Method for detecting trophoblast residues in NK cell product and application of method

Abstract

The invention relates to a method for detecting trophoblast residues in an NK cell product and application of the method. The method comprises the following steps: extracting nucleic acid molecules of the NK cell product, and detecting whether trophoblast specific gene segments exist in the nucleic acid molecules or not. According to the method, the residual condition of the trophoblasts in the NK cell final product is detected by a molecular biological method, specifically, detection can be performed in a manner of amplifying specific genes of the trophoblasts through qPCR or amplifying STR loci of the NK cells through PCR, the method is simple, convenient and rapid, the accuracy rate is high, and the detection precision is improved by combining multiple methods, so that the accuracy of a detection result is ensured.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for detecting residual trophoblasts in NK cell products and an application thereof. Background Art

[0002] Natural killer cells (NK cells) are immune cells that can kill tumor cells. In vitro expansion of NK cells and then reinfusion into the body is one of the important means of clinical tumor cell immunotherapy. Using the trophoblast method to expand NK cells is one of the important in vitro expansion methods. Trophoblasts are constructed by genetic engineering methods. The NK cells obtained by this method are of high purity and large multiples. For example, CN110684730A discloses a preparation method for efficiently expanding NK cells using trophoblasts. The method uses a plasmid vector expressing three molecules, mbIL-21, 4-1BBL, and MICA, and a recombinant lentivirus to infect K562 to prepare NK cells. The NK cell expansion multiple is as high as 890 times, and the purity of the prepared NK cells reaches 92.2%. In addition, the expansion multiples between different PBMC cells are highly reproducible.

[0003] However, due to (1) the involvement of gene transfection, the cost and steps are relatively complicated; (2) the need to use K562 cells (a type of tumor cell), users have certain concerns about its safety; (3) when registering NK cells or CAR-NK cells for drugs, they need to be subject to safety evaluation. Trophoblasts are complete cells, and the evaluation content involved is very complicated. Although they are treated with radiation or mitomycin to eliminate the proliferation ability of trophoblasts before use, some users still question their safety and require reliable safety test reports.

[0004] In summary, how to detect the residual rate of trophoblasts in the final product of NK cells expanded by the trophoblast method to prove whether the final product of NK cells is safe has become one of the urgent problems to be solved in this field. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a method for detecting residual trophoblasts in NK cell products and its application. The residual trophoblasts in the final NK cell product are detected by molecular biological methods. The method is simple, rapid and highly accurate. Combining multiple methods improves the detection accuracy, thereby ensuring the accuracy of the detection results.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for detecting residual trophoblasts in an NK cell product, the method comprising: extracting nucleic acid molecules of the NK cell product, and detecting whether trophoblast-specific gene fragments are present in the nucleic acid molecules.

[0008] The most common trophoblast cell vector on the market is K562, which primarily expresses interleukin-21 (IL-21), along with multiple stimulatory molecules such as CD48, mIL-15, CD16A, and OX40L, to stimulate the expansion of NK cells and inhibit the proliferation of T cells. This invention, based on the differences between trophoblast cells and NK cells, uses molecular biological methods to rapidly obtain test results, making confirmation of results simple and rapid. Compared with existing technologies, the test accuracy is higher, the test dimensions are more extensive, and the test results are more accurate.

[0009] Preferably, the nucleic acid molecule comprises a DNA molecule and / or an RNA molecule.

[0010] Preferably, the NK cell product is prepared by trophoblast stimulation method.

[0011] Preferably, the starting cells of the trophoblast cells include K562 cells.

[0012] Preferably, the method includes any one of the following two methods or a combination of both:

[0013] (I) Extract RNA from the NK cell product, reverse transcribe it, and amplify trophoblast-specific genes using qPCR to obtain an amplification curve to confirm whether the NK cell product contains trophoblast residues;

[0014] (II) DNA of the NK cell product was extracted and the STR loci of the NK cells were amplified by PCR. After electrophoresis separation, data analysis and result comparison, the presence of residual trophoblasts in the NK cell product was confirmed.

[0015] STR identification is a method for cell line authentication using short tandem repeats (STRs). STR sequences, composed of short, tandemly repeated sequences of 3 to 7 base pairs in length, are widely present in the human genome and can serve as highly polymorphic markers, often referred to as the "DNA fingerprint" of cells. This method distinguishes cell lines by quantifying the number of copies of repeat sequences within a PCR-amplified region. This is followed by capillary electrophoresis separation and fluorescence detection, and ultimately, comparison with a specialized cell line STR database to infer the cell line to which the sample belongs, or the name of any cross-contaminating cell line.

