Background of AAV-DC-CTLTM Turmor Cell Targeting Therapy - A new breakthrough in tumor therapy
On 3rd October, 2011, the Canadian scientist, member of Institute of Medicine and National Academy of Science in the US, Professor Ralph M. Steinman, was awarded as one of the Nobel Laureates in Medicine according to the “Discovery of the Dendritic cell (DC cell) and its role in the adaptive immune system”. In the early 21st century, under the supervision of Professor Steinman in Stanford Cancer Institute in the US, Professor Yong LIU combined his research in Recombinant Adeno-associated Virus (rAAV) with the work of Professor Steinman to develop a type of rAAV transfected DC cells which could carry one or several type of tumor-associated epitopes determinant gene(s). By in vitro elicitation of T lymphocytes from patients, cytotoxic T lymphocytes (CTL) would be generated to target specific anti-tumor related antigen positive cancer cells. With subsequent related clinical research, such technology, which was named as “AAV-DC-CTLTM Tumor Cell Targeting Therapy Technology”, was proven to be successful and has obtained protection from 5 international patents. The cytotoxic T lymphocytes (A-CTLTM, in which A represents specific antigen) generated by this technology possess powerful function which can efficiently and specifically eliminate one or several type of specific tumor-related antigen positive cancer cells and PSMA positive angiogenesis tumor endothelial cells. Nevertheless, there is no harm to any normal cells.
The typical examples of clinical application of AAV-DC-CTLTM Tumor Cell Targeting Therapy Technology have been revealed by Professor Steinman and the late CEO of Apple Computer, Steve Jobs, during their advanced phase of pancreatic cancer. By applying this therapeutic technology, the lifespan of Professor Steinman was extended by 4 years. Together with the application with other treatments, Steve Jobs could survive for 6 more years.
After performing the clinical research in Stanford Cancer Institute for several years, by 2010, it was proven that besides direct targeting elimination of cancer cells, A-CTLTM specific cytotoxic T lymphocytes could also block blood vessels produced by tumor angiogenesis, facilitating indirect anti-cancer effect and enhancing clinical efficacy significantly. Such research outcome was commented as the most important breakthrough in cancer biotherapy by the American Society of Clinical Oncology (ASCO) in the first half of 2010.
The clinical research in the US showed that A-CTLTM specific cytotoxic T lymphocytes not only possibly reverse the drug-resistance of gynecological and prostate gland cancers due to hormonal therapy, but also probably reverse the resistance of cancer cells against other molecular targeted drugs, including IRESSA and Avastin. As such, Professor Liu was rewarded research funding from Roche USA to perform the related research work.
According to the estimation from International Agency for Research on Cancer (IARC), cancer incidence rate will increase by 3-5% annually in the future. By 2020, there would be 20 millions of new cases. Meanwhile, death cases would reach 12 millions. In China, the occurrence of cancer is showing a rapid growth obviously. Due to the serious toxic side-effects of traditional radiochemotherapy, the quality of life of cancer patients is greatly demolished. Furthermore, experimental results show that only old and weak cancer cells can be eliminated by radiochemotherapy. In addition, the problems of metastasis and drug-resistance still cannot be avoided. Although molecular targeted drugs possess the characteristic of high specificity to cancer cells and the quality of life of patients can be well improved, the shortcoming is that after applying molecular targeted drugs for a certain time frame, cancer cells would undergo mutational pathways to develop drug resistance. Since the new “people-oriented” concept in medical treatment development has been widely accepted, the promotion of clinical use of AAV-DC-CTLTM Tumor Cell Targeting therapy can facilitate high specificity cancer cell elimination, low or even no toxic side-effects, without drug resistance, higher chance of recovery, greater improvement in the quality of life of patients at advanced stage of cancer, and the possibility of cancer patients having good quality of living and working life even living along with tumors for long time. Therefore, AAV-DC-CTLTM Tumor Cell Targeting Therapy technology offers cancer patients not only an improved therapeutic method, but also a huge leap in the overall concept of cancer treatment.
