, establish a stably transfected target cell line, and measure the biological cytotoxicity mediated by CAR-T cells. Select a suitable effect-target ratio to co-incubate target cells and CAR-T cells in a suitable system. After co-culturing for a period of time, the reporter enzyme activity (representing the number of surviving target cells) released into the culture medium is measured by a kit, and the killing rate of effector cells against target cells can be calculated.

[0047] In the practice of the present invention, cytokine (IFN-γ) secretion is detected by ELISA method, specifically: the collected cell supernatant after co-culture is used to quantitatively detect the IFN-γ content in the sample using the Human IFN-γ ELISA Set kit based on the principle of antigen-antibody binding.

[0048] Example 1 Quality Control Standardized Cell Construction

[0049] The viral vector carrying the nucleotide fragment expressing four antigens (CEA, CD19, BCMA, CD70) and Luc-GFP was transfected into 293T cells in batches at an infection coefficient of 10 MOI. After culture, the corresponding antibodies were selected to label the cells and the co-positive and stably expressed cell population was sorted using a flow cytometry instrument. Finally, 293T-CEA-CD19-BCMA-CD70-Luc-GFP cells were obtained. The cells were expanded and a library was established. After flow cytometry sorting, the positive rate of each antigen expression was tested as shown below. Figure 1-5 As shown, CEA antigen expression was 98.33%, strong expression ( Figure 1 CD19 antigen expression was 99.61%, strong expression ( Figure 2 BCMA antigen expression was 97.65%, strong expression ( Figure 3 CD70 antigen expression was 99.94%, strong expression ( Figure 4 GFP expression was 99.32%, strong expression ( Figure 5 shown).

[0050] Example 2 Target Verification

[0051] (1) CEA target verification

[0052] 1) Comparison and verification with conventional target cells

[0053] CEA-CAR-T was used as effector cells for in vitro efficacy evaluation, and 293T-CEA-CD19-BCMA-CD70-Luc-GFP and 293T-Luc-GFP were used as target cells. In the control experiment, DLD1-CEA-Luc-GFP and DLD1-Luc-GFP were used as target cells, and the experimental results of the two were compared.

[0054] The results of cell killing experiments showed (Table 2 and Figure 6 (As shown) At E / T = 8:1 for 24 h, the killing rate of CEA-CAR-T against 293T-CEA-CD19-BCMA-CD70-Luc-GFP was consistent with that against DLD1-CEA-Luc-GFP.

[0055] Table 2 Comparison of cell killing results of CEA target cells with multi-target target cells and conventional target cells

[0056]

[0057] The results of cytokine secretion experiments showed that (Table 3 and Figure 7 As shown in the figure, under the same target ratio as the control experiment, 293T-CEA-CD19-BCMA-CD70-Luc-GFP can secrete higher IFN-γ; in addition, the results of both experiments show that the signal-to-noise ratio of using 293T-CEA-CD19-BCMA-CD70-Luc-GFP and 293T-Luc-GFP as target cells is better than that of conventional target cells.

[0058] Table 3 Comparison of cytokine secretion experimental results and signal-to-noise ratio between CEA target cells and conventional target cells

[0059]

[0060] 2) Comparison and verification with single target cells

[0061] CEA-CAR-T cells were used as effector cells for in vitro efficacy evaluation, and 293T-CEA-CD19-BCMA-CD70-Luc-GFP and 293T-Luc-GFP were used as target cells. In the control experiment, 293T-CEA-Luc-GFP and 293T-Luc-GFP were used as target cells, and the experimental results of the two were compared.

[0062] The results of cell killing experiments showed (Table 4 and Figure 8 As shown in the figure, at E / T = 8:1 for 24 hours, the killing rate of CEA-CAR-T on 293T-CEA-CD19-BCMA-CD70-Luc-GFP was consistent with that on 293T-CEA-Luc-GFP.

[0063] Table 4 Comparison of cell killing results of CEA target cells with multi-target cells and single-target cells

[0064]

[0065] The results of cytokine secretion experiments showed (Table 5 and Figure 9 As shown in the figure, under the same target ratio as the control experiment, 293T-CEA-CD19-BCMA-CD70-Luc-GFP could secrete higher IFN-γ.

[0066] Table 5 Comparison of cytokine secretion experimental results and signal-to-noise ratio between CEA target cells with multiple targets and single target cells

[0067]

[0068] (2) CD19 target validation

[0069] CD19-CAR-T cells were used as effector cells for in vitro efficacy evaluation, and 293T-CEA-CD19-BCMA-CD70-Luc-GFP and 293T-Luc-GFP were used as target cells. In the control experiment, NALM6-Luc-GFP and K562-Luc-GFP were used as target cells, and the experimental results of the two were compared.

[0070] The results of cell killing experiments showed (Table 6 and Figure 10 As shown in the figure, at E / T=8:1 for 24h, the killing rate of CD19-CAR-T against 293T-CEA-CD19-BCMA-CD70-Luc-GFP was consistent with that against NALM6-Luc-GFP.

[0071] Table 6 Cell killing results of CD19 target of multi-target target cells

[0072]

[0073]

[0074] The results of the cytokine secretion experiment showed (Table 7 and Figure 11 As shown in the figure, under the same target ratio as the control experiment, 293T-CEA-CD19-BCMA-CD70-Luc-GFP could secrete higher IFN-γ.