[0016] Preferably, the trophoblast-specific gene in method (I) comprises a cytokine-specific gene fragment inserted into the trophoblast.

[0017] Preferably, the cytokine includes any one of IL-21, CD48, mIL-15, CD16A or OX40L, or a combination of at least two thereof, more preferably IL-21.

[0018] Preferably, method (I) further comprises the steps of using trophoblast cells as a positive control, extracting RNA, and amplifying trophoblast-specific genes by qPCR after reverse transcription.

[0019] Preferably, the STR loci in method (II) include any one or a combination of at least two of D19S433, D5S818, D21S11, D18S51, D6S1043, D3S1358, D13S317, D7S820, D16S539, CSFIPO, Penta D, D2S441, vWA, D8S1179, TPOX, PentaE, TH01, D12S391, D2S1338, FGA or the sex gene AMEL.

[0020] The combination of the 22 STR loci provided by the present invention can be used to identify cell lines and monitor cross-contamination between cell lines.

[0021] Preferably, the STR loci in method (II) are a combination of D5S818, D21S11, D18S51, D6S1043, D3S1358, D13S317, D7S820, D16S539, CSFIPO and Penta D.

[0022] The above STR loci provided by the present invention have a specific pattern in K562 cells, which can be used for comparison with NK cells. Through steps such as electrophoresis separation and data analysis, the presence of trophoblasts in NK cells can be quickly determined.

[0023] Preferably, the comparison of the results in method (II) includes comparison with data in ATCC, DSMZ or JCRB cell banks.

[0024] Preferably, method (II) further comprises using trophoblast cells as a positive control, extracting DNA, and PCR amplifying the STR loci of the trophoblast cells.

[0025] In a second aspect, the present invention provides an application of a product for STR identification in detecting trophoblast residues in NK cell products, wherein the STR identification sites include any one or a combination of at least two of D19S433, D5S818, D21S11, D18S51, D6S1043, D3S1358, D13S317, D7S820, D16S539, CSFIPO, Penta D, D2S441, vWA, D8S1179, TPOX, Penta E, TH01, D12S391, D2S1338, FGA or the sex gene AMEL.

[0026] Preferably, the NK cell product is prepared by trophoblast stimulation method.

[0027] Preferably, the starting cells of the trophoblast cells include K562 cells.

[0028] Preferably, the loci identified by STR are a combination of D5S818, D21S11, D18S51, D6S1043, D3S1358, D13S317, D7S820, D16S539, CSFIPO and Penta D.

[0029] Preferably, the product for STR identification comprises a primer combination.

[0030] Preferably, the nucleic acid sequence of the primer combination includes the sequences shown in SEQ ID NO.1 to SEQ ID NO.42.

[0031] The nucleic acid sequences of the primer combinations provided by the present invention are shown in Table 1.

[0032] Table 1

[0033]

[0034]

[0035] In a third aspect, the present invention provides a kit for detecting residual trophoblasts in NK cell products, the kit comprising a primer combination, the nucleic acid sequence of the primer combination comprising the sequences shown in SEQ ID NO.1 to SEQ ID NO.42.

[0036] Preferably, the kit further comprises a DNA extraction reagent and / or a PCR amplification reagent.

[0037] Preferably, the NK cell product is prepared by trophoblast stimulation method.

[0038] Preferably, the starting cells of the trophoblast cells include K562 cells.

[0039] In a fourth aspect, the present invention provides the use of any one or a combination of at least two of the method for detecting residual trophoblasts in NK cell products as described in the first aspect, the application as described in the second aspect, or the kit as described in the third aspect in the quality control of NK cell products.

[0040] Preferably, the NK cell product is prepared by trophoblast stimulation method.

[0041] Preferably, the starting cells of the trophoblast cells include K562 cells.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The present invention uses molecular biological methods to detect residual trophoblasts in the final NK cell product. Specifically, this can be done by amplifying trophoblast-specific genes through qPCR or STR loci through PCR to verify the safety of the final NK cell product. Compared with existing technologies, the present invention has higher detection accuracy, more detection dimensions, and more accurate test results.

[0044] The STR locus combination provided by the present invention can be used to identify NK cell lines and determine whether NK cell products are contaminated by other cell lines with high detection accuracy. A further preferred STR locus combination can specifically distinguish NK cells from trophoblasts, thereby enabling rapid detection of the presence of trophoblasts in NK cell products. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the STR typing pattern of K562 cells at the preferred STR loci in Example 2.

[0046] Figure 2 This is the STR typing profile of the NK cell product at the preferred STR loci in Example 2.

