Human NSCLC cell lines and their uses

Abstract

The present invention provides human non-small cell lung cancer (NSCLC) cell lines, reagents and kits containing the human NSCLC cell lines of the present invention, and methods for evaluating the efficacy of anti-NSCLC drugs using the human NSCLC cell lines to measure drug efficacy or obtain pharmacological data for the drugs, as well as methods for producing animal models.

Description

[Technical Field]

[0001] The present invention relates to human non-small cell lung cancer (NSCLC) cell lines and their use in evaluating the efficacy of anti-NSCLC drugs. The present invention further discloses animal models comprising human NSCLC cell lines, methods for their preparation, and uses of the animal models in testing the efficacy of drugs or obtaining pharmacological data for said drugs. The present invention further relates to kits comprising the human NSCLC cell lines of the present invention. The present invention further discloses methods for evaluating the efficacy of anti-NSCLC drugs using human NSCLC cell lines. [Background technology]

[0002] Lung cancer is one of the most common malignant tumors in the world and the leading cause of death from malignant tumors in urban populations in China. Non-small cell lung cancer (NSCLC) includes squamous cell carcinoma (SCC), adenocarcinoma, and large cell carcinoma. Compared with small cell carcinoma, cancer cells grow and divide more slowly, and spread and metastasize more slowly. NSCLC accounts for approximately 80% of all lung cancer cases. Approximately 75% of patients are diagnosed with the disease at the mid- or late-stage stage, and the 5-year survival rate is low.

[0003] A cell line refers to a population of cells that grows after the first passage of a primary cell culture. It also refers to a culture of cells that can be passaged continuously over an extended period of time. However, very few cells survive and can be passaged continuously, i.e., very few cells can become cell lines.

[0004] EGFR (epidermal growth factor receptor) is a receptor for EGF cell proliferation and signal transduction. Studies have shown that EGFR is highly or abnormally expressed in many solid tumors. EGFR is associated with tumor cell proliferation, angiogenesis, tumor invasion, metastasis, and apoptosis inhibition. Mutations in the EGFR tyrosine kinase domain primarily occur in exons 18-21, with mutations in exons 19 and 21 accounting for 90% of all EGFR mutations. EGFR mutations can be detected using PCR and direct sequencing techniques, for example. EGFR plays an important role in tumor cell proliferation, growth, repair, and survival. EGFR is overexpressed in many epithelial tumors, including non-small cell lung cancer, breast cancer, glioma, head and neck cancer, cervical cancer, bladder cancer, and gastric cancer. Furthermore, abnormal EGFR expression is closely associated with neovascularization, tumor invasion and metastasis, chemotherapy resistance, and poor prognosis. Approximately 80–90% of EGFR mutations are small exon 19 deletions or the L858R mutation in exon 21, but other TKI-sensitive EGFR mutations can occur in exons 12, 19, 20, and 21. Mutations associated with TKI resistance, such as T790M in exon 20, can also develop in small tumor cell subclones and require identification (Dario de Biase et al., “Next-Generation Sequencing of Lung Cancer EGFR Exons 18–21 Allows Effective Molecular Diagnosis of Small Routine Samples (Cytology and Biopsy),” December 23, 2013, Vol. 8, Issue 12, e83607, https: / / doi.org / 10.1371 / journal.pone.0083607).Methods for detecting EGFR mutations in NSCLC cells are well known in the art (see Huili Chu et al., "Direct sequencing and amplification refractory mutation system for epidermal growth factor receptor mutations in patients with non-small cell lung cancer," published online August 29, 2013, https: / / doi.org / 10.3892 / or.2013.2709, pp. 2311-2315, and Ching-Hsiung Lin et al., "Rapid detection of epidermal growth factor receptor mutations with multiplex PCR and primer extension in lung cancer," J Biomed Sci. 2010;17(1):37, published online May 12, 2010, doi:10.1186 / 1423-0127-17-37, http: / / www.jbiomedsci.com / content / 17 / 1 / 37).

