A cell line for treating melanoma and its multivalent whole-cell vaccine and use method
By using a multi-titer whole-cell vaccine composed of two isogenic human melanoma cell lines, ZJMM-45 and FLFMM-34, combined with low-dose interferon, the shortcomings of existing vaccines in stimulating specific immune responses were solved, and the results were significantly improved. Improves survival and immune response in melanoma patients.
Patent Information
- Application Number
- CN202210706286.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-06-21
- Publication Date
- 2025-02-07
- Estimated Expiration
- 2042-06-21
AI Technical Summary
The existing melanoma vaccines are insufficient in stimulating patients' specific immune responses, especially in the Chinese population. Due to the diversity of HLA phenotypes and the differences in melanoma characteristics, the efficacy is uncertain.
A multi-titer whole-cell vaccine composed of two allogenic human melanoma cell lines ZJMM-45 and FLFMM-34 was used to activate the patient's immune response through the combination of mixed cell lines and low-dose interferon.
It significantly improved the 5-year survival rate of melanoma patients, and compared with the treatment with high-dose interferon alone, it prolonged the patient's survival and increased the specific anti-tumor immune response of the vaccine.
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Figure CN115074328B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biological vaccine technology, and in particular to a cell line for treating melanoma and a multivalent whole-cell vaccine thereof and a use method thereof. Background Art
[0002] Malignant melanoma (MM) is a malignant tumor originating from melanocytes of the skin, accounting for about 1% to 3% of malignant tumors and 80% of patients who die from skin malignancies. The 5-year survival rate is only 14%, and the 5-year survival rate of metastatic MM is less than 5%. In addition, the incidence rate has a significant increasing trend, and the age of onset is also tending to be younger. MM is not only highly invasive, highly metastatic, and has a high mortality rate, but more importantly, MM has a strong tolerance to various treatments, which directly determines the great harm of this tumor to the human body.
[0003] Interferon-α (IFN-α) was first used in clinical trials in the mid-1980s. Results suggested that IFN-α could inhibit tumor cell proliferation and stimulate immune responses, so IFN-α was initially used in patients with advanced disease. Several meta-analyses have shown that high-dose IFN-α can prolong disease-free survival in patients with high-risk melanoma. IFN-α is a member of the type I interferon family and has multiple antitumor activities, including inhibition of proliferation and angiogenesis, and enhancement of antitumor immune responses by increasing NK cell cytotoxicity and tumor antigen processing. In 1995, high-dose IFN-α became the first exogenous cytokine approved by the FDA for adjuvant treatment of advanced melanoma. However, compared with low-dose, long-term use of high-dose IFN-α often produces a variety of side effects, the most common of which are fatigue, hematotoxicity, and hepatotoxicity.
[0004] In recent years, immunotherapy for MM has been one of the research hotspots. With the rapid development of modern tumor immunology, immunotherapy for the occurrence, development and metastasis of malignant melanoma has become a hot topic of current research. The purpose of treating MM is to activate the body's specific immune response to MM through vaccines, including autologous tumor cell vaccines, allogeneic inactivated whole cell vaccines, DC cell vaccines, and antigen peptide vaccines. However, tumor vaccines have encountered many challenges in clinical practice. These mechanisms mainly include immune tolerance and immune escape induced by the tumor itself or difficulty in stimulating immune response. For example, when inactivated tumor cells are used or they are crushed and used as tumor vaccines to treat malignant melanoma, the immunogenicity of the vaccine tumor prepared by a single melanoma cell line is too low to induce the body to produce an effective anti-tumor immune response, and the patient's immune response cannot be effectively induced or activated, resulting in immune failure. Allogeneic vaccines can overcome the defect of insufficient cell number of autologous tumor vaccines and can maximize the expression spectrum of tumor-specific antigens. However, in order to activate specific immune responses, this type of vaccine must also meet the restrictions of HLA and meet the multiple needs of different patients due to HLA diversity.