AAV-DC-CTLTM Tumor Cell Targeting therapy belongs to the aspect of cancer biotherapy technology. In 2000, the report of the annual meeting of “International cancer bio-/immunotherapy and gene therapy” concluded that “Biotherapy is currently known as the only possible mean to eliminate cancer completely. The 21st century would be the era of cancer biotherapy.” However, nowadays, due to the uncertainty in efficacy and the greater side-effects in the use of LAK cell, CIK cells, DC cell immunotherapy, cancer biotherapy cannot gain wide recognition by specialists in oncology. Thus, cancer biotherapy can only work as an adjuvant therapy. Generated by AAV-DC-CTLTM Tumor Cell Targeting therapy technology, A-CTLTM specific cytotoxic T lymphocytes obtained possess precise clinical efficacy and ratable characteristics. With the in-depth clinical application research and recruitment of larger sample pools in multiple clinical trial centers, more significant and complementary scientific data can be obtained. It is expected that the launching of AAV-DC-CTLTM Tumor Cell Targeting therapy would make cancer biotherapy become the 4th practical cancer treatment after surgery, radiotherapy and chemotherapy, implying tremendous clinical and academic significance.
What is AAV-DC-CTLTM(ACTLTM)?
Studies have shown that the immune system can recognize tumor cells, generate antitumor immunity, prevent and inhibit of tumor cell growth. However, the tumor cell can adopt itself through a variety of mechanisms to avoid the destruction from immune system. Immunotherapy, with the assistant of various drugs, to stimulate and enhance immune function, in order to kill cancer cells as the most effective and promising cancer treatment.
There are varieties of immunotherapy treatment currently available, most of these immunotherapy has been practicing clinically. And it has become the most promising and least invasive cancer treatment typically after major surgery, radiotherapy and chemotherapy. Besides cytokines, most of these adoptive immunotherapies have been widely applied clinically.
Adoptive immunotherapy: peripheral vein is extracted from patient or other healthy donor, or Peripheral Blood Mononuclear Cells(PBMC) are collected. To be cultured externally, with antigen positive cancer cells killer and transfused back to patients for cancer treatment. These are clinically commonly practicing treatment: Lymphokine-Activated Killer cells (LAK), Tumor-Infiltrating Lymphocytes (TIL), Cytokine-Activated Killer cells (CIK) and Dendritic Cells (DC) .
AAV-DC-CTLTM, that is, by taking a small amount of peripheral blood from patients with, separation of immune cells in vitro to produce a large number to identify and attack specific types of tumor-specific killer t- cells and transfuse back to patients, in order to achieve the purpose of targeted cancer treatment. As to carry a tumor-associated Antigen determinants of recombinant adeno-associated virus gene transfection of dendritic cells in vitro stimulation in patients with t- lymphocytes, specific killing a certain number or types of tumor-associated Antigen positive tumor cell-specific killer T- lymphocytes (Antigen Cytotoxic Lymphocyte, ACTL. ACTLTM specific killer T cells not only can directly targeted killing tumor cells, but also to terminate and stop the neovascularization tumor, play an indirect role of killing tumor cells.
The study result was awarded by United States Cancer Institute (ASCO) in 2010. As the most important breakthroughs in the field in the first half of the year, and has received 5 international patent protections.
How does ACTLTM fight cancer?
AAV-DC-CTLTM Tumor Cell Targeting Therapy technology makes use of the non-pathogenic wild-type Adeno-associated virus (AAV), through gene recombination technique, to construct a recombinant AAV carrying specific tumor-associated epitope determinant gene(s). After the infection of the recombinant AAV to the peripheral blood monocular cells of the cancer patent and cytokine induction aftwards, monocytes would transform to the powerful antigen presenting DC cells. Subsequently, the DC cells obtained through such technology can in vitro elicit the T lymphocytes extracted from patient and produce effective anti-tumor cytotoxic T lymphocytes (CTL). Since the CTL produced possess tumor antigen specificity, i.e. targeting ability, therefore, one or several types of specific anti-tumor related antigen positive tumor cells and PSMA positive angiogenesis tumor endothelial cells would be eliminated. As such, antigen negative cells would not be affected.
CTL is the CD8+ subset of T cells, which is a type of specific T cell responsible to directly kill certain types of virus and cancer cells. Together with Natural Killer cells, CTL cells form the front line defense against virus and cancer in the organism. The killing mechanism carried out by CTL includes:
What are the indications of AAV-DC-CTLTM(ACTLTM)?