[0005] The present invention discloses novel NSCLC tumor cell lines harboring one or more EGFR mutations in exons 19, 20, and 21, which are of great significance for studying the mechanisms of cell carcinoma and tumor metastasis and evaluating the efficacy of drugs for NSCLC. The establishment of different tumor cell lines for cancer research can provide useful data for the research and development of new drugs. Tumor cell lines harboring EGFR mutations are useful tools for evaluating anti-NSCLC drugs both in vitro and ex vivo. For example, tumor cell lines can be subcutaneously implanted to form animal models for evaluating drug efficacy, which can be more effective by simulating the patient's internal environment and reflecting the patient's response. Therefore, the related art requires NSCLC cell lines harboring one or more EGFR mutations in exons 19, 20, and 21 that maintain stable genetic characteristics during proliferation and grow into tumors in animal models, as well as wild-type NSCLC cell lines of the same origin that can be used as references. The human NSCLC cell line of the present invention, and the reagent, kit, or animal model containing said cell line will prove useful in drug research and development. Summary of the Invention

[0006] The present invention provides human NSCLC (hNSCLC) cell lines, namely, LD1-0025-200636, LD1-0025-200694, LD1-0006-215676, and LD1-0025-200717.

[0007] Specifically, human NSCLC (hNSCLC) cell line LD1-0025-200636 was established directly from a patient with EGFR WT NSCLC, LD1-0025-200694 was established directly from a patient with EGFR L858R NSCLC, LD1-0006-215676 was established directly from a patient with EGFR double mutation (L858R / T790M), and LD1-0025-200717 was established directly from a patient with EGFR triple mutation (19del / T790M / C797S).

[0008] The human NSCLC cell lines of the present invention are capable of growing in vitro and developing into tumors in animal models while maintaining their genetic characteristics.

[0009] The present invention further includes progeny cell lines of LD1-0025-200636, LD1-0025-200694, LD1-0006-215676, and LD1-0025-200717.

[0010] In an embodiment, the present invention relates to a method for generating an hNSCLC cell line of the present invention from a patient's NSCLC sample through serial passage. In an embodiment, the hNSCLC cell line of the present invention is passaged for 10 or more generations, particularly 20 or more generations, more particularly 30 or more generations, more particularly 50 or more generations, even more particularly 70 or more generations, and even more particularly more than 100 generations. Preferably, the hNSCLC cell line of the present invention has been passaged for 10, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 generations.

[0011] The present invention also relates to a tissue or organ sample comprising the hNSCLC cell line of the present invention. In particular, the tissue or organ sample is derived from an animal model into which the hNSCLC cell line of the present invention has been transplanted or injected. In an embodiment, the tissue or organ is derived from the lung of the animal model, preferably from the lung of an immunocompromised mouse. In an embodiment, the tissue or organ sample comprises the metastatic hNSCLC cell line of the present invention, in particular, the tissue or organ sample comprises the metastatic hNSCLC cell line of the present invention in the brain or bone of a mammal.

[0012] In one aspect, the present invention relates to a reagent comprising the hNSCLC cell line of the present invention, which can be used for the evaluation of anti-hNSCLC drugs.

[0013] In another aspect, the present invention relates to a kit comprising an hNSCLC cell line of the present invention or a tissue or organ sample comprising an hNSCLC cell line of the present invention, which in embodiments can be used to evaluate the efficacy of anti-NSCLC drugs or to conduct drug research and development to obtain pharmacological or other relevant data.

[0014] In another aspect, the present invention discloses an animal model comprising the hNSCLC cell line of the present invention. In one embodiment, the animal model is obtained by subcutaneously implanting or injecting the hNSCLC cell line of the present invention into an animal. In one embodiment, the animal model is an immune-compromised mammal, preferably an immune-compromised mouse.

[0015] The present invention further discloses a method for evaluating the efficacy of a drug using the hNSCLC cell line of the present invention, or a reagent or kit comprising the hNSCLC cell line of the present invention. In an embodiment, the hNSCLC cell line of the present invention, a reagent comprising the hNSCLC cell line of the present invention, or a kit of the present invention is used to obtain data for drug research and development. In an embodiment, data obtained from the hNSCLC cell line of the present invention, a reagent comprising the hNSCLC cell line of the present invention, or a kit of the present invention is used to establish a computer model for evaluating the efficacy of a drug, particularly by processing the data via computer software to establish a computer model for evaluating the efficacy of an anti-NSCLC drug.

[0016] Biomaterial deposit information The present invention provides a human NSCLC cell line LD1-0025-200636, with accession number CCTCC No. C202005, which was deposited at the China Center for Typical Culture Collection (CCTCC) (Address: Wuhan University, Wuhan, China 430072) on June 3, 2020.