[0005] Therefore, although allogeneic whole-cell vaccines have been studied by researchers abroad, the different HLA phenotypes and MM characteristics of the Chinese population determine the uncertainty of the clinical efficacy of this type of vaccine for MM in the Chinese population. For example, more than half of MM abroad can be found to have BRAF (V600E) mutations, while the Han population does not have such a positive rate. Most of the MM patients abroad develop MM in exposed areas, which is closely related to ultraviolet rays, while MM in the Han population mostly occurs in the extremities and mucosa. These clinical characteristics suggest that the clinical efficacy of cell vaccines for different races may be different. Therefore, given the obvious differences in disease characteristics between the Chinese melanoma population and the Caucasian melanoma population, it is urgent to develop a new MM vaccine to increase the specific anti-tumor immune response of malignant melanoma vaccines, in order to establish a new treatment strategy for the treatment of MM in the Han population. Summary of the invention
[0006] In view of the above problems, the present invention provides a cell line for treating melanoma and a multivalent whole-cell vaccine and a method of use thereof, which can increase the specific anti-tumor immune response of malignant melanoma vaccines and improve the survival of patients.
[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0008] A cell strain for treating melanoma, the cell strain consisting of ZJMM-45 homologous human melanoma cell strain with a preservation number of CCTCC NO: C2022105 and FLFMM-34 homologous human melanoma cell strain with a preservation number of CCTCC NO: C2022100, and the cell strain is used for treating melanoma.
[0009] The present invention also discloses a multivalent whole-cell vaccine for treating melanoma. The multivalent whole-cell vaccine comprises a ZJMM-45 homologous human melanoma cell strain and a FLFMM-34 homologous human melanoma cell strain. The ZJMM-45 homologous human melanoma cell strain has a preservation number of CCTCC NO: C2022105 and is classified as a homologous human melanoma cell strain ZJMM-45. The FLFMM-34 homologous human melanoma cell strain has a preservation number of CCTCC NO: C2022100 and is classified as a homologous human melanoma cell strain FLFMM-34.
[0010] The above-mentioned FLFMM-34 homologous human melanoma cell line and ZJMM-45 homologous human melanoma cell line are both preserved at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), and the preservation time is May 31, 2022.
[0011] Furthermore, the multivalent whole-cell vaccine is used to treat melanoma
[0012] Furthermore, the ZJMM-45 and FLFMM-34 cell lines in the multivalent whole-cell vaccine are mixed at a molar ratio of 1:1.
[0013] Further, the preparation method is as follows:
[0014] a. Mix two human melanoma cells, ZJMM-45 and FLFMM-34, in the logarithmic growth phase at a ratio of 1:1, and then filter with a 200-mesh cell sieve;
[0015] b. At 37°C, the filtered cells are irradiated continuously for 4 days under 60Co with a total dose of 80 Gy and a continuous dose of 20 Gy per day. The irradiation is stopped when the daily irradiation dose is reached. After the cumulative irradiation dose is reached, the cell activity is tested, and the cells are confirmed to be inactivated and then packaged and frozen to obtain a whole-cell vaccine for inhibiting or preventing melanoma.
[0016] Furthermore, each of the multivalent whole-cell vaccines contains 2.0-3.0×10 7 cells and used after aliquoting.
[0017] Further, the preparation method of ZJMM-45 comprises the following steps:
[0018] a. Isolation of axillary lymph node metastasis cells: Melanoma tissues were obtained from affected lymph nodes of patients clinically diagnosed with malignant melanoma lymph node metastasis by the Dermatology Department of Xijing Hospital. After lymph node tissue resection, pathological examination was performed and the diagnosis was confirmed as melanoma lymph node metastasis.
[0019] Culture: Under sterile conditions, fresh lymph node tissue tumor specimens were immersed in serum-free culture medium supplemented with antibiotic solution (penicillin 100U / ml, streptomycin 100U / ml), fat, connective tissue and necrotic tumor tissue were removed, and then washed twice and cut into 1mm 3 The tissue blocks were digested with 0.2% complex collagenase at 37°C. After 3 hours, the tissue blocks dispersed and lost their shape. DMEM containing 10% fetal bovine serum was added to dilute the digestion solution.
