Abstract
We devised an innovative type of immunocell therapy called BRM (biological response modifier)-activated killer (BAK) therapy, which utilizes most of non-MHC (major histocompatibility complex) restricted lymphocytes, CD56+ cells including γδ T cells and NK cells. Peripheral blood lymphocytes were selected by immobilizing them with anti-CD3 monoclonal antibody, cultured for 2 weeks with serum-free medium containing IL-2, and then were reactivated by 1,000 U/ml of IFN-α for 15 min. The patients were infused with about 6×109 BAK cells by intravenous drip infusion at 1-month intervals. All advanced solid cancer patients who had received chemotherapy but for whom it was not effective or have refused chemotherapy were included in the present study. A good marker of impairment of host immune response by chemotherapy is an immunosuppressive acidic protein (IAP) level in serum above 580 μg/ml; survival rates were compared with the high (>580 μg/ml) and the low (≤580 μg/ml) serum IAP groups. We enrolled in this study 23 immunosuppressed patients whose IAP levels in serum were over 580 μg/ml, and 42 immunoreactive solid cancer outpatients whose IAP level in serum were under 580 μg/ml and whose performance statuses were over 80% on the Karnofsky scale. After giving informed consent, patients were treated with BAK therapy on an outpatient basis at our hospital. The ethical review board of the Miyagi Cancer Center approved this pilot study. Treated with BAK therapy, the mean survival of immunosuppressed patients was 4.6 months. On the other hand, survival for one of immunoreactive advanced postoperative patients (stage IV) and inoperable lung cancer patients (stage IIIb) was 24.7 months. The difference in survival between the 2 groups was significant (P<0.01). This shows that BAK therapy is not indicated for an advanced cancer patient whose serum IAP is over 580 μg/ml, perhaps due to extensive chemotherapy. Overall response to BAK therapy was complete response (CR) in 5 cases, partial response (PR) in 1 case, and prolonged no change (NC) in 26 cases, with an effectiveness rate at 76.2% in 42 advanced stage IIIb and IV cancer patients. BAK therapy has a life-prolonging effect without any adverse effects and maintains satisfactory quality of life (QOL) for advanced cancer patients.
Keywords: Immunocell therapy, CD56+ cells, Immunosuppressive acidic protein, Lung cancer, Quality of life
Introduction
Lymphokine-activated killer (LAK) therapy was developed as a means of providing cancer patients with adoptive immunotherapy [17]. From a pool of peripheral mononuclear cells obtained from a patient, a large quantity of LAK cells are isolated and incubated in a test tube with interleukin-2 (IL-2). The activated cells along with IL-2 are subsequently returned to the patient. Patients treated with LAK cells experience adverse effects, however, and the outcome of the treatment is not satisfactory.
In our previous report [9], we introduced a new type of immunocell therapy called BRM-activated killer (BAK) therapy, which uses lymphocytes cultured and activated by immobilized anti-CD3 antibody, IL-2, and IFN-α. We have shown that most of these activated and proliferated lymphocytes are CD56+ cells, which are found in about half of γδ T cells and NK cells (non-MHC-restricted killer cells). The CD56 antigen is identical to the neural cell adhesion molecule (NCAM) [15], and was the first cell-cell adhesion molecule to be identified, isolated, and sequenced [6]. Structurally, the extracellular portion of NCAM comprises five IgG-like domains [22]. CD56 is expressed during embryonic development at a variety of sites in the nervous system and other tissues; its expression is more restricted in the adult brain. Our previous paper showed that CD56+ cells produced β-endorphin, which exhibits very potent analgesic and sedative activities. Studies by Gilman et al. [12] have shown that β-endorphin affects lymphocyte function, and suggest that β-endorphin may play an important role in the immune system [1, 4, 20]. It has also been shown that the immune system, the psychoneurological system, and the endocrine system interact in the body, and that CD56+ cells are multifunctional, integrated neuro-immune-endocrine (NIE) cells. As a QOL marker, we adopted the Face scale, a brief, nonverbal method for assessing patient mood, because QOL varies depending on each patient's subjective point of view [16]. Immunosuppressive acidic protein (IAP), initially reported by Tamura et al. [21], was purified from the ascitic fluid of patients with advanced cancer. Sakamoto et al. have shown that IAP suppresses several immune responses in vivo as well as in vitro, and an IAP level above 580 μg/ml is a good marker of impairment of the host immune response [18, 19]. IAP, which is one of the human serum α1-acid glycoproteins, is also a good tumor-marker protein [7].