[0017] The present invention provides a human NSCLC cell line LD1-0025-200694, with the accession number CCTCC No. C2020102, which was deposited at the China Center for Typical Culture Collection (CCTCC) (Address: Wuhan University, Wuhan, China 430072) on June 3, 2020.

[0018] The present invention provides a human NSCLC cell line LD1-0006-215676, with accession number CCTCC No. C2020104, which was deposited at the China Center for Typical Culture Collection (CCTCC) (Address: Wuhan University, Wuhan, China 430072) on June 3, 2020.

[0019] The present invention provides a human NSCLC cell line LD1-0025-200717, with accession number CCTCC No. C2020103, which was deposited at the China Center for Typical Culture Collection (CCTCC) (Address: Wuhan University, Wuhan, China 430072) on June 3, 2020. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 shows the results of cell proliferation tests for LD1-0025-200636 (EGFR WT cell line, FIG. 1A), LD1-0006-215676 (FIG. 1B), and LD1-0025-200717 (FIG. 1C). [Figure 2A] Figure 2A shows EGFR mutations in exons 20 and 21 encoding T790M and L858R detected in the hNSCLC cell line LD1-0006-215676. [Figure 2B] FIG. 2B shows EGFR mutations in exon 20 encoding T790M and C797S, and the exon 19 deletion 746_750del (“19 deletion”) detected in the hNSCLC cell line LD1-0025-200717. [Figure 3]A is the inhibition curve for LD1-0025-200636 (EGFR WT cell line). B is the inhibition curve for LD1-0025-200694 (EGFR L858R) cell line. C is the inhibition curve for LD1-0006-215676 cell line. D is the inhibition curve for LD1-0025-200717 cell line. DETAILED DESCRIPTION OF THE INVENTION

[0021] Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter sought to be protected belongs. In some cases, for clarity and / or ease of reference, this specification defines terms that have a universally understood meaning, and such definitions contained herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art.

[0022] As used herein, the singular forms "a," "an," and "said" include the plural forms unless the context clearly dictates otherwise. Also, the term "and / or" as used herein should be understood to mean and cover all possible combinations of one or more of the associated listed items. Furthermore, the terms "comprise," "include," and / or "contain," when used herein, specify the presence of said features, integers, steps, operations, components, ingredients, and / or units, but do not exclude that one or more other features, integers, steps, operations, components, ingredients, and / or groups thereof are also present or additional.

[0023] The present invention provides a human NSCLC cell line LD1-0025-200636, with accession number CCTCC No. C202005, which was deposited at the China Center for Typical Culture Collection (CCTCC) (Address: Wuhan University, Wuhan, China 430072) on June 3, 2020.

[0024] In an embodiment, the present invention provides a human NSCLC cell line LD1-0025-200694, with accession number CCTCC No. C2020102, deposited at the China Center for Typical Culture Collection (CCTCC) (Address: Wuhan University, Wuhan, China 430072) on June 3, 2020. Cell line LD1-0025-200694 contains an EGFR mutation in exon 21 encoding L858R.

[0025] In an embodiment, the present invention provides a human NSCLC cell line LD1-0006-215676, with accession number CTCCC No. C2020104, deposited at the China Center for Typical Culture Collection (CCTCC), Wuhan University, Wuhan, China 430072, on June 3, 2020. Cell line LD1-0006-215676 contains EGFR mutations in exons 20 and 21, encoding T790M and L858R.

[0026] In an embodiment, the present invention provides a human NSCLC cell line LD1-0025-200717, with accession number CTCCC No. C2020103, deposited at the China Center for Typical Culture Collection (CCTCC) (Address: Wuhan University, Wuhan, China 430072) on June 3, 2020. Cell line LD1-0025-200717 contains EGFR mutations in exon 20 encoding T790M and C797S, and exon 19 deletion 746_750del.

[0027] Specifically, human NSCLC (hNSCLC) cell line LD1-0025-200636 was established directly from an EGFR WT NSCLC patient, LD1-0025-200694 was established directly from an EGFR L858R NSCLC patient, LD1-0006-215676 was established directly from an EGFR double-mutated (L858R / T790M) patient, and LD1-0025-200717 was established directly from an EGFR triple-mutated (19del / T790M / C797S) patient.