[0020] Collection: Collect the diluted digestion fluid, remove the supernatant at 1000r / min for 5min, rinse once by centrifugation in serum-free medium, transfer the cells to serum-free basal medium prepared with MCDB153 and L-15 in a volume ratio of 4:1, add additives to the medium: 10ng / ml EGF, 10ng / ml ITS, culture for 5 days, and add 7% fetal bovine serum after 5 days to continue culture.
[0021] b. Establish cell lines by in vitro culture: After differential adherence purification, subculture, subculture twice a week to 70 generations through 0.25% trypsin digestion, and name the cell ZJMM-45. ZJMM-45 cells of different generations were frozen in liquid nitrogen, and the 50th generation cells were taken for identification and cell biological characteristics analysis. It was identified that ZJMM-45 met the genetic and biological characteristics of melanoma cell lines in terms of staining karyotype analysis, mutant gene detection, melanoma-specific marker expression, cell proliferation, migration and invasion ability.
[0022] MCDB153 and L-15 are ITS universal culture additive and EGF epidermal growth factor respectively.
[0023] Further, the preparation method of FLFMM-34 comprises the following steps:
[0024] a. Isolation of axillary lymph node metastasis cells: Melanoma tissues were obtained from affected lymph nodes of patients clinically diagnosed with malignant melanoma lymph node metastasis by the Dermatology Department of Xijing Hospital. After lymph node tissue resection, pathological examination was performed and the diagnosis was confirmed as melanoma lymph node metastasis.
[0025] Culture: Under sterile conditions, fresh lymph node tissue tumor specimens were cultured with phosphate buffered saline (PBS) plus antibiotic solution (penicillin 100U / mL, streptomycin 100U / mL). After rinsing with antibiotics in PBS, the tissue was cut into approximately 1 mm 3 The fragments were then transplanted into a 25 cm 2 The cells were cultured in a culture flask containing keratinocyte-SFM medium supplemented with 10% fetal bovine serum (FBS), bovine pituitary extract, recombinant human epidermal growth factor and glutamine.
[0026] b. Establishing a cell line by in vitro culture: After differential adherence purification, the cells were subcultured and digested with 0.25% trypsin, and subcultured twice a week until the 110th generation. The cells were named FLFMM-34.
[0027] FLFMM-34 cells of different generations were cryopreserved in liquid nitrogen, and the 80th generation cells were taken for identification and cell biological characteristics analysis.
[0028] It was identified that FLFMM-34 met the genetic and biological characteristics of melanoma cell lines in terms of staining karyotype analysis, mutant gene detection, melanoma-specific marker expression, cell proliferation, migration and invasion ability.
[0029] Furthermore, the cell lines of ZJMM-45 and FLFMM-34 are derived from Chinese Han women.
[0030] Furthermore, the multivalent whole-cell vaccine is used in combination with BCG and IFN-α1b.
[0031] Preferably, the interferon is IFN- α 1b. According to the patient's specific condition and drug tolerance, give 300-600ugIFN- α 1b, once every other day, maximum single dose is 1000ug.
[0032] Furthermore, the multi-valent whole-cell vaccine is administered 1-22 times according to the patient's specific condition and survival status, and more than 90% of patients are vaccinated 3-13 times. Each patient is subcutaneously injected with one vaccine, about 1 ml, containing 2.0-3.0×10 7 inactivated cells.