AAV-DC-CTLTM can be applied to various of cancer patients who meet the following conditions:
1) Tumor tissue MHC-I Class (HLA-I Antigen ) -Positive.
2) Normal function of liver and kidney.
3) No severe anaemia.
4) Stopped chemotherapy and radiation therapy, no low peripheral white blood cell count.
5) Large size of tumor has been removed.
6) No severe allergies.
7) Tumor associated Antigen by at least one of the following checked with positive serology or pathology can be treated:
( Please note that even if serologic testing is negative, does not means a pathologic Immunohistochemistry testing is also negative. Many cases serological test are negative, but immunohistochemical detection shown tumor tissue tested with positive.)
Respiratory tract tumors
1. Nasopharyngeal carcinoma: LMP-1 Antigen; Cytokeratin 19 (CK19；K19) Antigen; carcinoembryonic Antigen (CEA)
2. Larynx: Cytokeratin 19 (CK19；K19) ; Carcinoembryonic Antigen (CEA)
3. Bronchial gland carcinoma: CEA (CEA) ； HER2/neu(c-erb B2) Antigen; Cytokeratin 19 (CK19; K19)
4. Non-small cell lung cancer
5. Undifferentiated Carcinoma of the lung
6. Carcinoid tumor of the lung: CEA (CEA) ； HER2/neu(c-erb B2) Antigen; carcinoma - Testicular Antigen (CT)SPANX Antigen; Cytokeratin 19 (CK19；K19) Antigen
1. Tongue cancer: Cytokeratin 19 (CK19；K19；) Antigen; carcinoma - Testicular Antigen (CT)SPANX Antigen ; Cancer Carcinoembryonic Antigen (CEA)
2. Oesophagus: squamous cell carcinoma: Cytokeratin 19 (CK19；K19) Antigen; carcinoembryonic Antigen (CEA) Adenocarcinoma: CEA (CEA) ； HER2/neu(c-erb B2) Antigen; Cytokeratin19 (CK19；K19) Antigen
3. Stomach cancer: CEA (CEA) ； HER2/neu(c-erb B2) Antigen; prostate specific membrane (PSMA) Antigen; Cytokeratin 19 (CK19；K19) Antigen
4. Primary liver cancer: AFP (AFP) Antigen; carcinoembryonic Antigen (CEA) ; Prostate specific membrane (PSMA)Antigen; cancer testis Antigen (CT)SPANX Antigen; Cytokeratin 19 (CK19；K19) Antigen
5. Bile duct cancer: adenocarcinoma: CEA (CEA) ； HER2/neu(c-erb B2) Antigen; Cytokeratin19(CK19；K19) Antigen Squamous cell carcinoma: Cytokeratin 19 (CK19；K19) Antigen; carcinoembryonic Antigen (CEA)
6. Gallbladder cancer: adenocarcinoma: CEA (CEA) ； HER2/neu(c-erb B2) Antigen; carcinoma – Testis Antigen (CT)SPANX Antigen; Cytokeratin 19 (CK19；K19) AntigenSquamous cell carcinoma: Cytokeratin 19 (CK19；K19) Antigen ; Carcinoembryonic Antigen (CEA)
7. Pancreatic cancer: CEA (CEA) ； HER2/neu(c-erb B2) Antigen; prostate specific membrane (PSMA) Antigen; cancer testis Antigen (CT)SPANX Antigen; Cytokeratin 19 (CK19；K19) Antigen
8. Colorectal cancer: CEA (CEA) ； HER2/neu(c-erb B2) Antigen; prostate specific membrane (PSMA)Antigen, cancer testis Antigen (CT)SPANX Antigen; Cytokeratin 19 (CK19；K19) Antigen
Urinary system tumors
1. Renal cell carcinoma: CEA (CEA) Cytokeratin, and 19 (CK19；K19) Antigens, tumor - Testicular Antigen (CT)SPANX Antigen; prostate specific membrane (PSMA) Antigen
2. Bladder cancer: CEA (CEA) ; Sperm proteins 17(SP17) Antigen; carcinoma - Testicular Antigen (CT)SPANX Antigen; Cytokeratin 19 (CK19；K19) Antigen
3. Prostate cancer: prostate specific antigen (PSA) ; Prostate specific membrane (PSMA) Antigen, and prostate acid Phosphatase in Antigen (PAP) ; Carcinoembryonic Antigen (CEA)
4. Carcinoma of the penis: Cytokeratin 19 (CK19；K19) Antigen; carcinoembryonic Antigen (CEA)