[0028] Therefore, in embodiments, the present invention provides patient-derived xenograft (PDX) models generated from NSCLC patients, including EGFR WT NSCLC patients, EGFR L858R NSCLC patients, EGFR double mutant (L858R / T790M) patients, and EGFR triple mutant (19del / T790M / C797S) patients. PDX models are the most reliable in vivo models of human cancer because they retain characteristics of the primary patient tumor, such as gene expression profiles and drug responses.

[0029] Treatment of non-small cell lung cancer (NSCLC) patients with activating epidermal growth factor receptor (EGFR) mutations using epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) represents a breakthrough in targeted NSCLC therapy. However, the inevitable emergence of acquired resistance limits long-term patient benefit in clinical settings. The emergence of the EGFR C797S mutation, induced by the third-generation EGFR TKI osimertinib as second-line treatment on top of the T790 mutation, is particularly challenging and ultimately leads to treatment failure. The lack of natural cell lines or patient-derived xenografts (PDX) harboring the EGFR triple mutations 19del, T790M, and C797S has significantly delayed understanding the biology of acquired resistance to osimertinib and the development of effective strategies to overcome C797S resistance.

[0030] Therefore, the present inventors successfully established PDXs from NSCLC patients with EGFR mutations 19del and T790M. The patients were resistant to osimertinib due to the presence of C797S. Furthermore, the inventors generated cell lines from such EGFR triple-mutated PDXs. Therefore, the PDXs and their corresponding cell lines provide valuable research models for understanding and overcoming acquired resistance to osimertinib caused by the EGFR C797S mutation.

[0031] The availability of cell lines and patient-derived PDXs harboring the C797S mutation is crucial for developing effective drugs or methods to overcome osimertinib resistance caused by the acquisition of the C797S mutation. Unfortunately, with the exception of engineered EGFR mutant cell lines expressing the 19del, T790M, and C797S triple-mutated EGFR, these patient-derived cell lines and PDXs are not available in our research community. In vivo models, cell-derived xenografts (CDXs) can be established using engineered EGFR mutant cell lines. However, CDX models are limited by poor intratumor heterogeneity and a poor record of predicting clinically effective treatments (Whittle et al., 2015 and references therein). In addition, engineered triple-mutated EGFR mutant cell lines cannot fully simulate the complex natural mechanisms of osimertinib resistance, as drug resistance can be caused by many factors, including the C797S mutation.

[0032] In embodiments, the present invention relates to progeny cell lines derived from the hSCLC cell lines of the present invention.

[0033] In an embodiment, the present invention relates to a method for generating an hNSCLC cell line of the present invention from a patient's NSCLC sample through serial passage. In an embodiment, the hNSCLC cell line of the present invention is passaged for 10 or more generations, particularly 20 or more generations, more particularly 30 or more generations, more particularly 50 or more generations, even more particularly 70 or more generations, and even more particularly more than 100 generations. Preferably, the hNSCLC cell line of the present invention has been passaged for 10, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 generations. In an embodiment, the hNSCLC of the present invention is obtained by a method comprising the following steps: (a) transplanting tumor tissue obtained from a patient into an immunocompromised animal or injecting tumor cells obtained from a patient into an immunocompromised animal; and (b) dissecting a tissue or organ containing tumor cells from the immunocompromised animal to collect the tumor cells. (c) Remove non-tumor tissue and necrotic tumor tissue, and reduce the tumor to 1-2 mm. 3(d) The cells are cut into fragments, and then the precipitate is suspended in digestion buffer and incubated at 37°C. (d) Single cells are collected using a 70µM filter and centrifuged at 1000 rpm for 3 minutes. (e) The cells are suspended and centrifuged in 10ml of Histopaque solution to collect the cells from the middle layer. (f) The cells are cultured using a conditional reprogramming cell culture kit (available from Shanghai Lidi). (g) The cells are passaged for 10-100 generations. (h) A stable cell line is obtained. In an embodiment, the patient sample is derived from in situ NSCLC tissue or metastatic NSCLC tissue.

[0034] The present invention further relates to a tissue or organ sample comprising the hNSCLC cell line of the present invention. In particular, the tissue or organ sample is derived from a mammal comprising the hNSCLC cell line of the present invention. In an embodiment, the tissue or organ is derived from the lung of an immune-compromised mammal, preferably from the lung of an immune-compromised mouse. In an embodiment, the tissue or organ sample comprises a metastatic hNSCLC cell line of the present invention, wherein the tissue or organ sample is derived from the brain or bone of an animal.

