WO2022094847A1

WO2022094847A1 - Cell lysis solution of engineering bacterium and use thereof in tumor therapy

Info

Publication number: WO2022094847A1
Application number: PCT/CN2020/126718
Authority: WO
Inventors: 刘陈立; 王作伟; 盛方芊; 郭旋; 曾正阳; 董宇轩; 卢伟琪; 李扬; 黄雄亮; 郑海; 刘为荣
Original assignee: 中国科学院深圳先进技术研究院
Priority date: 2020-11-05
Filing date: 2020-11-05
Publication date: 2022-05-12

Abstract

A cell lysis solution of an engineering bacterium, particularly a cell lysis solution of a facultative anaerobic bacterium that knocks out the dapA gene or the dapE gene, and the use thereof in tumor therapy.

Description

Cell lysate of engineered bacteria and its application in tumor therapy

technical field

The present invention relates to the field of tumor targeted therapy, in particular to a cell lysate of engineered bacteria, in particular to a cell lysate of facultative anaerobic bacteria knocking out dapA gene or dapE gene and its application in tumor therapy.

Background technique

Cancer is the leading cause of death worldwide. Compared with normal cells, cancer cells have the characteristics of infinite proliferation, transformation and easy metastasis. In addition to uncontrolled division (multipolar division), cancer cells also locally invade surrounding normal tissues and even metastasize to other organs via the circulatory system or lymphatic system in the body. The history of cancer treatment development shows that traditional cancer treatment methods, such as surgery, chemotherapy, radiation therapy, hormone therapy, bone marrow/stem cell transplantation, etc., all have certain defects, such as surgical treatment is prone to recurrence and some tumors are difficult to operate, etc. Problems; chemotherapy and radiotherapy will cause serious side effects to patients, resulting in ineffective treatment; hormone therapy suppresses inflammation, increases body fat mass, reduces muscle mass, seriously affects blood sugar levels, and greatly increases the incidence of diabetes and infectious diseases The probability of bone marrow/stem cell transplantation is difficult to match, and immune rejection is prone to occur. The difficulty of cancer treatment stems from the complex and changeable etiology of cancer. Not only changes at the gene level of the body, but also changes in the external environment are one of the important factors in the development of cancer. In the process of treating tumors with traditional methods, normal tissues and organs will be severely toxic, and cancer cells will develop multidrug resistance, which cannot completely and effectively remove cancer cells. In recent years, multiple studies have found that gene therapy, non-invasive radiofrequency treatment of cancer, insulin-boosting therapy, dietary therapy, and bacterial therapy can not only prevent cancer cells from developing multidrug resistance, but also enhance the efficacy of traditional therapies. Among them, bacterial therapy is a promising cancer treatment method that overcomes the shortcomings of traditional treatment methods.

The history of using live bacteria to treat cancer goes back more than 150 years. In 1868, the German physician W. Bush first applied bacteria to treat sarcoma that could not be treated by surgery. Within a week of receiving treatment, the tumor volume was reduced by half and the size of the cervical lymph nodes was reduced. Unfortunately, the patient died of sepsis caused by bacterial infection 9 days later. In 1883, German surgeon Friedrich Fehleisen identified erysipelas as a cause of Streptococcus pyogenes infection. Subsequently, Friedrich Fehleisen and Willian B Coley, a surgeon from New York Hospital, independently conducted experiments to prove that Streptococcus pyogenes can make patients' tumors regress. However, the results were controversial because the experimental results were difficult to reproduce and did not meet the clinical standards of the time. In 1935, Connell observed that filtrates from Clostridium enzymes could regress metastases. In 1947, scientists first injected spores of Clostridium histolytica into mice transplanted with sarcomas and observed lysis of cancer cells and regression of tumor tissue. However, the survival rate of mice was low due to the acute toxicity caused by the bacteria. In 1959, BCG (attenuated Mycobacterium bovine tuberculosis) was successfully used in cancer immunotherapy. In 2002, the attenuated Salmonella VNP20009 (msbB-, purI-) underwent a phase I clinical trial, and the results showed that the strain could colonize tumor tissue, but the effect on tumor treatment was not obvious.