1. Breast cancer: breast milk protein BA46 Antigen; carcinoembryonic Antigen (CEA) ； HER2/neu(c-erb B2) Antigen; Breast cancer associated genes 1 Protein (Bcg1) Antigen; carcinoma - Testicular Antigen (CT)SPANX Antigen; prostate specific membrane (PSMA) Antigen; Cytokeratin 19 (CK19；K19) Antigen
2． Ovarian cancer: carcinoembryonic Antigen (CEA);HER2/neu (c-ERB B2) Antigen; sperm protein 17 (SP17) Antigen; Tumor - Testicular Antigen (CT)SPANX Antigen; Cytokeratin 19 (CK19；K19) Antigen
3. cervical cancer: human papillomavirus ( HPV ） E6 Or / E7 Antigen ( Most patients with positive ) ; Cell cornerProtein 19(CK19；K19) Antigen; sperm proteins 17(SP17) Antigen; carcinoembryonic Antigen (CEA) 。 In addition, for adenocarcinoma:HER2/neu (c-ERB B2) Antigen
4. Endometrial cancer: CEA (CEA) ； HER2/neu(c-erb B2) Antigen; Cytokeratin 19(CK19； K19) Antigen; sperm proteins 17(SP17)
1. Myeloma: sperm proteins 17(SP17) Antigen, HM1 ， 24 Antigen
2. Multiple myeloma: HM1 ， 24 Antigen
Nervous system tumor
1.Tumor - Testicular Antigen (CT)SPANX Antigen
Malignant neoplasm of skin
1. Malignant melanoma tumor - Testicular Antigen (CT)SPANX Antigen; Cytokeratin 19 (CK19；K19) Antigen ; Carcinoembryonic Antigen (CEA)
2. Skin cancer: Cytokeratin 19 (CK19；K19) Antigen ; Carcinoembryonic Antigen (CEA)
Clinical summary of AAV-DC-CTLTM(ACTLTM)
1． Recombinant of Adeno-Associated virus (rAAV) :
Our laboratory which practicing with GMP Standard for massive and commercial production of high-purity, high titer of activity and in accordance with the clinical application of tumor associated Antigen determinants carried of recombinant Adeno-Associated Virus (rAAV) , has overcome the previous defects of which DC stimuli cannot be massively produced.
2． Treatment technology procedures:
As shown in Figure II, Blood (50-150 ml) from peripheral vein is extracted from patient, or Peripheral Blood Mononuclear Cells(PBMC) are collected directly by blood cell separator. After culture, PBMC cells are separated into lymphocytes and monocytes. Lymphocytes are further cultured for subsequent use. Monocytes are infected by rAAV and cytokine is used to induce DC cells maturation. Mature DC cells are then co-cultured with lymphocytes to produce lymphocyte-induced antigen specific cytotoxic T lymphocytes (A-CTLTM) which can kill antigen positive cancer cells. Finally the activated A-CTLTM cells are transfused back to patient for cancer treatment. The whole process takes 12-14 days.
3． Treatment procedures:
To complete a transfusion within every 12-14 days, 2-3 infusion per month, 6-9 infusions within a 3 months period as a complete and recommended course. Continue the treatment if positive feedback found.
What are the adverse reactions of AAV-DC-CTLTM(ACTLTM)?
Preliminary study of clinical trials showed that, this patented treatment technology has no obvious adverse reactions, the main side effects are as follows:
1), Flu-like symptoms: the main part in tumor patients ( About 45%) within a few hours after the transfusion, generally without treatment, symptoms may go away after a few hours. Main symptoms are: fever (37.0c-39.0c) and muscle soreness. If intolerable patients with symptoms, general antipyretic drugs can be given to ease the symptoms.
2) Excited: mainly occurs within a few days after the transfusion. Patients feel good about themselves, and feel the strength increase. About 50% of patients reported.
3) Fatigue: occurs mainly in patients with advanced cancer, especially patients treated with chemotherapy, radiotherapy or patients with anemia. Transfusion red blood cells or supportive therapy can alleviate the symptoms.