As all patients refused chemotherapy, we could not carry out a double-blind randomized controlled study based on chemotherapy. We performed this pilot translational study as evidence-based medicine (EBM) for the grouping of serum IAP levels. The present paper reports the comparison of the survival months with BAK therapy of 23 immunosuppressed patients whose IAP levels in serum were over 580 μg/ml and 42 stage IV and IIIb immunoreactive solid cancer patients whose IAP levels were under 580 μg/ml. This study shows that BAK therapy has a life-prolonging effect without any adverse effects and that it maintains satisfactory QOL for advanced cancer patients. Consequently, our goal is to provide such patients with immunotherapy that allows them to live with cancer without necessarily trying to completely eliminate the tumor tissue, so these patients can spend their last days as they wish.
Materials and methods
Patients
As we could not carry out a double-blind randomized controlled study for advanced cancer patients who had received chemotherapy but for whom it was not effective and who had refused further chemotherapy, all advanced solid cancer patients whose life expectancy was limited to several months were included in the present study. Twenty-three immunosuppressed terminal-stage patients whose IAP levels were over 580 μg/ml (Table 1) and 42 outpatients with solid cancer whose IAP levels were under 580 μg/ml and whose performance statuses were over 80% on the Karnofsky scale (Table 2) were enrolled in this translational pilot study. Patient characteristics of the two groups is shown in Table 3. The ethical review board of the Miyagi Cancer Center approved this pilot study in advance, and informed written consent was obtained from all participants.
Table 1.
Immunosuppressed solid cancer patients with BAK therapy (Serum IAP levels >580 μg/ml)
| Patient number | Gender | Age | IAP (μg/ml) | Primary lesion | Survival (months) |
|---|---|---|---|---|---|
| 101 | Female | 54 | 742 | Lung cancer | 5 |
| 102 | Male | 54 | 630 | Pancreatic carcinoma | 9 |
| 103 | Female | 42 | 902 | Uterus myosarcoma | 3 |
| 104 | Male | 48 | 1,096 | Colon cancer | 4 |
| 105 | Male | 62 | 889 | Rectum cancer | 4 |
| 106 | Male | 68 | 714 | Pancreatic carcinoma | 2 |
| 107 | Female | 80 | 720 | Lung cancer | 2 |
| 108 | Male | 67 | 794 | Lung cancer | 1 |
| 109 | Female | 76 | 1,100 | Lung cancer | 10 |
| 110 | Female | 49 | 660 | Ovarian cancer | 8 |
| 111 | Male | 48 | 1,150 | Gastric cancer | 1 |
| 112 | Female | 58 | 730 | Pharyngeal cancer | 3 |
| 113 | Male | 50 | 740 | Colon cancer | 7 |
| 114 | Female | 51 | 770 | Breast cancer | 8 |
| 115 | Female | 47 | 1,010 | Breast cancer | 5 |
| 116 | Male | 59 | 720 | Cholangio carcinoma | 6 |
| 117 | Female | 51 | 720 | Chorionic carcinoma | 3 |
| 118 | Male | 70 | 1,000 | Pharyngeal cancer | 3 |
| 119 | Female | 43 | 1,445 | Breast cancer | 3 |
| 120 | Female | 58 | 1,200 | Accinic cell carcinoma | 5 |
| 121 | Male | 57 | 600 | Lung cancer | 4 |
| 122 | Male | 62 | 720 | Gastric cancer | 2 |
| 123 | Male | 38 | 600 | Renal cell carcinoma | 8 |
Table 2.