[0035] In another aspect, the present invention relates to a reagent comprising an hNSCLC cell line of the present invention or a tissue or organ sample comprising an hNSCLC cell line of the present invention, which in embodiments can be used to evaluate anti-NSCLC drugs or conduct drug research and development to obtain pharmacological or other relevant data.

[0036] In another aspect, the present invention relates to a kit comprising an hNSCLC cell line of the present invention or a tissue or organ sample comprising an hNSCLC cell line of the present invention. In an embodiment, the kit can be used to evaluate anti-NSCLC drugs or conduct drug research and development to obtain pharmacological or other relevant data. In an embodiment, the kit further comprises a reagent or tool for detecting the presence of a human NSCLC cell line of the present invention.

[0037] In another aspect, the present invention discloses an animal model comprising the hNSCLC cell line of the present invention. In one embodiment, the animal model is obtained by subcutaneously implanting or injecting the hNSCLC cell line of the present invention into an animal. In one embodiment, the animal is an immune-compromised mammal. In one embodiment, the immune-compromised mammal is a mouse.

[0038] The present invention further discloses a method for evaluating the efficacy of a drug using the hNSCLC cell line of the present invention, or a reagent, kit, or animal model comprising the hNSCLC cell line of the present invention. In an embodiment, the hNSCLC cell line of the present invention, or a reagent, kit, or animal model comprising the hNSCLC cell line of the present invention is used to obtain data for drug research and development. In an embodiment, data obtained from the hNSCLC cell line of the present invention, or a reagent, kit, or animal model comprising the hNSCLC cell line of the present invention is used to establish a computer model for drug screening, particularly to process the data via computer software to obtain a computer model of the efficacy of an anti-NSCLC drug.

[0039] The anti-NSCLC drug to be evaluated can be administered by any suitable route, oral or parenteral. For example, the candidate drug is administered to the animal model of the present invention by oral administration or intramuscular injection (e.g., intramuscular, subcutaneous, or intravenous infusion), topical administration, inhalation, transdermal delivery such as a skin patch, implant, suppository, etc. Those skilled in the art will select an appropriate administration route as needed.

[0040] The anti-NSCLC drugs evaluated in the present invention may be known anti-tumor drugs or combinations thereof, novel anti-tumor drugs or combinations thereof, or novel combinations of known anti-tumor drugs. In the methods of the present invention, the drugs to be screened may be used in solid, semi-solid, or liquid form.

[0041] The animal models of the present invention are administered the anti-NSCLC drug by oral administration or intramuscular injection (e.g., intramuscular, subcutaneous, or intravenous infusion), topical administration, inhalation, transdermal delivery such as a skin patch, implant, suppository, etc. Those skilled in the art will select an appropriate administration route as needed.

[0042] In an embodiment, the cell proliferation of the hNSCLC cell line of the present invention is tested through CTG (CELL TITER-GLO). A CTG test kit is commercially available. In an embodiment, the genomic analysis of the hNSCLC cell line of the present invention is tested through Sanger sequencing, which is a method known to those skilled in the art.

[0043] The present invention further provides a method or use of the hNSCLC cell line of the present invention, or a reagent, kit, or animal model comprising the hNSCLC cell line of the present invention, in obtaining drug efficacy data for creating a drug efficacy database for hNSCLC cells containing the EFGR mutation in exons 19-21. In an embodiment, the drug efficacy database includes cell growth inhibition rates of one or more candidate drugs. In an embodiment, the present invention provides a computer-readable medium for implementing the following steps:

[0044] (i) Calculate the growth inhibition rate of the hNSCLC cell line of the present invention after contacting it with one or more candidate drugs according to the following formula: Inhibition rate (%)=((V コントロール群 -V 媒体群 )-(V 薬剤治療群 -V 媒体群 )) / (V コントロール群 -V 媒体群 )×100%

[0045] (ii) Generate an inhibition curve using XLfit (IDBS) and compare the corresponding IC 50 Calculate.