Preliminary clinical trials findings & research of AAV-DC-CTLTM (ACTLTM)
Current research and clinical trials has not been done long enough time, and cases are limited, which these cases cannot be taken for statistical analysis, therefore, there has no exact numbers of efficiency of recovery rate and survival rate to determine the effectiveness of AAV-DC-CTLTM tumor cell targeting therapy. However, with the preliminary observations which has indicated that, the overall treatments were satisfactory. As shown in table-A, vast majority of stage IV cancer patients during and after chemotherapy, radiation therapy, hormone therapy, or other treatment or relapsed. Yet, with the AAV-DC-CTLTM treatment, most clinical symptoms with these patients has different degree of eases, such as prostate cancer or breast cancer have bone metastases occur in patients with bone pain significantly eased or disappeared. After receiving the AAV-DC-CTLTM treatment, the majority of serum tumor markers in patients has a different degree of decline, or returning to a normal level. More importantly, the majority of patients’ CT, PET-CT and physical examination such as bone scans show that metastatic lesions with different degrees of reducing or even disappear, especially for a lesion of lymph nodes metastasis. With the preliminary observations, patients who find with positive feedbacks who lymph node metastasis lesions will disappear firstly. During the treatment period, and the quality of life of patients is guaranteed, mortality rates is significantly lower than any other treatment methods. No withdrawal nor intolerance were found with these patients during the treatment. Therefore, we strongly believe that it should show a higher effective rate for those patients who are found in an earlier stage. Study shown on the data table of acceptable patients in clinical trials below.
Note: Patients shown below who has received at least more than three months of AAV-DC-CTLTM tumor cell targeting therapy treatment. Within that, 2 cases of pulmonary metastasis of cervical cancer patients survive for as long as 9 years, and consecutively no new lesions was found in a 5 years period. And, who has already discontinued receiving any treatments.
Table-A: Patients accepted research-clinical trials:
What are the advantages of AAV-DC-CTLTM(ACTLTM)?
AAV-DC-CTLTM tumor cell targeting therapy has the advantages of which the Dendritic Cells (DC) Treatment technologies do not have or cannot have.
1. Adeno-associated virus (AAV) Non-pathogenic:
Adeno-Associated Virus (AAV): shorthand for the AAV, belonging to the Parvovirus, non-enveloped linear single-strand DNA viruses. Latent infection in the crowd is 60-80%, AAV has the long-term potential to pathogenicity of human body and does not have any obvious advantages. After worldwide scientific research on Adeno-associated virus (AAV) for 20 years, it is shown that AAV is a non-pathogenic virus and there is no pathological change occurred in human even after infection. Meanwhile, the safety of AAV has already been recognized by medical professionals. The National Institutes of Health (NIH) and the Food and Drug Administration in the US announced that AAV is the safest virus vector in gene therapy. The joint investigation and practical research carried out among the University of Arkansas for Medical Science and other universities in the US has shown that AAV not only is non-pathogenic but also possesses the ability to inhibit the growth of cervical cancer and the replication of human papillomavirus, hepatitis B virus and HIV virus.. AAV is specific to the consolidation in the human 19 on the long arm of the chromosome, has a certain predictability, avoiding the risk of mutagenicity of stochastic integration into other viral vectors. AAV has wide infection of host cells, splinter cell and still infected cells, cultivate growth, good stability, concentration, and purification and inactivation. Relative to other carriers of the virus, its immunogenicity is weaker, and good for repeated vaccination.
United States National Institutes of Health (NIH) And the Food and Drug Administration (FDA) has announced AAV is the safest viral vectors for gene therapy. Under the United States National Institutes of Health, bio-safety regulations for biosafety management in the institutions, for gene therapy of DNA and gene carriers, on the basis of their pathogenicity in healthy adults are divided into four risk classes.
Risk 1 class (RG1): organisms and not related to the disease in healthy adults.
Risk 2 classes (RG2): organisms associated with human disease. But few cause serious diseases, and can usually be preventive or therapeutic interventions.
Risk 3 classes (RG3): organisms associated with serious or fatal human disease, but may be preventive or therapeutic interventions.
Risk 4 classes (RG4): organisms can cause serious or fatal human disease, and are usually not subject to preventive or therapeutic interventions.