Immunoreactive advanced solid cancer patients with BAK immunocell therapy (serum IAP levels ≤580 μg/ml)
| Patient number | Gender | Age | Primary lesion | Metastatic lesion | Performance status (Karnofsky scale) | IAP (μg/ml) |
|---|---|---|---|---|---|---|
| Postoperative cancer patients (stage IV) | ||||||
| Metastatic or recurrent lung cancer | ||||||
| 1 | Male | 66 | Renal cell carcinoma | Lung | 90% | 415 |
| 2 | Male | 45 | Hemangiosarcoma of spleen | Lung, Liver | 80% | 520 |
| 3 | Female | 69 | Renal cell carcinoma | Lung | 100% | 470 |
| 4 | Male | 53 | Colon cancer | Lung | 90% | 280 |
| 5 | Male | 35 | Lung cancer (Adenocarcinoma) | Recurrence | 80% | 580 |
| 6 | Female | 71 | Rectum cancer | Lung | 90% | 340 |
| 7 | Female | 54 | Colon cancer | Liver, lung | 80% | 330 |
| 8 | Female | 56 | Lung cancer (Adenocarcinoma) | Recurrence | 80% | 360 |
| 9 | Male | 55 | Lung cancer (Adenocarcinoma) | recurrence | 80% | 360 |
| 10 | Female | 43 | Renal cell carcinoma | Lung | 80% | 580 |
| 11 | Male | 67 | Lung cancer (Adenocarcinoma) | Brain | 80% | 450 |
| 12 | Male | 67 | Lung cancer (Adenocarcinoma) | Lymph nodes | 100% | 520 |
| 13 | Female | 80 | Lung cancer (Adenocarcinoma) | Recurrence | 90% | 410 |
| 14 | Male | 65 | Esophageal carcinoma | Lung | 100% | 279 |
| Metastatic or recurrent, other cancers | ||||||
| 15 | Female | 50 | Breast cancer | Lymph nodes | 100% | 290 |
| 16 | Female | 62 | Thyroid carcinoma | Neck | 100% | 360 |
| 17 | Female | 43 | Breast cancer | Bone | 80% | 250 |
| 18 | Female | 38 | Breast cancer | Bone | 80% | 350 |
| 19 | Male | 57 | Multiple liver tumor (endocrine cell carcinoma) | - | 90% | 430 |
| 20 | Female | 58 | Breast cancer | Recurrence | 100% | 190 |
| 21 | Female | 52 | Breast cancer | Uterine | 80% | 420 |
| 22 | Male | 63 | Prostatic cancer | Bone | 90% | 200 |
| 23 | Male | 58 | Gastric cancer | Lymph nodes | 90% | 240 |
| 24 | Male | 52 | Colon cancer | Liver | 90% | 370 |
| 25 | Male | 61 | Scirrhous gastric cancer | Colon, Peritoneum | 80% | 130 |
| 26 | Male | 71 | Appendical carcinoma | Abdominal wall | 80% | 280 |
| 27 | Male | 60 | Gastric cancer | Lymph nodes | 90% | 250 |
| 28 | Female | 30 | Ovarial cancer | Lymph nodes | 80% | 410 |
| 29 | Female | 69 | Ovarial cancer | Lymph nodes | 90% | 580 |
| 30 | Female | 51 | Ovarial cancer | Liver, Spleen | 80% | 300 |
| 31 | Male | 71 | Gastric cancer | Colon | 80% | 417 |
| 32 | Female | 51 | Breast cancer | Lymph nodes | 90% | 320 |
| 33 | Female | 71 | Colon cancer | Liver | 80% | 580 |
| 34 | Female | 50 | Breast cancer | Lymph nodes | 100% | 280 |
| 35 | Male | 52 | Penile cancer | Lymph nodes | 80% | 490 |
| 36 | Female | 54 | Tubal cancer | Recurrence | 80% | 570 |
| 37 | Male | 54 | Esophageal cancer | Lymph nodes | 100% | 250 |
| 38 | Female | 47 | Ovarial cancer | Recurrence | 90% | 279 |
| Inoperable lung cancer patients (stage IIIb) | ||||||
| 39 | Male | 62 | Squamous cell carcinoma | Lymph nodes | 100% | 420 |
| 40 | Male | 66 | Adenocarcinoma | Lymph nodes | 80% | 410 |
| 41 | Male | 61 | Adenocarcinoma | Lymph nodes | 80% | 370 |
| 42 | Male | 52 | Squamous cell carcinoma | Lymph nodes | 100% | 431 |
Table 3.