Although VNP20009 did not achieve good clinical results, in view of the immunomodulatory function of Salmonella, researchers believe that various modifications may be used to make Salmonella suitable for tumor treatment. The reason why Salmonella needs to be modified is that wild-type Salmonella is virulent and can cause symptoms such as fever, vomiting, diarrhea, and abdominal cramps, and in severe cases, bacteremia can be life-threatening. With the rapid development of molecular biology technology, the Salmonella genome can be re-edited through genetic modification strategies to make it suitable for tumor therapy.

Chinese patent application CN104031146A discloses that the bacterial lysate is combined with tumor cells and inactivated to prepare a vaccine.

Chinese patent application CN111315868A discloses the construction of bacteria expressing cytotoxins, which are derived from bacteria targeting tumors, thereby specifically killing tumors.

Wang Zhirui's paper "Immune regulation of bacterial lysates" studies the immune regulation of lysates of a variety of bacteria, confirming that the bacterial lysates of Bifidobacterium bifidum, Staphylococcus aureus, Streptococcus pneumoniae, and Haemophilus influenzae have Immunomodulatory effects in vitro, and demonstrated that sonicated bifidobacteria lysates had direct inhibitory effects on colon cancer cell lines HT-29 and THC-8908. It can be seen that Wang Zhirui's paper mainly partially proves that a variety of bacteria have immunomodulatory effects and inhibitory effects on cell lines, but does not effectively indicate that the bacteria have obvious tumor therapeutic effects.

In the present invention, on the basis of transforming the genome of the Salmonella strain, the transformed strain is mechanically broken to obtain a cell lysate. Using the transformed strain cell lysate for tumor treatment can further improve the safety of tumor treatment.

SUMMARY OF THE INVENTION

In order to solve the problems of the prior art, the purpose of the present invention is to provide a bacterial cell lysate obtained by engineering facultative anaerobic bacteria, and its application in tumor treatment.

In one aspect, the present invention provides a cell lysate of bacteria, which are facultative anaerobic bacteria knocking out essential genes in the metabolic pathway of facultative anaerobic bacteria.

In the cell lysate of the present invention, the facultative anaerobic bacteria include: Enterobacteriaceae (for example, Escherichia coli, Pneumococcus, Proteus, Enterobacter, Salmonella, Shigella), Staphylococcus Genus, Streptococcus, Pneumococcus, Bacillus anthracis and Diphtheria.

In the cell lysate of the present invention, 2,6-diaminopimelic acid (alias: 2,6-diaminopimelic acid (alias: 2,6-diaminopimelic acid) is additionally added to the culture medium when the facultative anaerobic bacteria are cultured in vitro after knocking out essential genes. 2,6-Diaminopimelic acid) or its analogs.

In the cell lysate of the present invention, the essential gene to be knocked out is the dapA gene, or the dapE gene.

In the cell lysate of the present invention, the facultative anaerobic bacteria is Salmonella typhi, and the source of the strain of Salmonella typhi includes human, chicken, dog or bovine.

In the cell lysate of the present invention, the cell lysate inhibits tumor growth and reduces tumor volume when used for in vivo tumor treatment.

In the cell lysate of the present invention, the tumors include: blood cancer (chronic leukemia, acute leukemia), bone cancer, lymphoma (non-Hodgkin's lymphoma, Hodgkin's lymphoma), intestinal cancer (colon cancer, rectal cancer) cancer), liver cancer, stomach cancer, pelvic cancer (cervical cancer, ovarian cancer, endometrial cancer, ovarian cancer), lung cancer, breast cancer, pancreatic cancer, bladder cancer, prostate cancer.

In the cell lysate of the present invention, the cell lysate is administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, intracerebral administration or nasal administration.

In another aspect, the present invention provides the application of the above-mentioned cell lysate of the present invention in tumor therapy, wherein the cell lysate is used in combination with other cancer treatment methods, including:

(a) the cell lysate therapy combined with surgical therapy;

(b) the cell lysate therapy combined with radiotherapy;