Adeno-Associated Virus (AAV) 1 to 4 and replication-defective recombinant AAV (GM did not code the potential cancer gene, or toxin-producing molecule) is currently the only one belonging to RG1 class carriers of the virus. Previous common adenovirus, lentivirus, herpes virus, smallpox virus belongs to RG2 class, art belongs to the GR3 class. It fully endorsed the AAV safety. At home and abroad the AAV vector research more and more, especially gene therapy based on clinical treatment. ( Figure III)
Figure III: NIH and FDA announced that AVV is the safest virus vector in gene therapy, which is non-harmful to human body.
United States Medical University of Arkansas United States of other universities of the epidemiological and laboratory studies have shown that AAV Not only non-pathogenic, and inhibits the growth of cervical cancer and human papilloma virus, viruses such as HIV and hepatitis b virus replication.
2． AAV-DC-CTLTM precise treatment efficacy. The functional mechanism of A-CTLTM specific cytotoxic T lymphocytes in cancer treatment is clear. A-CTLTM cells possess powerful function to efficiently and continuously eliminate the targeted cancer cells. The evaluation of clinical treatment can be made objectively by medical imaging, level of serum tumor markers, quality of life and lifespan of patient. Study confirms AAV DC features raised, this is AAV-DC-CTLTM advantages and characteristics of the treatment. DC stimulation and inducing tumor cell-specific killer t lymphocytes (CTL).
3． RAAV infection DC precursor - cells, mononuclear cell proliferation: after infection of rAAV, mononuclear cell expansion, with more DC will help AAV-DC-CTLTM treatment.
4． Raising DC CD1a, andCD40, andCD80 and CD83 expression: the CD Molecular level, enhanced DC Function.
5． Raising DC IL-12 expression levels, and the lower IL-10 levels: high IL-12, low IL-10, DC main cellular immune response launched, primarily inducing specific cytotoxic activity of t lymphocytes (CTL) .
6． Recombinant AAV infection of DC and lymphocytes after mixing, promoting CD8+T cells cut CD4+t- cells: get more killing of tumor cells CTL。
7． Raising IFN-gamma level of expression: so that the CTL killing activity of tumor cell enhanced.
8． AAV-DC-CTLTM treatment used in time to meet the needs of clinical treatment: past DC immune from cell culture treated to return to lost to cancer patients need to take about 3-8 weeks, unable to meet the continuing demand for treatment in cancer patients, Especially severe in patients with cancer. AAV-DC-CTLTM targeting therapy of tumor cells from cell culture to lose to patients only need 12-14 day, fully meets the needs of clinical treatment of the frequency.
9． Determine specific killer t lymphocytes (CTL) for anti-tumor cell function be simpler: it is AAV-DC-CTLTM one of the unique features of the targeting therapy of tumor cells, only in optical observation under a microscope, to determine accurately CTL Already have anti-tumor cell function, very consistent with clinical requirements and applications.
10. AAV-DC-CTLTM In the treatment, excess of lymphocytes CIK Cells, play a non-targeted treatment. Antigen-specific CTL and CIK in patients with both body and achieve the goal of two kinds of treatment.
11． AAV-DC-CTLTM have fewer adverse treatment reaction: AAV-DC-CTLTM has mild side effects of the treatment technique as described above, has not yet been found of serious adverse reactions and induce autoimmune reactions not only extremely low chance, but very weak. But in the past the DC immunotherapy technologies, organizations dissolved or tumor antigens as tumor protein, not only side effects significantly and are induced by severe autoimmune reactions. Adeno-Virus or retrovirus as the carrier to stimulate Dendritic Cells (DC) therapy risks greater, because these viruses are pathogenic, can cause severe inflammation, and there is a dangerous new Neoplasms induced by.
12． AAV-DC-CTLTM gene in the treatment of pollution rate very low, this is AV-DC immune treatment of one of the unique features. Effective CTL, and back to the before lost to cancer patients, infection of the DC , carrying tumor epitope gene of AAV viral genes are basically complete degradation and elimination.13． AAV-DC-CTLTM treatment against tumor angiogenesis endothelial cells, most of the patients with gastrointestinal tumors, breast cancer, and renal cell carcinoma tumor angiogenesis of prostate-specific membrane antigen expression in endothelial cells (PSMA). Carry PSMA epitope gene of rAAV for direct anti-angiogenesis endothelial cells in the tumors of AAV-DC-CTLTMtreatment by anti-tumor destruction of tumor angiogenesis to achieve purpose.