[0033] Beneficial effects:
[0034] The present invention is processed in vitro by non-autologous homologous antigens of 2 melanoma cell lines ZJMM-45 and FLFMM-34 to make a multivalent cell vaccine, with thousands of antigenic proteins, compared with the vaccine prepared by a single melanoma cell line, it has more diverse non-autologous homologous antigens, can cover a variety of MM specific antigens to activate specific immune responses, and ZJMM-45 and FLFMM-34 are derived from the Han Chinese population in China, and the antigens of the vaccine are closer to the HLA typing of the Han Chinese population, so it is more suitable for Chinese population melanoma. Through clinical trials, 174 patients were treated with the whole cell vaccine and adjuvant BCG combined with low-dose interferon, and the 5-year survival rate was significantly improved compared with the previous high-dose interferon treatment. It can be seen that the vaccine composition is not only easy to use, but also has a good therapeutic effect, indicating that it plays an important role in the treatment of melanoma and has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS
[0035] Figure 1 :Survival curve of 174 melanoma patients (including stage I-IV) treated with this vaccine;
[0036] Figure 2 : Survival curve of high-risk melanoma patients treated with high-dose interferon in the literature (solid line represents the high-dose interferon group). DETAILED DESCRIPTION
[0037] The present invention will be described in detail below with reference to specific embodiments and accompanying drawings:
[0038] Example 1: Preparation of ZJMM-45 and FLFMM-34 cell lines
[0039] 1. Preparation of ZJMM-45 cell line
[0040] a. Isolation of axillary lymph node metastasis cells: Melanoma tissue was derived from the affected lymph nodes of a 17-year-old female patient who was clinically diagnosed with malignant melanoma lymph node metastasis by the Dermatology Department of Xijing Hospital. After lymph node tissue resection and histopathological examination, it was confirmed to be melanoma lymph node metastasis.
[0041] Culture: Under sterile conditions, fresh lymph node tissue tumor specimens were immersed in serum-free culture medium supplemented with antibiotic solution (penicillin 100U / ml, streptomycin 100U / ml), fat, connective tissue and necrotic tumor tissue were removed, and then washed twice and cut into 1mm 3 The tissue blocks were digested with 0.2% complex collagenase at 37°C. After 3 hours, the tissue blocks dispersed and lost their shape. DMEM containing 10% fetal bovine serum was added to dilute the digestion solution.
[0042] Collection: Collect the diluted digestion fluid, remove the supernatant at 1000r / min for 5min, rinse once by centrifugation in serum-free medium, transfer the cells to serum-free basal medium prepared with MCDB153 and L-15 in a volume ratio of 4:1, add additives to the medium: 10ng / ml EGF, 10ng / ml ITS, culture for 5 days, and add 7% fetal bovine serum after 5 days to continue culture.
[0043] b. Establishment of cell line by in vitro culture: After differential adherence purification, the cells were subcultured and digested with 0.25% trypsin and subcultured twice a week until the 70th generation. The cells were named ZJMM-45.
[0044] ZJMM-45 cells of different generations were cryopreserved in liquid nitrogen, and cells of the 50th generation were taken for identification and cell biological characteristics analysis. It was found that ZJMM-45 met the genetic and biological characteristics of melanoma cell lines in terms of staining karyotype analysis, mutant gene detection, melanoma-specific marker expression, cell proliferation, migration and invasion ability.
[0045] 2. Preparation of FLFMM-34 cell line
[0046] a. Isolation of axillary lymph node metastasis cells: Melanoma tissue was derived from the affected lymph nodes of a 47-year-old female patient who was clinically diagnosed with malignant melanoma lymph node metastasis by the Dermatology Department of Xijing Hospital. After lymph node tissue resection and histopathological examination, it was confirmed to be melanoma lymph node metastasis.
[0047] Culture: Under sterile conditions, fresh lymph node tissue tumor specimens were rinsed with phosphate buffered saline (PBS) plus antibiotic solution (penicillin 100U / mL, streptomycin 100U / mL). After rinsing with antibiotics in PBS, the tissue was cut into approximately 1 mm 3 The fragments were then transplanted into a 25 cm 2 The cells were cultured in a culture flask containing keratinocyte-SFM medium supplemented with 10% fetal bovine serum (FBS), bovine pituitary extract, recombinant human epidermal growth factor and glutamine.
[0048] b. Establishing a cell line by in vitro culture: After differential adherence purification, the cells were subcultured and digested with 0.25% trypsin, and subcultured twice a week until the 110th generation. The cells were named FLFMM-34.