Patient characteristics of two groups
| Characteristic | Immunosuppressed patients (serum IAP Levels >580 μg/ml) | Immunoreactive patients (serum IAP Levels ≦580 μg/ml) |
|---|---|---|
| Number | 23 | 42 |
| Gender | ||
| Male | 12 (52.2%) | 22 (52.4%) |
| Female | 11 (47.8%) | 20 (47.6%) |
| Age (years) | ||
| <60 | 16 (69.6%) | 24 (57.1%) |
| ≥60 | 7 (30.4%) | 18 (42.9%) |
| Stage | ||
| IIIb | 0 | 4 |
| IV | 23 | 38 |
| CD56+ cells (%) in PBL | 19.0±9.93 | 18.4±6.45 |
Serum-free medium for BAK cell preparation
In our previous paper [9], the BAK cells were cultured with HyMedium (Nipro, Tokyo) plus 2% human serum in a 1l bag. However, we developed a new serum-free ALyS medium (Dr Takeshi Sato, Cell Science & Technology Institute, Sendai) containing ascorbate-2-glucose and 4 trace heavy metals [23]. As shown in Table 4, Artificial Lymphocyte Stimulation (ALyS) medium is superior to conventional medium with 2% serum on cell proliferating activity and increased activity for CD56+ cells. Therefore, a minor modification was made for preparation of BAK cells (Table 5).
Table 4.
Cell-proliferating activity of serum-free ALyS medium. Fresh peripheral blood leukocyte (PBL) were cultured with immobilized anti-CD3 antibody and IL-2 for 2 weeks
| Medium | Total cells | CD16+ cells | CD56+ cells |
|---|---|---|---|
| ALyS | 9.5×109/2 bags | 6.7×109 (70.2%) | 6.9×109 (72.2%) |
| HyMedium + 2% serum | 3.2×109/2 bags | 2.0×109 (61.7%) | 2.0×109 (61.1%) |
| (Fresh PBL) | 1.6×107 | (15.3%) | (17.2%) |
Table 5.
Revised BRM activated killer (BAK) cell preparation
| 20 ml of patients' heparinized peripheral blood |
| ↓ |
| Ficoll-Paque centrifugation at 350 g for 25 min |
| ↓ |
| Peripheral blood mononuclear cells (PBMC, 3–5x107) in ALyS medium (CSTI, Sendai) containing 10% auto-serum plus rh IL-2 (700 U/ml) |
| The 225-cm2 culture flask was incubated with anti-CD3 antibody (OKT3, Ortho Diagnostics, 5 μg/ml) overnight at room temperature |
| ↓ |
| 30 ml of ALyS medium was added to the coated flask and cultured in CO2 incubator at 37°C for 2 days |
| ↓ |
| 60 ml of ALyS medium was added and cultured for 2–3 days |
| ↓ |
| Split to 3 flasks and culture nonadherent cells for 1–2 days |
| ↓ |
| Transfer into gas-permeable bag containing 1 l of ALyS medium with 175 U/ml of IL-2 |
| ↓ |
| Culture for 2–3 days and split 2 bags |
| ↓ |
| Culture for 2–3 days and split 4 bags |
| ↓ |
| Sterilization test and endotoxin assay |
| ↓ |
| Reactivation by IL-2 (1,000 U/ml) and IFN-α (1,000 U/ml) for 15 min |
| ↓ |
| Washed 2 times by centrifugation in saline with 0.1% human albumin |
| ↓ |
| Harvest lymphocytes (0.6–1x1010) in transfusion bag containing 200 ml of saline with 2.5% human albumin |
Evaluation of clinical effects of solid cancer therapy
Response to treatment was assessed in terms of the decrease in the total tumor area measured with radiological methods (CT and/or MRI). Complete response (CR) was defined as disappearance of all measurable disease for a minimum of 4 weeks. Partial response (PR) was defined as a 50% or greater reduction in the tumor area for at least 4 weeks. No change (NC) was defined as a decrease of less than 50% or as an increase of less than 25% for at least 4 weeks. Progressive disease (PD) was defined as an increase of 25% or more in the tumor area. We propose a new criterion, prolonged no change (prolonged NC), defined as a less than 50% reduction or less than 25% increase in the tumor area for at least 6 months, to evaluate the effectiveness of therapy without tumor shrinkage. We proposed that all patients meeting criteria CR, PR, or prolonged NC be classified as responding to solid tumor therapy.