14． Reversal of drug resistance of tumor cells, AAV-DC-CTLTM gynecological and prostate cancer treatment has the potential to reverse not only against the hormone treatment, drug resistance, and possibly reverse tumor cells on Iressa, drug resistance of bio-targeted therapy such as AVASTIN
Case 1: Advanced metastatic breast cancer patient
The Result of PET-CT Scan of a metastatic breast cancer case before AAV-DC Treatment
This image shows positive uptake in the sternum and mediastinal lymph node.Before treatment, metastasis has been spread to sternum and mediastinal lymph node.
The Result of PET-CT scan of the breast cancer patient after AAV-DC treatment for 10 months
The image shows positive response to the treatment. The CTL cells were elicited by the DC cells transfected by AAV/BA46 and AAV/Her-2 virus. This patient has survived for more than 4 years.
Case 2: Advanced metastatic breast cancer patient
A metastatic breast cancer case with AAV-DC treatment using CTL cells elicited by AAV/Her2-neu, AAV/CEA and AAV/BA46 virus transfected DC cells
Before treatment, metastasis has been spread to vertebral column and the patent was suffering from severe pain.
After treatment for 4 months
After the transfection of DC cells by AAV-BA46, AAV-CEA and AAV-Her2, the BA46-CTL, CEA-CTL and Her2-CTL specific cytotoxic T lymphocytes were produced by in vitro elicitation of T lymphocytes. With the treatment for 4 months, the metastatic lesion in the vertebral column reduced in size noticeably and pain was obviously relieved.
Case 3: Advanced metastatic cervical cancer patient
Cervical cancer cases with lung metastases treated with cytotoxic T lymphocytes (CTL) stimulated by the pulsed dendritic cells (DC)
The isotope-labeled CTL can be found in the lung after I.V. of 4 hrs. It is showed that the CTL cells have moved to the lung rapidly, and could last for 24-120 hours.Two cases of advanced cervical cancer patients with lung metastases were examined. Isotope-labeled CTL could efficiently reach the lesion in lung after entering the body in 4 hours. It is proved that CTL cells activated by AAV-DC-CTLTM possess targeting and specific characteristics. These 2 patients have been living for 10 years. After the follow up observation for 5 years, no new tumor lesion has been found.
Case 4: A cervical cancer patient
A cervical cancer case treated with the CTL cells stimulated by AAV/HPV-E6-infected DC cells.
Before treatment After treatment for 7months
After the trasfection of DC cells by AVV-HPV-E6, HPV-E6-CTL specific cytotoxic T lymphocytes were generated by in vitro elicitation for subsequent treatment. The lesion disappeared after treatment for 7 months.
Case 5: An advanced colon cancer patient
One colon cancer case treated for 4 months with the CTL cells elicited by AAV/CEA Virus-infected DCs.
After the trasfection of DC cells by AAV-CEA, CEA-CTL specific cytotoxic T lymphocytes were generated by in vitro elicitation for subsequent treatment. Obvious efficacy could be observed after treatment for 4 months.
Case 6: A non-small cell lung cancer patient (Stage IV) patient
One lung cancer (adeocarcinoma) case treated for 8 months with the CTL cells elicited by AAV/CEA Virus-transfected DC cells.
The patient in this case received neither chemotherapy nor radiotherapy. After the onset of cancer, the patient immediately received the biological treatment of CEA-CTL specific cytotoxic T lymphocytes, which was generated by in vitro transfection of DC cell by AAV-CEA. This figure on the left shows the patient’s conditions before treatment, while the figure on the right shows the fellow up conditions after treatment for 5 months. This patient has been survived for more than 5 years.
Case 7: Nasopharyngeal cancer patients with lung metastasis
One nasopharyngeal cancer case with lung metastasis treated with the CTL elicited by AAV-K19 and AAV-LMP-1 virus-transfected DC(1) cells.
One nasopharyngeal cancer case with lung metastasis treated with the CTL elicited by AAV-K19 and AAV-LMP-1 virus-tranfected DC(2) cells.