[0049] FLFMM-34 cells of different generations were cryopreserved in liquid nitrogen, and the 80th generation cells were taken for identification and cell biological characteristics analysis. It was found that FLFMM-34 met the genetic and biological characteristics of melanoma cell lines in terms of staining karyotype analysis, mutant gene detection, melanoma-specific marker expression, cell proliferation, migration and invasion ability.
[0050] The above-mentioned FLFMM-34 homologous human melanoma cell line and ZJMM-45 homologous human melanoma cell line have been preserved at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), and the preservation time is May 31, 2022.
[0051] Example 2: Vaccine preparation
[0052] The 50th generation ZJMM-45 and 80th generation FLFMM-34 cells frozen in Example 1 were thawed and then incubated in keratinocyte-SFM medium supplemented with 10% fetal bovine serum (FBS), bovine pituitary extract, recombinant human epidermal growth factor and glutamine at 5% CO. 2 , and cultured at 37°C for 6 days to prepare the whole-cell vaccine.
[0053] a. ZJMM-45 and FLFMM-34 human melanoma cells of the same type cultured in the logarithmic growth phase were mixed at a molar ratio of 1:1, and then filtered through a 200-mesh cell sieve;
[0054] b. At 37°C, the filtered cells were irradiated continuously for 4 days at 60Co with a total dose of 80 Gy and a continuous dose of 20 Gy per day. The irradiation was stopped when the daily irradiation dose was reached. After the cumulative irradiation dose was reached, the cell activity was detected. After confirming that the cells were inactivated, they were packaged and frozen (freezing solution: 90% DMEM + 10% DMSO), and the whole-cell vaccine for inhibiting or preventing melanoma was obtained. The prepared whole-cell vaccine contained 2.0-3×10 7 cells.
[0055] Example 3: Vaccine treatment trial
[0056] 1. Experimental Design
[0057] 1.1 Trial type: single-center, non-randomized, open-label study
[0058] 1.2 Sample size estimation: Based on the pre-assumed 95% confidence interval, P ≤ 0.05 was considered a significant difference. The study included 200 subjects. Considering the 15% dropout rate, the sample size of this study was appropriate.
[0059] 2. Subject selection and withdrawal
[0060] 2.1 Inclusion criteria are:
[0061] 1) Histopathologically confirmed malignant melanoma stage I-IV;
[0062] 2) No other systemic diseases;
[0063] 3) Aged ≥ 18 years;
[0064] 4) White blood cell count ≥3,000 μL; platelet count ≥100,000 μL;
[0065] (5) The informed consent form has been signed.
[0066] 2.2 Exclusion criteria are as follows: 1) Physical examination failure or poor compliance; 2) Severe abnormalities or disorders of kidney, liver, heart or other organs; 3) Pregnant or lactating women; 4) Other malignant tumors; 5) Previous treatment with the vaccine in this study; 6) Patients treated with glucocorticoids; 7) Patients treated with cytotoxic drugs within 4 weeks; 8) HIV(+) patients; 9) Patients with depression; 10) Patients with hypersensitivity (especially allergic to eggs); 11) Severe liver and kidney dysfunction before surgery; 12) Not the first breast surgery; 13) Severe systemic infection; 14) Unable to cooperate with the study for any reason, such as the following: language comprehension, mental illness, etc.; 15) Participated in other clinical trials within 3 months before enrollment in the study; 16) The researcher believes that it is not appropriate to be included in this experiment.
[0067] 2.3 Trial termination: 1) Patients who are found not to meet the inclusion criteria after enrollment; 2) Patients who are not suitable to continue the trial due to serious adverse events, complications or special physiological changes; 3) Patients whose blind is broken during a blind trial; 4) Patients who request to withdraw from the trial on their own for reasons other than efficacy.
[0068] 3. Experimental steps
[0069] 3.1 According to the inclusion and exclusion criteria, a total of 174 patients were screened, including 13 patients in stage I, 118 patients in stage II, and 43 patients in stage III+IV. The age range was 3-83 years old, with an average age of 54.6 years old, including 79 males and 95 females.