[0047] Figure 3 This is the qPCR standard curve in Example 3.

[0048] Figure 4 This is the qPCR amplification curve in Example 3.

[0049] Figure 5 This is a curve chart showing the change in cell survival rate of trophoblasts over time in Comparative Example 1.

[0050] Figure 6 This is the flow cytometry analysis result of the NK cell product in Comparative Example 2. DETAILED DESCRIPTION

[0051] To further illustrate the technical means and effects of the present invention, the present invention is further described below with reference to the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0052] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0053] Reagents used in the following examples:

[0054] Cell DNA extraction: cell lysis buffer (Tris-HCl, EDTA, SDS, proteinase K), phenol:chloroform:isoamyl alcohol mixture (25:24:1), NaAc (pH 5.2), ethanol, TE buffer (Tris-EDTA, pH 8.0);

[0055] PCR amplification and detection: Master Mix (Taq DNA polymerase, dNTPs, MgCl2 Tris-HCl buffer), ddH2O, Hi-Di formamide, LIZ internal standard, POP-4 polymer required for electrophoresis detection, qPCR Master Mix (hot-start Taq DNA polymerase, dNTPs, MgCl2, Tris-HCl buffer).

[0056] Example 1

[0057] This example provides a kit for detecting residual trophoblasts in an NK cell product, comprising a primer combination, a DNA extraction reagent, and a PCR amplification reagent. The primer combination is used to amplify D19S433, D5S818, D21S11, D18S51, D6S1043, D3S1358, D13S317, D7S820, D16S539, CSFIPO, Penta D, D2S441, vWA, D8S1179, TPOX, Penta E, TH01, D12S391, D2S1338, FGA, and the sex gene AMEL, the nucleic acid sequences of which are shown in Table 1. DNA extraction reagents included cell lysis buffer (Tris-HCl, EDTA, SDS, proteinase K), a phenol:chloroform:isoamyl alcohol mixture (25:24:1), NaAc (pH 5.2), ethanol, and TE buffer (Tris-EDTA, pH 8.0). PCR amplification reagents included Master Mix (Taq DNA polymerase, dNTPs, MgCl2 buffer Tris-HCl), ddH2O, and Hi-Di formamide, LIZ internal standard, and POP-4 polymer required for electrophoresis detection.

[0058] Example 2

[0059] This example provides a method for detecting residual trophoblasts in an NK cell product using STR identification, comprising the following steps:

[0060] (1) DNA of the NK cell product was extracted using the DNA extraction reagent provided in Example 1. The extraction steps were as follows: After cell collection, the cells were washed twice with PBS, lysis buffer was added, and incubated at 56°C for 2 hours. An equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) was added, mixed, and centrifuged. The upper aqueous phase was taken and chloroform extraction was repeated once. 3M NaAc (pH 5.2) and pre-cooled anhydrous ethanol were added, and the supernatant was discarded after standing. The precipitate was washed twice with 70% ethanol, centrifuged, dried, dissolved in TE buffer, and the concentration was determined.

[0061] (2) Referring to Table 2, a PCR system was prepared using the PCR amplification reagents and primer combinations provided in Example 1. Amplification was performed according to the PCR program provided in Table 3. STR loci were detected on a genetic analyzer (ABI3100) and compared with data from cell banks such as ATCC, DSMZ, and JCRB.

[0062] Table 2

[0063] Components Dosage Template DNA 1-2 μL 2×PCR Master Mix 12.5μL STR primer mix (10 μM) 2.5 μL <![CDATA[Sterile ddH2O]]> Make up to 25 μL

[0064] Table 3

[0065]

[0066] (3) If the test results show that NK cell DNA does not have tri-allelic phenomena at each locus and no human cell cross-contamination is found in the cells, it indicates that there are no residual engineered trophoblasts.

[0067] The results of this method are shown in Table 4. The DNA from the NK cell product showed no tri-allelic mutations at any locus, indicating no cross-contamination with human cells and no residual engineered trophoblasts. However, the D21S11 locus showed tri-allelic mutations in K562 cells. Since these K562 cells were irradiated, this STR mutation may have been caused by irradiation.

[0068] Table 4

[0069]

[0070] The preferred D5S818, D21S11, D18S51, D6S1043, D3S1358, D13S317, D7S820, D16S539, CSFIPO, and Penta D sites were amplified using the kit provided in Example 1. The detection steps were carried out with reference to Example 2. The results were as follows: Figure 1 、 Figure 2 As shown, Figure 1 This is the STR typing profile of K562 cells. Figure 2 This is the STR profiling profile of the NK cell product. No human cell cross-contamination was found in the NK cell product, indicating that there are no residual engineered trophoblasts in the NK cell product.