After 10-month treatment, the patient was treated with CTL stimulated by AAV-K19 plus AAV/LMP-1 virus-tranfected DC cells generated in vitro. The size of metastatic lesion has reduced obviously. After treatment for 7 months, the radius of the lesion reduced to 2 mm and it is shown to be surrounded by pulmonary fibrosis tissue.
Case 8: An advanced prostate cancer patient with widespread metastasis in the body
The result of PET-CT scan of a metastatic prostate cancer case before AAV-DC treatment
The result of PET-CT scan of the prostate cancer patient after AAV-DC treatment for 3 months
The CTL cells were elicited by the AAV/PSA and AAV/PSMA virus-tranfected DC cells.The patient has received surgery and followed by hormonal therapy at the beginning. However, the treatment failed shortly afterwards and metastasis occurred in lymph nodes and bones. Subsequently, the patient received chemotherapy and radiotherapy but the efficacy was not obvious with more severe metastasis of cancer cells. Through the joint consultation of medical experts, it was expected that the patient would only able to survive in 3 months. By using PSA-CTL and PSMA-CTL specific cytotoxic T lymphocytes generated by in vitro elicitation of T lymphocyte by DC cells transfected by AAV-PSA and AAV-PSMA, the clinical conditions of the patient was improved and hormonal therapy became effective again. The patient has been surviving for almost 6 years. The figure on the left shows the patent conditions before treatment. The figure on the right shows the follow up conditions after treatment for 6 months. The results from PET-CT scan shows that 98% of metastatic lesions disappeared.
The results of clinical research
1、 The clinical research in 4 cases with metastatic cervical cancer
Four patients received the treatment of HPV-E6-CTL specific cytotoxic T lymphocyte generated by in vitro elicitation of CD cells prior transfected by AAV-HPV-E6. After treatment for 6 months, the serum tumor marker SCC obviously reduced. Two of the patients have been surviving over 5 years.
2、The clinical research in 4 cases with colon cancer
Four patients received the treatment of CEA-CTL specific cytotoxic T lymphocytes generated by in vitro elicitation of CD cells prior transfected by AAV-CEA. After treatment for 6 months, the serum tumor marker CEA obviously reduced.
3. The clinical research in 4 cases with metastatic non-small cell lung cancer
Four patients received the treatment of CEA-CTL and/or Her2-CTL specific cytotoxic T lymphocytes generated by in vitro elicitation of CD cells prior transfected by AAV-CEA and/or AAV-Her2. After treatment for 3 months, the serum tumor marker CEA obviously reduced.
4、The clinical research in 12 cases with metastatic prostate gland cancer
Twelve patients received the treatment of PSA-CTL and PSMA-CTL specific cytotoxic T lymphocytes generated by in vitro elicitation of CD cells prior transfected by AAV-PSA and AAV-PSMA. After treatment for 1 month, the serum tumor marker PSA in 8 patients obviously reduced.
5、The clinical research in the reversal of molecular targeted drug-resistance in metastatic colon cancer
After the failure of sole treatment by VEGF in 6 colon cancer patients, PSMA-CTL specific cytotoxic T lymphocytes, which were generated by in vitro elicitation of CD cells prior transfected by AAV-PSMA, was applied as the treatment afterwards. After treatment for 1 month, the serum tumor marker CEA reduced obviously.
After the failure of sole treatment by VEGF in 4 colon cancer patients, PSMA-CTL specific cytotoxic T lymphocytes, which were generated by in vitro elicitation of CD cells prior transfected by AAV-PSMA, was applied as the treatment. The treatment of VEGF became effective again and the serum tumor marker CEA reduced obviously.
Clinical Treatment Processes
(Blood 50-150 ml) from peripheral vein is extracted from patient, or Peripheral Blood Mononuclear Cells(PBMC) are collected directly by blood cell separator. After culture, PBMC cells are separated into lymphocytes and monocytes. Lymphocytes are further cultured for subsequent use. Monocytes are infected by rAAV and cytokine is used to induce DC cells maturation. Mature DC cells are then co-cultured with lymphocytes to produce lymphocyte-induced antigen specific cytotoxic T lymphocytes (A-CTLTM) which can kill antigen positive cancer cells. Finally the activated A-CTLTM cells are transfused back to patient for cancer treatment. The whole process takes 12-14 days.