[0046] (iii) the name of the one or more candidate drugs, the growth inhibition rate of the hNSCLC cell line of the present invention, the inhibition curve, and the corresponding IC 50Optionally, a computer is established that has access to a database including: [Example]

[0047] Example 1. Construction of PDX models The LD1-0025-200636 PDX model was established from an EGFR WT NSCLC patient, the LD1-0025-200694 PDX model was established from an EGFRL 858R NSCLC patient, and the LD1-0006-215676 PDX model was established from a pleural effusion sample from an EGFR double-mutated (L858R / T790M) patient.

[0048] The LD1-0025-200717 PDX model was established from a biopsy sample obtained from a patient diagnosed with NSCLC and resistant to osimertinib (AZD9291). The patient had an EGFR triple mutant (19del / T790M / C797S). Details of the patient's treatment history are as follows:

[0049] [Table 1-1]

[0050] EGFR mutations gradually progressed from 19del to 19del / T790M, and then to 19del / T790M / C797S. No other artificial model has been able to mimic the effects of this treatment history. Naturally derived PDX and cell lines provide valuable research tools and models for developing effective drugs or approaches to combat acquired resistance to osimertinib.

[0051] Table 1: WES (whole exon group sequencing) results of cell line PDX models are as follows. All mutations are conserved in the corresponding PDX models. [Table 1-2]

[0052] Example 2. Cell line establishment PDX tumors were harvested and immersed in HBSS. The tumor tissue was washed with HBSS and non-tumor and necrotic tumor tissue was removed in a biosafety cabinet. The tumors were then cut into 1-2 mm pieces. 3 The cells were digested into 100 μm fragments, and the precipitate was suspended in digestion buffer and incubated at 37°C for 2-4 hours. Single cells were collected using a 70 μm filter and centrifuged at 1000 rpm for 3 minutes. The cells were suspended and centrifuged in 10 ml of Histopaque solution, and the cells were collected from the middle layer. The cells were suspended and cultured using a conditional reprogramming cell culture kit. The cells were serially passaged for more than 10 generations until a stable cell line was obtained.

[0053] Example 3. Cell proliferation and genome analysis Cell proliferation was tested by CTG, and genomic analysis was tested by Sanger sequencing.

[0054] LD1-0025-200636: Cells showed a 4.0-fold proliferation and good cell viability (Figure 1A).

[0055] LD1-0006-215676: Cells showed a 3.3-fold increase in proliferation and good cell viability (Figure 1B).

[0056] LD1-0025-200717: Cells showed a 3.7-fold increase in proliferation and good cell viability (Figure 1C).

[0057] The results of genome analysis of the cell line of the present invention are shown in FIG.

[0058] EGFR mutations in the hNSCLC cell lines of the present invention were confirmed by Sanger sequencing. After 9-50 generations, mutations in the hNSCLC cell lines of the present invention were re-detected by Sanger sequencing to identify all remaining mutations in related cell lines.

[0059] Example 4 This study was conducted to investigate the authenticity of the newly established cell lines using short tandem repeat (STR) DNA analysis. The protocol is as follows.

[0060] 1. Genomic DNA was extracted from cell pellets and PDX tissues.

[0061] 2. Samples were amplified along with positive and negative controls using the GenePrint 10 System (Promega).

[0062] 3. The amplified products were processed using an ABI 3730x1 Genetic Analyzer.

[0063] 4. Data were analyzed using GeneMapper 4.0 software, and cells were compared with their corresponding PDX tissues.

[0064] Detailed information on the cell lines used in the STR analysis and the results are shown in Tables 2 and 3 below.

[0065] Table 2: Detailed information of cell lines used in STR analysis [Table 2]

[0066] Table 3: Profile of concordance between primary cell lines and PDX

[0067] [Table 3-1]

[0068] [Table 3-2]

[0069] [Table 3-3]

[0070] [Table 3-4]

[0071] This example demonstrates the authenticity of the newly established cell lines.

[0072] Example 5. Drug Efficacy Study Cell lines of the present disclosure (i.e., LD1-0025-200636, LD1-0025-200694, LD1-0006-215676, and LD1-0025-200717) were cultured in 96-well round-bottom ultra-low attachment plates at 2×10 4 Cells were seeded at 1000 cells / well. Drugs were added to the cell lines, and the cells were cultured at 37°C and 5% CO2 for 6 days. ATP levels were measured using CellTiter-Glo. The inhibition rate corresponding to the drug concentration was calculated. The formula for calculating the inhibition rate is as follows:

[0073] Inhibition rate (%)=((V コントロール群 -V 媒体群 )-(V 薬剤治療群 -V 媒体群 )) / (V コントロール群 -V 媒体群 )×100%

[0074] Inhibition curves were generated using XLfit (IDBS) and IC50 values ​​were calculated.