[0075] Table 7 Cytokine secretion results and signal-to-noise ratio of CD19 target of multi-target target cells

[0076]

[0077] (3) BCMA target verification

[0078] BCMA-CAR-T cells were used as effector cells for in vitro efficacy evaluation, and 293T-CEA-CD19-BCMA-CD70-Luc-GFP and 293T-Luc-GFP were used as target cells. In the control experiment, MM.1S-Luc-GFP and K562-Luc-GFP were used as target cells, and the experimental results of the two were compared.

[0079] The results of cell killing experiments showed (Table 8 and Figure 12 As shown in the figure, at E / T = 8:1 for 24 hours, the killing rate of BCMA-CAR-T on 293T-CEA-CD19-BCMA-CD70-Luc-GFP was consistent with that on MM.1S-Luc-GFP.

[0080] Table 8 Cell killing results of BCMA target in multi-target target cells

[0081]

[0082] The results of the cytokine secretion experiment showed (Table 9 and Figure 13 As shown in the figure, under the same effect-target ratio as the control experiment, the IFN-γ secreted by 293T-CEA-CD19-BCMA-CD70-Luc-GFP was also consistent with the secretion amount of MM.1S-Luc-GFP.

[0083] Table 9 Cytokine secretion results and signal-to-noise ratio of BCMA target in multi-target target cells

[0084]

[0085]

[0086] (4) CD70 target verification

[0087] CD70-CAR-T cells were used as effector cells for in vitro efficacy evaluation, and 293T-CEA-CD19-BCMA-CD70-Luc-GFP and 293T-Luc-GFP were used as target cells. In the control experiment, 786-O-Luc-GFP and A549-Luc-GFP were used as target cells, and the experimental results of the two were compared.

[0088] The results of cell killing experiments showed (Table 10 and Figure 14 As shown in the figure, at E / T=8:1 for 24h, the killing rate of CD70-CAR-T against 293T-CEA-CD19-BCMA-CD70-Luc-GFP was consistent with that against 786-O-Luc-GFP.

[0089] Table 10 Cell killing results of CD70 target on multi-target target cells

[0090]

[0091] The results of the cytokine secretion experiment showed (Table 11 and Figure 15 As shown in the figure, under the same target ratio as the control experiment, 293T-CEA-CD19-BCMA-CD70-Luc-GFP can secrete higher IFN-γ; in addition, the results of both experiments show that the signal-to-noise ratio of using 293T-CEA-CD19-BCMA-CD70-Luc-GFP and 293T-Luc-GFP as target cells is better than that of conventional target cells.

[0092] Table 11 Cytokine secretion results and signal-to-noise ratio of BCMA target in multi-target target cells

[0093]

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A target cell, characterized in that The target cells include a nucleotide fragment a expressing the antigen CEA, a nucleotide fragment b expressing the antigen CD19, a nucleotide fragment c expressing the antigen BCMA, and a nucleotide fragment d expressing the antigen CD70; the basal cells of the target cells are 293T cells; the nucleotide sequence of the nucleotide fragment a expressing the antigen CEA is shown in Genebank accession number NM_004363.6; the nucleotide sequence of the nucleotide fragment b expressing the antigen CD19 is shown in Genebank accession number NM_001770.6; the nucleotide sequence of the nucleotide fragment c expressing the antigen BCMA is shown in Genebank accession number NM_001192.3; the nucleotide sequence of the nucleotide fragment d expressing the antigen CD70 is shown in Genebank accession number NM_001252.

5.

2. The target cell according to claim 1, characterized in that The target cell also includes a nucleotide fragment that expresses a marker protein.

3. The target cell according to claim 1, wherein the nucleotide fragment a and / or the nucleotide fragment b and / or the nucleotide fragment c and / or the nucleotide fragment d are respectively linked to different / the same expression vector; when linked to different expression vectors, the types of the expression vectors are the same or different.

4. The method for preparing target cells according to claim 1, characterized in that: The preparation method comprises: selecting appropriate infection coefficients for the nucleotide fragment a, the nucleotide fragment b, the nucleotide fragment c, and the nucleotide fragment d, respectively, and transfecting them into target cells a; the infection coefficients of the nucleotide fragment a, the nucleotide fragment b, the nucleotide fragment c, and the nucleotide fragment d are all 8-12 MOI; and the target cells a are 293T cells.

5. The preparation method according to claim 4, characterized in that The nucleotide fragment a, the nucleotide fragment b, the nucleotide fragment c, and the nucleotide fragment d are sequentially transfected into the target cell a.

6. The preparation method according to claim 4, characterized in that The preparation method further comprises: using the nucleotide sequence of the marker protein to transfect into the target cell a.

7. The preparation method according to claim 4, characterized in that The preparation method further includes: after transfection is completed and culture is performed, the target cells are labeled with corresponding antibodies, and then a cell population that expresses positive antigens CEA, CD19, BCMA, and CD70 is sorted out.

8. Use of the target cells according to any one of claims 1 to 3 as or in the preparation of a reagent or device for evaluating in vitro drug efficacy of cells.

9. The use according to claim 8, characterized in that The in vitro cell efficacy evaluation reagent or device is a reagent or device for testing the killing effect of CAR-T cells and / or a reagent or device for testing the secretion amount of CAR-T cell factors, and the CAR-T cells target at least one of CEA, CD19, BCMA, and CD70.

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