Face scale as a QOL marker
During the course of BAK therapy, we adopted the Face scale as a QOL indicator, a brief nonverbal assessment of patient mood [9]. The Face scale contains 10 drawings of faces, arranged in serial order by rows, with each face depicting a different mood. Subtle changes in the eyes, eyebrows, and mouth are used to represent slightly different levels of mood. The faces are arranged in order of worsening mood and numbered from 1 to 10, with 1 representing the most positive mood and 10 representing the most negative mood. As the examiner pointed at the faces, the following instructions were given to each patient: "The faces below range from very happy at the top to very sad at the bottom. Point to the face that best represents the way you felt today."
Results
Comparison of survival of the immunosuppressed high IAP and immunoreactive low IAP groups
As a marker of general immunological state of patients, serum IAP levels were assayed. Comparison of the survival curves in BAK therapy between the high IAP immunosuppressed terminal group (>580 μg/ml) and the low IAP immunoreactive stage IV and IIIb group (≤580 μg/ml) was performed. Overall survival time of patients correlated with the serum IAP levels using the Kaplan-Meier method as shown in Fig. 1. The difference in survival between the groups (4.6 vs 24.7 months) was significant (P<0.01). This shows that BAK therapy is not indicated for a terminal patient whose IAP is over 580 μg/ml, perhaps due to extensive chemotherapy. Other immunological paramenters, such as number of leukocytes, CD4+/CD8+ ratio, and % of CD56+ cells (Table 3), were not different for the two groups.
Fig. 1.
Comparison of the Kaplan-Meier survival curves in the low IAP group (<580 μg/ml, n=42) versus the high IAP group (>580 μg/ml, n=23). The survival rate for patients in the low IAP group was significantly higher than that for patients in the high IAP group (P<0.01)
Prognosis of advanced cancer patients in low IAP group with BAK therapy
Thirty-eight advanced postoperative stage IV patients and 4 inoperable stage IIIb lung cancer patients whose life expectancy was estimated to be a few months and who had refused chemotherapy were included in this study. The patients were infused with about 6×109 BAK cells by intravenous drip infusion, at 1-month intervals at an outpatient clinic. As shown in Table 6, the proportion of CD56+ cells increased from 18.4±6.45% to 41.4±14.1% with culture. No adverse effects were observed during treatment. During the course of BAK therapy, we adopted the Face scale as a QOL indicator. The QOL of all patients either remained satisfactory or improved. The prognosis and clinical responses of all patients are shown in Table 6.
Table 6.