(c) The cell lysate therapy is combined with chemical drugs: chemotherapy drugs include alkylating agents (nimustine, carmustine, lomustine, cyclophosphamide, ifosfamide, pyruvate mustard, etc.) , antimetabolites (deoxyfluridine, docefluridine, 6-mercaptopurine, cytarabine, fluoroguanosine, tegafur, gemcitabine, carmofur, hydroxyurea, methotrexate, Fortin, amcitabine, etc.), antitumor antibiotics (actinomycin, arubicin, epirubicin, mitomycin, pelomycin, pingyangmycin, pirarubicin, etc.), plant Anticancer drugs (irinotecan, harringtonine, hydroxycamptothecin, vinorelbine, paclitaxel, taxotere, topotecan, vincristine, vindesine, vinblastine, etc.), hormones (a Tamestane, Anastrozole, Anlutamide, Letrozole, Formestane, Metgestrol, Tamoxifen, etc.) immunosuppressants and other anticancer drugs such as asparaginase, carboplatin, cisplatin , Dacarbazine, Oxaliplatin, Lexadine, Keplatin, Mitoxantrone, Procarbazine;

(d) said cell lysate therapy in combination with biological therapy; and

(e) The cell lysate therapy is combined with traditional Chinese medicine treatment.

The cell lysate of the present invention is a cell lysate of tumor-targeted engineered bacteria, which can be further attenuated in the actual treatment process compared with intact live bacteria. The genetically engineered bacterial cell lysate of the present invention, such as the Salmonella typhimurium cell lysate, has the ability to inhibit the growth of tumors, and simultaneously improves the safety of the Salmonella typhimurium used in tumor treatment.

Description of drawings

Figure 1 shows the tumor volume of the experimental animals at the indicated times after administration of different samples in the SL7207 (ΔdapA) strain cell lysate internal experiment.

Figure 2 shows the body weight of the experimental animals at the indicated time after administration of different samples in the SL7207 (ΔdapA) strain cell lysing liquid internal experiment.

Figure 3 shows the survival rate curve of each sample experimental group after administration of different samples in the internal experiment of SL7207 (ΔdapA) strain cell lysing liquid. The test time is 100%, so it coincides as one line.

Figure 4 shows the tumor volume of the experimental animals at the indicated times after administration of different samples in the SL7207 (ΔdapE) strain cell lysate internal experiment.

Figure 5 shows the body weight of the experimental animals at the indicated time after administration of different samples in the SL7207 (ΔdapE) strain cell lysing liquid internal experiment.

Figure 6 shows the survival rate curve of each sample experimental group after administration of different samples in the internal experiment of SL7207 (ΔdapE) strain cell lysing liquid. The test time is 100%, so it coincides as one line.

Detailed ways

While various modifications can be made to the invention and the invention can have various forms, specific examples will be described and explained in detail below. However, it should be understood that these are not intended to limit the present invention to the specific disclosure, and the present invention includes all modifications, equivalents or substitutes thereof without departing from the spirit and technical scope of the present invention.

When Salmonella typhimurium was used in the mouse tumor model treatment test, it was found that the proliferation rate of wild-type Salmonella strains or auxotrophic Salmonella strains (for example: SL7207) in vivo was much greater than the clearance rate of the body, resulting in a large number of bacteria proliferating in mice. Causes severe bacteremia, produces serious side effects, until the death of mice, the safety is seriously lacking.

Hereinafter, the cell lysate of engineered bacteria according to specific embodiments of the present invention, especially the cell lysate of facultative anaerobic bacteria with knockout of dapA gene or dapE gene and its application in tumor therapy will be explained in more detail .

In one or more embodiments of the present invention, the present invention provides a cell lysate of bacteria, which are facultative anaerobic bacteria knocking out essential genes in the metabolic pathway of facultative anaerobic bacteria.

In one or more embodiments of the present invention, the facultative anaerobic bacteria used in the cell lysate of the present invention include: Enterobacteriaceae bacteria (eg, Escherichia coli, Pneumococcus, Proteus, Enterobacter, Typhoid Bacillus, Salmonella, Shigella), Staphylococcus, Streptococcus, Pneumococcus, Bacillus anthracis and Bacillus diphtheriae. Especially Salmonella.

In one or more embodiments of the present invention, the facultative anaerobic bacteria described in the present invention need to be additionally supplemented with 2,6-diaminopimelic acid (alias: 2,6-Diaminopimelic acid; 2,6-Diaminopimelic acid) or an analog thereof.

In one or more embodiments of the present invention, the essential gene to be knocked out is the dapA gene, or the dapE gene. Described essential gene is not limited to dapA gene or dapE gene, also includes dapB, dapD, argD, dapF, murE, murF and/or lysA etc.