[0075] In vitro efficacy study of AZD9291 against NSCLC cell lines In vitro efficacy testing of AZD9291 against the disclosed NSCLC cell lines (i.e., LD1-0025-200636, LD1-0025-200694, LD1-0006-215676, and LD1-0025-200717) was conducted. The results are shown in Figure 3, where A is the inhibition curve for the LD1-0025-200636 (EGFR WT) cell line, B is the inhibition curve for the LD1-0025-200694 (EGFR L858R) cell line, C is the inhibition curve for the LD1-0006-215676 (EGFR L858R / T790M) cell line, and D is the inhibition curve for the LD1-0025-200717 (EGFR 19del, T790M & C797S mutant) cell line.

[0076] Table 4: IC of AZD9291 in four hNSCLC cell lines 50 The mean inhibition for each dose is shown (triplicates). Error bars, SEM. [Table 4]

[0077] Within PDX models, tumor cells grow in a physiologically relevant tumor microenvironment that mimics the oxygen, nutrient, and hormone levels found at the patient's primary tumor site. Furthermore, the transplanted tumor tissue maintains the genetic and epigenetic abnormalities found in the patient's body. This study found that PDX models exhibited responses to anticancer drugs (i.e., AZD9291) similar to those seen in actual patients who provided tumor samples. Therefore, this example demonstrates the advantages of PDX models for testing the therapeutic response of NSCLC drugs.

[0078] Example 6. Construction of a CDX (cell line-derived xenograft) animal model NCG mice are triple immunodeficient, lacking functional / mature T, B, and NK cells, and exhibiting reduced macrophage and dendritic cell function. These animal models are capable of accepting xenograft cells, tissues, and human immune system components. The characteristics of the NCG mouse model make it ideal for oncology research.

[0079] Cells of the NSCLC cell lines of the present disclosure (i.e., LD1-0025-200636 or LD1-0025-200717) were digested into single cells with trypsin. Cells were counted and 5×10 6 The cells were transferred to a centrifuge tube. The cells were washed with PBS, centrifuged, and the supernatant removed. The precipitate was suspended in 100 μL of PBS, and the test tube was immediately placed on ice. The cell suspension was mixed with 100 μL of Matrigel on ice, and the mixture was subcutaneously inoculated into NCG mice. After cell transplantation, the animals were examined daily for morbidity and tumor progression. It was observed that CDX models of the NSCLC cell lines of the present invention (i.e., LD1-0025-200636 and LD1-0025-200717) were successfully established.

[0080] The generation of CDX and PDX models in this disclosure suggests that the cell lines of this disclosure can be used as promising tools for preclinical drug development.

[0081] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or alterations thereto will be suggested to those skilled in the art and are within the spirit and scope of this application and the appended claims.

Claims

1. Human NSCLC cell line LD1-0025-200717, with CCTCC No. C2020103, containing EGFR mutations in exon 20 encoding T790M and C797S, and exon 19 deletion 746_750del.

2. A progeny cell line derived from the human NSCLC cell line of claim 1.

3. A reagent comprising the human NSCLC cell line of claim 1 or the progeny cell line of claim 2.

4. A kit comprising the human NSCLC cell line of claim 1, the progeny cell line of claim 2, or the reagent of claim 3.

5. A method of using the cell line of claim 1, the progeny cell line of claim 2, the reagent of claim 3, or the kit of claim 4 to generate NSCLC in an immune-compromised non-human mammal for the purpose of evaluating the efficacy of anti-NSCLC drugs.

6. 10. A method for producing a non-human animal model, comprising injecting or implanting the human NSCLC cell line of claim 1, the progeny cell line of claim 2, or the reagent of claim 3 into the body of the non-human animal.

7. (i) contacting one or more candidate drugs with the human NSCLC cell line of claim 1, the progeny cell line of claim 2, the reagent of claim 3, or the kit of claim 4; and (ii) measuring the inhibition of cell proliferation.

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