Prognosis of immunoreactive advanced solid cancer patients with BAK therapy
| Patient number | CD56+ cell (%) with culture | Face scale (QOL marker) | Time since BAK therapy started | Course | Response | ||||
|---|---|---|---|---|---|---|---|---|---|
| Before | Change | After | Start | Change | Latest | ||||
| 1 | 23 | ↗ | 57 | 5 | ↗ | 4 | 3 years 3 months | Deceased | Prolonged NC |
| 2 | 25 | ↗ | 61 | 4 | ↗ | 2 | 3 years 1 month | Deceased | Prolonged NC |
| 3 | 22 | ↗ | 71 | 4 | ↗ | 1 | 4 years | Alive | CR |
| 4 | 22 | ↗ | 43 | 7 | → | 7 | 1 year 10 months | Deceased | NC |
| 5 | 30 | ↗ | 39 | 2 | → | 2 | 1 year 6 months | Deceased | NC |
| 6 | 13 | ↗ | 33 | 7 | → | 7 | 1 year 4 months | Deceased | Prolonged NC |
| 7 | 11 | ↗ | 39 | 6 | → | 6 | 1 year 5 months | Deceased | Prolonged NC |
| 8 | 15 | ↗ | 29 | 6 | → | 6 | 1 year 3 months | Deceased | NC |
| 9 | 30 | ↗ | 35 | 7 | ↗ | 6 | 1 year 6 months | Deceased | Prolonged NC |
| 10 | 16 | ↗ | 45 | 4 | → | 4 | 2 years 1 month | Deceased | Prolonged NC |
| 11 | 37 | ↗ | 55 | 3 | ↗ | 2 | 1 year | Deceased | prolonged NC |
| 12 | 18 | ↗ | 38 | 2 | → | 2 | 1 year 4 months | Alive | Prolonged NC |
| 13 | 18 | ↗ | 18 | 2 | → | 2 | 1 year 4 months | Alive | Prolonged NC |
| 14 | 25 | ↗ | 80 | 3 | → | 3 | 1 year | Alive | Prolonged NC |
| 15 | 18 | ↗ | 37 | 6 | ↗ | 3 | 4 years 8 months | Deceased | Prolonged NC |
| 16 | 13 | ↗ | 35 | 4 | → | 4 | 4 years 8 months | Alive | Prolonged NC |
| 17 | 11 | ↗ | 38 | 6 | ↗ | 5 | 3 years | Deceased | Prolonged NC |
| 18 | 10 | ↗ | 26 | 7 | ↗ | 3 | 2 years 7 months | Deceased | Prolonged NC |
| 19 | 11 | ↗ | 52 | 4 | → | 4 | 2 years 2 months | Deceased | PR |
| 20 | 20 | ↗ | 22 | 5 | → | 5 | 1 year 11 months | Deceased | Prolonged NC |
| 21 | 22 | ↗ | 46 | 6 | ↗ | 5 | 1 year 8 months | Deceased | Prolonged NC |
| 22 | 14 | ↗ | 33 | 6 | ↗ | 2 | 3 years | Deceased | Prolonged NC |
| 23 | 16 | ↗ | 28 | 7 | ↗ | 5 | 1 year 4 months | Deceased | NC |
| 24 | 15 | ↗ | 25 | ND | ND | 1 year 7 months | Deceased | Prolonged NC | |
| 25 | 22 | ↗ | 35 | 4 | → | 4 | 1 year 5 months | Deceased | NC |
| 26 | 12 | ↗ | 40 | 4 | ↗ | 3 | 1 year 1 month | Deceased | NC |
| 27 | 14 | ↗ | 36 | 5 | ↗ | 4 | 2 years 7 months | Alive | Prolonged NC |
| 28 | 11 | ↗ | 43 | 4 | → | 4 | 1 year 8 months | Deceased | NC |
| 29 | 24 | ↗ | 41 | 4 | → | 4 | 2 years 5 months | Alive | Prolonged NC |
| 30 | 11 | ↗ | 33 | 6 | → | 6 | 2 years 6 months | Alive | Prolonged NC |
| 31 | 11 | ↗ | 37 | 6 | ↗ | 2 | 2 years 2 months | Alive | Prolonged NC |
| 32 | 20 | ↗ | 46 | 3 | ↗ | 2 | 2 years | Alive | Prolonged NC |
| 33 | 22 | ↗ | 56 | 3 | ↗ | 2 | 1 year | Deceased | NC |
| 34 | 16 | ↗ | 35 | 6 | ↗ | 5 | 1 year 8 months | Alive | Prolonged NC |
| 35 | 29 | ↗ | 58 | 4 | ↗ | 3 | 1 year 8 months | Alive | NC |
| 36 | 21 | ↗ | 42 | 1 | → | 1 | 1 year 8 months | Alive | NC |
| 37 | 14 | ↗ | 31 | 5 | ↗ | 4 | 1 year 5 months | Alive | Prolonged NC |
| 38 | 18 | ↗ | 30 | 1 | → | 1 | 1 year | Alive | CR |
| 39 | 27 | ↗ | 52 | 2 | ↗ | 1 | 4 years 7 months | Alive | CR |
| 40 | 23 | ↗ | 79 | 4 | → | 4 | 2 years 2 months | Alive | Prolonged NC |
| 41 | 11 | ↗ | 22 | 3 | ↗ | 2 | 2 years 1 month | Alive | CR |
| 42 | 14 | ↗ | 40 | 2 | → | 2 | 1 year | Alive | CR |
If we insert the evaluation criterion of prolonged no change (prolonged NC) in tumor size during 6 months or longer that ranges from less than 50% shrinkage to 25% or less increase, between PR and NC, we see even more positive results. Overall response to BAK therapy was CR in 5 cases, PR in 1 case and prolonged NC in 26 cases with an effectiveness rate of 76.2% in 42 advanced stage IIIb and IV cancer patients. This shows that BAK therapy has a life-prolonging effect without any adverse effects for advanced cancer patients.