In one or more embodiments of the present invention, the facultative anaerobic bacterium is Salmonella typhi, and the source of the strain of Salmonella typhi includes human, chicken, dog or bovine.

In one or more embodiments of the present invention, the cell lysate inhibits tumor growth and reduces tumor volume when used for in vivo tumor therapy.

In one or more embodiments of the invention, the tumor comprises: blood cancer (chronic leukemia, acute leukemia), bone cancer, lymphoma (non-Hodgkin lymphoma, Hodgkin lymphoma), intestinal cancer (colon cancer, rectal cancer), liver cancer, stomach cancer, pelvic cancer (cervical cancer, ovarian cancer, endometrial cancer, ovarian cancer), lung cancer, breast cancer, pancreatic cancer, bladder cancer, prostate cancer. Solid tumors among them are preferred, and bladder cancer is more preferred.

In one or more embodiments of the invention, the cell lysate is administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, intracerebral administration, or nasal administration. Those skilled in the art can select a specific route of administration considering the specific condition of the patient and the location of the tumor.

In one or more embodiments of the present invention, the present invention provides the application of the above-mentioned cell lysate of the present invention in tumor treatment, wherein the cell lysate is used in combination with other cancer treatment methods.

In one or more embodiments of the present invention, the cell lysate therapy of the present invention may be combined with surgical therapy. The surgical therapy may be tumor resection surgery.

In one or more embodiments of the present invention, the cell lysate therapy of the present invention may be combined with radiation therapy. The radiation therapy may be a radiation therapy method that a physician can specifically select and employ.

In one or more embodiments of the present invention, the cell lysate therapy of the present invention can be combined with a combination of chemical drugs. The chemotherapeutic drugs may include, for example, alkylating agents (nimustine, carmustine, lomustine, cyclophosphamide, ifosfamide, mustard, etc.), antimetabolites (deoxyfluorouracil, etc.) glycosides, docefluridine, 6-mercaptopurine, cytarabine, fluoroguanosine, tegafur, gemcitabine, carmofur, hydroxyurea, methotrexate, eufradin, amcitabine, etc.), Antitumor antibiotics (actinomycin, arubicin, epirubicin, mitomycin, pelomycin, pingyangmycin, pirarubicin, etc.), plant anticancer drugs (irinotecan, harringtonine, hydroxycamptothecin, vinorelbine, paclitaxel, taxotere, topotecan, vincristine, vindesine, vinblastine, etc.), hormones (ateametan, anastrozole, Lumide, Letrozole, Formestane, Metgestrol, Tamoxifen, etc.) Immunosuppressants and other anticancer drugs such as Asparaginase, Carboplatin, Cisplatin, Dacarbazine, Oxaliplatin , Lexadine, Coplatin, Mitoxantrone, Procarbazine. The specific selection and dose selection of the chemotherapeutic drugs can be determined by the physician according to the specific situation of the patient.

In one or more embodiments of the present invention, the cell lysate therapy of the present invention may be combined with biological therapy. The biological therapy is other biological therapy for tumor treatment other than the engineered bacterial cell lysate of the present invention.

In one or more embodiments of the present invention, the cell lysate therapy of the present invention can be combined with traditional Chinese medicine treatment. Chinese medicine treatment can be specifically formulated by the doctor according to the specific condition and physical condition of the patient.

In one or more embodiments of the present invention, the present invention utilizes genetic engineering technology to obtain SL7207 (ΔdapA) and SL7207 (ΔdapE) transformed strains, and obtains a cell lysate of the transformed strains by high pressure crushing method.

The high pressure crushing of bacterial cells in the present invention specifically adopts a high pressure crushing cytometer. -40 minutes, most preferably 30 minutes, the solution used for high-pressure crushing is, for example, a balanced salt solution PBS or physiological saline, and the mixture after high-pressure crushing can be directly used for injection to the subject after mixing.

When applying the prior art Salmonella typhimurium to the mouse tumor model treatment test, it was found that the proliferation rate of wild-type Salmonella strains or auxotrophic Salmonella strains (for example: SL7207) in vivo is much greater than the elimination rate of the body, resulting in bacteria in small A large number of mice proliferated, causing severe bacteremia, resulting in severe lethal side effects, resulting in the death of mice. In the present invention, the cell lysate of the transformed strain is injected into the tumor-bearing mice by means of tail vein administration. When the cell lysate of the modified strain was used for tumor treatment, it was found that it had the ability to inhibit tumor growth, the weight of the mice recovered quickly, and there was no death during the treatment cycle. It shows that the modified strain cell lysate involved in the present invention has anti-tumor ability and also improves safety.