[0071] Example 3

[0072] This example provides a method for detecting residual trophoblasts in an NK cell product using qPCR, comprising the following steps:

[0073] (1) Select the specific gene fragment of the cytokine inserted into the trophoblast, design a unique primer, and test the stability of the primer sequence.

[0074] (2) The NK cell final product was selected as a negative sample, the cells were concentrated, RNA was extracted, and reverse transcription was performed. In addition, trophoblast cells were selected as a positive sample control and reverse transcribed in the same manner.

[0075] (3) Prepare the qPCR system according to Table 5: qPCR Master Mix contains: hot start Taq DNA polymerase (0.025-0.05 U / μL), dNTPs (0.2-0.4 mM each dNTP), MgCl2 (3-4 mM), Tris-HCl buffer (pH 8.3-8.8)

[0076] Table 5

[0077]

[0078]

[0079] (4) Perform PCR cycles according to Table 6:

[0080] Table 6

[0081]

[0082] (5) Analyze the sequence amplification curves of positive and negative samples to confirm whether there are any residual trophoblasts in the final NK cell product cultured with trophoblasts. The copy number of positive samples gradually increases with the cycle, while the copy number of negative samples is always 0, which is a qualified test result.

[0083] Using this method for detection, qPCR primers were designed for the cytokine IL-21 gene fragment inserted into trophoblasts (sequence from NCBI). The nucleic acid sequences are shown in SEQ ID NO.43 to SEQ ID NO.44, and the probe sequence is shown in SEQ ID NO.45. Its 5' end is labeled with FAM and its 3' end is labeled with BHQ1. The standard curve established is as shown in Figure 3 shown.

[0084] SEQ ID NO. 43: CATAGTAGACTCCAGCCTTGG.

[0085] SEQ ID NO. 44: GTCCAGGTTCTCCGGC.

[0086] SEQ ID NO. 45: CTGCGCAAACATGCCCTGCCG.

[0087] Detection of the amplification curve of NK cell products Figure 4As shown, ST1-ST6 are standard curve points, Sample 1 and Sample 2 are trophoblast samples of other types other than target trophoblasts, Sample 3, Sample 4, Sample 7, and Sample 8 are NK cell products expanded using different batches of target trophoblasts, Sample 5 and Sample 6 are target trophoblast samples, and NTC is a no-template control using purified water instead of nucleic acid samples. The results showed that no IL-21 gene fragment was amplified in the NK cell product group, and all positive target trophoblast samples produced amplification curves. In addition, no amplification curves were produced for trophoblast samples of other types other than target trophoblasts, indicating that this method has strong targeting and specificity.

[0088] Comparative Example 1

[0089] This comparative example provides a method for detecting residual trophoblasts in NK cell products, which detects the natural apoptosis of trophoblasts after de-proliferation treatment, comprising the following steps:

[0090] (1) After the proliferation ability of trophoblasts is reduced by irradiation, mitomycin treatment or other methods, the trophoblasts are cultured in NK culture medium at 37° C. and 5% CO 2 concentration.

[0091] (2) Observe and record the cell death rate and number of living cells daily.

[0092] (3) Observe the cell apoptosis curve. The cell viability data gradually decreases, and the rate of decrease gradually becomes gentle. Within 10-12 days, the viability gradually decreases to below 2%, and the number of living cells gradually decreases to below 99.9995% of the initial cell number, which is a qualified test result.

[0093] The results of the test using the method of Comparative Example 1 are as follows Figure 5 As shown, after using this method to de-proliferate trophoblasts, the viability dropped to below 2% after about 10 days, indicating that this method can eliminate the proliferation ability of trophoblasts and that the NK cell product is safe. However, this method is time-consuming and can only be used to evaluate whether a specific trophoblast de-proliferation treatment method can produce a safe NK cell product. It cannot provide a safety test report for a specific NK cell product.

[0094] Comparative Example 2

[0095] This comparative example provides a method for detecting residual trophoblasts in an NK cell product, which uses flow cytometry to detect cytokines in the NK cell product, comprising the following steps:

[0096] (1) Select one or more cytokines expressed by trophoblasts but not by NK cells, such as IL-21, CD48, mIL-15, CD16A, OX40L, etc., inserted into engineered trophoblasts.

[0097] (2) The NK cell final product was stained using the flow cytometry antibody corresponding to the cytokine, and the residual rate of the cytokine was detected using a flow cytometer. A cytokine expression rate lower than 0.1% was considered a qualified test result.