Discussion
It has been shown that, even with today's medical advances, systemic chemotherapy for patients with advanced stage (inoperable stage IIIb and IV) non–small cell lung cancer prolonged survival only by 5.8 months [5]. Treated with BAK therapy, however, mean survival duration of inoperable stage IIIb and stage IV recurrent and metastatic lung cancer was 23.8 months (n=18). All 4 inoperable stage IIIb lung cancer patients and 4 stage IV recurrent and metastatic lung cancer patients from our study are presently still living (Table 6). This favorable clinical response in lung cancer cases may be the result of the fact that the lungs are first exposed to BAK cells via the bloodstream using intravenous drip infusion.
In our previous study [10], BRM-activated γδ T cells were shown to produce antitumor cytokines (IFN-γ and TNF-α), which are the major cytotoxic cytokines of BAK cells, and we showed that CD56+ cells have stronger cytotoxic activity than CD56− cells. Bauer et al. [3] showed that a stress-inducible MICA (MHC class I chain-related A) protein on tumor cells was recognized by NKG2D, a MICA receptor, found in non–MHC-restricted killer, NK, and γδ T cells. Anti-CD3 antibody and IFN-α induce TRAIL (TNF-related apoptosis-inducing ligand) on NK and γδ T cells and induce apoptosis on tumor cells via death receptor DR4 and DR5 [2]. Kato et al. showed that tumor cells were targeted by human γδ T cells via nonpeptide pyrophosphate [13]. Granulysin, another cytotoxic molecule present in NK cells and CTL, is able to induce apoptosis in tumor cells [11], and its expression in NK cells has been negatively correlated with cancer progression in human patients [14]. Therefore, we assayed granulysin in serum of solid cancer patients with BAK therapy. Responding cases have a tendency to higher granulysin level (over 7.0 ng/ml).
In conventional CTL (cytotoxic T lymphocyte) immunotherapy, potent αβ T (αβ CD8+ killer T) cells, which recognize tumor antigens and MHC class I antigens, are purified and multiplied for therapy. However, αβ T cells kill both cancer cells and normal leukocytes, thus increasing the risk of causing adverse reactions in patients [8]. Therefore, we devised the BAK therapy by utilizing a new class of lymphocytes (CD56+ cells containing NK cells and γδ T cells) that kill cancer cells only, regardless of MHC antigens. CD56 is a member of the Ig superfamily, and CD56+ cells produce β-endorphin, an intracerebral hormone thought to make patients feel well and help to maintain good QOL due to its potent analgesic and sedative effects. CD56+ cells are considered to be neuro-immune-endocrine (NIE) cells, and are potent killer cells that produce antitumor cytokine, IFN-γ and TNF-α [10].
Acknowledgements
The authors wish to express their gratitude to Ms Eiko Ohkubo of the Miyagi Cancer Center Research Institute for editorial help, and to Dr Masataka Suzuki, Dr Ryuichi Katakura, Dr Mitsutaka Okuda, Dr Sadahiro Koinumaru, Dr Takashi Matsuda, Dr Tsuneaki Fujiya, Dr Yoichiro Kakugawa, Dr Junichi Mikuni, and Dr Shigenori Ujiie of Miyagi Cancer Center Hospital for their generous assistance in carrying out the clinical trial. This translational research was supported in part by a grant from the Sendai Institute of Microbiology.
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