The bacterial cell lysate of the present invention knocks out the essential genes on the metabolic pathway of the facultative anaerobic bacteria by engineering the facultative anaerobic bacteria. 2,6-diaminopimelic acid (alias: 2,6-diaminopulmonic acid; 2 , 6-Diaminopimelic acid) or its analogs. The essential gene to be knocked out can be, for example, the dapA gene, and/or the dapE gene.

The cells after knocking out the dapA gene and/or dapE gene cannot synthesize 2,6-diaminopimelic acid by themselves, so the medium contains 2,6-diaminopimelic acid at a concentration of 1-100 μg/ml , preferably 10-80 μg/ml, more preferably 30-70 μg/ml, most preferably 50 μg/ml.

Example

The following non-limiting examples are provided.

Example 1: In vivo characterization of SL7207 (ΔdapA) strain cell lysate for tumor therapy

6-8 weeks old C57BL/6 mice were purchased from Beijing Weitong Lihua Biotechnology Co., Ltd., weighing about 20 g, housed in SPF animals, and subcutaneously inoculated with 1×10 ⁶ mouse bladder cancer cells (MB49) , the feeding cycle was about ¹⁴ days, and the mouse bladder cancer ^subcutaneous tumor model was established. The experimental animals were divided into 5 groups, 8 mice in each group, respectively received PBS, SL7207 cells, SL7207 (ΔdapA), SL7207 (ΔdapA) cell lysate, and MG1655 cell lysate. A single injection of 1 x 10 ⁷ bacteria/125 μl bacterial cell lysate or 1 x 10 ⁷ bacterial cells was administered to the tail vein. Changes in tumor volume, body weight and survival rate of tumor-bearing mice were monitored.

Wherein, the bacterial cell lysate is prepared as follows:

(1) The SL7207 (ΔdapA) strain was cultured in LB (DAP+) medium to logarithmic phase;

(2) 5000rpm, centrifuge for 5min, remove the supernatant;

(3) Using filtered PBS, pH 7.2, resuspend the bacterial cells at a volume of 1×10 ⁷ bacteria/125 μl, centrifuge at 5000 rpm for 5 min. Wash the bacteria three times;

(4) Apply flow cytometer to count the number of cells;

(5) Use a high-pressure disrupting cytometer (Shanghai Litu Machinery Equipment Engineering Co., Ltd., FB-110X-PLUS) to disrupt the cells in an ice bath for 30 minutes to obtain a cell lysate.

The experimental results (as shown in Figures 1-3) are:

(1) Changes in tumor volume (A): Compared with the PBS group, the SL7207 (ΔdapA) cell lysate group could inhibit tumor growth; although the SL7207 group also had a better tumor growth inhibitory effect, the mice died 7 days later. sepsis; and the cell lysate of bacteria like MG1655 did not see obvious tumor suppressive effect.

(2) Changes in the body weight of mice (B): The body weight of the mice in the cell lysate group only took three days to recover to the level of the PBS group.

(3) Mice survival rate (C): During the experimental period, the mice in the PBS group and the experimental group did not die.

Experimental conclusion: During the experimental period, (1) the cell lysate of this strain has the effect of inhibiting tumor growth; (2) the body weight of the mice in the cell lysate group can be restored to the level of the control group in a short time. death, indicating that the bacterial lysate is relatively safe when used for tumor treatment.

Example 2: In vivo characterization of SL7207 (ΔdapE) strain cell lysate for tumor therapy

C57BL/6 mice were subcutaneously inoculated with 1×10 ⁶ mouse bladder cancer cells (MB49) to establish a mouse bladder cancer subcutaneous tumor model. The experiment was divided into five groups, 3 mice in each group, respectively received PBS, SL7207, SL7207 (ΔdapE), SL7207 (ΔdapE) cell lysate, MG1655 cell lysate, 3 mice in each group. A cell lysate of 1×10 ^{7 bacteria or 1×10 7} ^bacterial cells was injected into the tail vein. Changes in tumor volume, body weight and survival rate of tumor-bearing mice were monitored.