[0070] 3.2 Preparation of the vaccine before use: Before injection, reconstitute at 37°C, wash three times with saline, and suspend in 1.0-1.2 ml of saline for later use.
[0071] 3.3 Select the appropriate injection site (bilateral axillary or inguinal lymph nodes) according to the patient's condition.
[0072] 3.4 Give the patient a subcutaneous injection of 1 ml of whole-cell vaccine on the 1st day, 14th day, 1 month, 3 months, and every 3 months thereafter, and a subcutaneous injection of 0.5 ml of BCG. In addition, according to the patient's specific condition and drug tolerance, give 300-600ug IFN-α1b once every other day, with a maximum single dose of 1000ug. Continue treatment with vaccines and interferon until CR occurs or the patient dies or is terminated due to other reasons.
[0073] The patients were followed up for 5 years after the start of treatment, and their condition changes and survival status were recorded.
[0074] After 5 years of continuous follow-up, 133 of the 174 patients survived. The survival curve is shown in the figure below: Figure 1 As shown in Figure 2, the 5-year survival rate is 76.4%. According to the literature, the 5-year survival rate of high-risk melanoma patients treated with high-dose (HDI) interferon 10 million in the same period is 52%. The statistical survival curve is shown in Figure 2. Figure 2 As shown. (Literature source: Kirkwood JM, Ibrahim J G, Sondak VK, et al. High-and low-dose interferon alfa-2b in high-risk melanoma: first analysis of intergroup trial E1690 / S9111 / C9190[J]. J Clin Oncol, 2000, 18(12): 2444-2458.).
[0075] Combination Figure 1 and Figure 2 It can be seen that compared with the treatment with high-dose interferon alone, the 5-year survival rate of high-risk melanoma treated with whole-cell vaccine prepared by lymph node metastatic cells of Han women combined with low-dose interferon has been significantly improved to 76.4%, while increasing the specific anti-tumor immune response of the malignant melanoma vaccine. Compared with the treatment with high-dose interferon alone, the whole-cell vaccine of the present invention can significantly prolong the survival of patients, and the effect is significant for high-risk melanoma patients.
[0076] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art 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 be included in the scope of the claims of the present invention. The techniques, shapes, and structural parts not described in detail in the present invention are all known technologies.
Claims
1. A multivalent whole-cell vaccine for treating melanoma, characterized in that: The multivalent whole-cell vaccine comprises a ZJMM-45 homologous human melanoma cell line and a FLFMM-34 homologous human melanoma cell line, wherein the ZJMM-45 homologous human melanoma cell line has a preservation number of CCTCC NO: C2022105, and the FLFMM-34 homologous human melanoma cell line has a preservation number of CCTCC NO: C2022100; The preparation method of the multivalent whole-cell vaccine is as follows: a. Mix two human melanoma cells of the same strain, ZJMM-45 and FLFMM-34, in the logarithmic growth phase at a molar ratio of 1:1, and then filter with a 200-mesh cell sieve; b. Irradiate the filtered cells continuously for 4 days at a total dose of 80 Gy, with a continuous dose of 20 Gy per day under 60Co. After reaching the cumulative irradiation dose, test the cell activity, and after confirming that the cells have been inactivated, divide them into portions and freeze them to obtain a multi-valent whole-cell vaccine.
2. A multivalent whole-cell vaccine for treating melanoma according to claim 1, characterized in that: In the preparation of multivalent whole-cell vaccines, irradiation was performed at 37°C.
3. A multivalent whole-cell vaccine for treating melanoma according to claim 1, characterized in that: Each of the multivalent whole-cell vaccines contains 2.0-3.0×10 7 cells.
4. Use of the multivalent whole-cell vaccine according to any one of claims 1 to 3 in the preparation of a drug for treating melanoma, characterized in that: The multivalent whole-cell vaccine is used in combination with BCG and IFN-α1b.