[0098] This method was used to detect NK cell products, and the residual rate of IL-21 was detected by flow cytometry. The results were as follows Figure 6 As shown, the expression rate of IL-21 is 0.05%, and the NK cell product is safe. However, this method requires the use of flow cytometry antibodies corresponding to cytokines in engineered trophoblasts, which has high detection costs and lower detection accuracy than the two molecular biology methods provided by the present invention.

[0099] In summary, the present invention uses molecular biological methods to detect residual trophoblasts in the final NK cell product, proving the safety of the final NK cell product at the DNA and RNA levels. Compared with existing technologies, the present invention offers higher precision, more diverse detection dimensions, and more accurate results.

[0100] 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 method for detecting residual trophoblasts in NK cell products, characterized in that: The method comprises: extracting nucleic acid molecules of NK cell products, and detecting whether trophoblast-specific gene fragments exist in the nucleic acid molecules.

2. The method for detecting residual trophoblasts in NK cell products according to claim 1, characterized in that: The nucleic acid molecules include DNA molecules and / or RNA molecules; Preferably, the NK cell product is prepared by trophoblast stimulation method; Preferably, the starting cells of the trophoblast cells include K562 cells.

3. The method for detecting residual trophoblasts in NK cell products according to claim 1 or 2, characterized in that: The method includes any one of the following two methods or a combination of the two: (I) Extract RNA from the NK cell product, reverse transcribe it, and amplify trophoblast-specific genes using qPCR to obtain an amplification curve to confirm whether the NK cell product contains trophoblast residues; (II) DNA was extracted from the NK cell product, and the STR loci of the NK cells were amplified by PCR. After electrophoresis separation, data analysis, and result comparison, the presence of residual trophoblasts in the NK cell product was confirmed.

4. The method for detecting residual trophoblasts in NK cell products according to claim 3, characterized in that: The trophoblast-specific gene in method (I) comprises a cytokine-specific gene fragment inserted into the trophoblast; Preferably, the cytokine comprises any one or a combination of at least two of IL-21, CD48, mIL-15, CD16A or OX40L, more preferably IL-21; Preferably, method (I) further comprises the steps of using trophoblast cells as a positive control, extracting RNA, and amplifying trophoblast-specific genes by qPCR after reverse transcription.

5. The method for detecting residual trophoblasts in NK cell products according to claim 3, characterized in that: Method (II) wherein the STR loci include any one or a combination of at least two of D19S433, D5S818, D21S11, D18S51, D6S1043, D3S1358, D13S317, D7S820, D16S539, CSFIPO, Penta D, D2S441, vWA, D8S1179, TPOX, Penta E, TH01, D12S391, D2S1338, FGA, or the sex gene AMEL; Preferably, the STR loci in method (II) are a combination of D5S818, D21S11, D18S51, D6S1043, D3S1358, D13S317, D7S820, D16S539, CSFIPO and Penta D; Preferably, the comparison of the results in method (II) includes comparison with data in ATCC, DSMZ or JCRB cell banks; Preferably, method (II) further comprises using trophoblast cells as a positive control, extracting DNA, and PCR amplifying the STR loci of the trophoblast cells.

6. Application of a product for STR identification in detecting residual trophoblasts in NK cell products, characterized in that: The loci identified by the STR include any one or a combination of at least two of D19S433, D5S818, D21S11, D18S51, D6S1043, D3S1358, D13S317, D7S820, D16S539, CSFIPO, Penta D, D2S441, vWA, D8S1179, TPOX, Penta E, TH01, D12S391, D2S1338, FGA or the sex gene AMEL.

7. The use according to claim 6, characterized in that The loci identified by the STR are a combination of D5S818, D21S11, D18S51, D6S1043, D3S1358, D13S317, D7S820, D16S539, CSFIPO, and Penta D; Preferably, the product for STR identification comprises a primer combination; Preferably, the nucleic acid sequence of the primer combination includes the sequences shown in SEQ ID NO.1 to SEQ ID NO.

42.

8. A kit for detecting residual trophoblasts in NK cell products, characterized in that: The kit includes a primer combination, and the nucleic acid sequence of the primer combination includes the sequences shown in SEQ ID NO.1 to SEQ ID NO.

42.

9. The kit according to claim 8, characterized in that The kit also includes a DNA extraction reagent and / or a PCR amplification reagent.

10. Use of any one or a combination of at least two of the method for detecting residual trophoblasts in NK cell products according to any one of claims 1 to 5, the use according to claim 6 or 7, or the kit according to claim 8 or 9 in quality control of NK cell products.

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