Wherein, the bacterial cell lysate is prepared as follows:

(1) The SL7207 (ΔdapE) strain was cultured in LB (DAP+) medium to log phase;

(2) 5000rpm, centrifuge for 5min, remove the supernatant;

(3) Resuspend the cells with filtered PBS, centrifuge at 5000 rpm for 5 min. Wash the bacteria three times;

(4) Apply flow cytometer to count the number of cells;

(5) Use a high-pressure cytometer (Shanghai Litu Machinery Equipment Engineering Co., Ltd., FB-110X-PLUS) to break the cells in an ice bath for 30 min to break the cells to obtain a cell lysate.

Experimental results (as shown in Figure 4-Figure 6):

(1) Changes in tumor volume (A): Compared with the PBS group, the SL7207 (ΔdapE) cell lysate group can inhibit tumor growth; although the SL7207 group also has a better tumor growth inhibition effect, but the mice died 7 days later sepsis; and the cell lysate of bacteria like MG1655 did not see obvious tumor suppressive effect.

Claims

A cell lysate of bacteria, which are facultative anaerobic bacteria knocking out essential genes in the metabolic pathway of facultative anaerobic bacteria.
The cell lysate of claim 1, wherein the facultative anaerobic bacteria comprise: Enterobacteriaceae (eg, Escherichia coli, Pneumonia, Proteus, Enterobacter, Typhi, Salmonella, Shigella), Staphylococcus, Streptococcus, Pneumococcus, Bacillus anthracis and Bacillus diphtheriae.
The cell lysate according to claim 1, wherein 2,6-diaminopimelic acid or an analog thereof needs to be additionally added to the culture medium when the facultative anaerobic bacteria are cultured in vitro after knocking out essential genes.
The cell lysate according to claim 1 or 3, wherein the essential gene to be knocked out is the dapA gene or the dapE gene.
The cell lysate according to claim 2, wherein the facultative anaerobic bacteria is Salmonella typhi, and the source of the strain of Salmonella typhi comprises human, chicken, dog or bovine.
The cell lysate of claim 1 or 2, wherein the cell lysate is used for in vivo tumor treatment to inhibit tumor growth and reduce tumor volume.
The cell lysate of claim 6, wherein the tumor comprises: blood cancer (chronic leukemia, acute leukemia), bone cancer, lymphoma (non-Hodgkin's lymphoma, Hodgkin's lymphoma), intestinal cancer (colon cancer) , rectal cancer), liver cancer, stomach cancer, pelvic cancer (cervical cancer, ovarian cancer, endometrial cancer, ovarian cancer), lung cancer, breast cancer, pancreatic cancer, bladder cancer, prostate cancer.
The cell lysate of claim 6, which is administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, intracerebral administration, or nasal administration.
The application of the cell lysate according to any one of claims 1 to 8 in tumor therapy, wherein the cell lysate is used in combination with other cancer treatment methods, including:

(a) the cell lysate therapy combined with surgical therapy;

(b) the cell lysate therapy combined with radiotherapy;

(c) The cell lysate therapy is combined with chemical drugs: chemotherapy drugs include alkylating agents (nimustine, carmustine, lomustine, cyclophosphamide, ifosfamide, pyruvate mustard, etc.) , antimetabolites (deoxyfluridine, docefluridine, 6-mercaptopurine, cytarabine, fluoroguanosine, tegafur, gemcitabine, carmofur, hydroxyurea, methotrexate, Fortin, amcitabine, etc.), antitumor antibiotics (actinomycin, arubicin, epirubicin, mitomycin, pelomycin, pingyangmycin, pirarubicin, etc.), plant Anticancer drugs (irinotecan, harringtonine, hydroxycamptothecin, vinorelbine, paclitaxel, taxotere, topotecan, vincristine, vindesine, vinblastine, etc.), hormones (a Tamestane, Anastrozole, Anlutamide, Letrozole, Formestane, Metgestrol, Tamoxifen, etc.) immunosuppressants and other anticancer drugs such as asparaginase, carboplatin, cisplatin , Dacarbazine, Oxaliplatin, Lexadine, Keplatin, Mitoxantrone, Procarbazine;

(d) said cell lysate therapy in combination with biological therapy; and

(e) The cell lysate therapy is combined with traditional Chinese medicine treatment.