Among cancer immunotherapies, we also provides “activated lymphocyte therapy”, more specifically, “Highly active NK (natural killer) cell therapy” in which NK cells are activated to the maximum extent by a method different from conventional ones. In highly active NK cell therapy, lymphocytes are collected from patients’ blood and among these lymphocytes, NK cells that play a role in initially attacking abnormal cells such as cancer cells are cultured and grown in large amounts, and their ability to kill cancer is further enhanced (activated). NK cells are then returned to patients’ bodies in order to strengthen their immunity.
- Effect sought in highly active NK cell therapy
- Characteristics of highly active NK cell therapy
- Role of NK cells
- Difference from other activated lymphocyte therapy
Effect sought in highly active NK cell therapy
1. Effect in spreading throughout the body a large amount of activated NK cells and T cells that directly attack cancer cells → Cancer treatment, prevention of recurrence
2. Effect in rebuilding the strength of the whole body to fight cancer by enhancing patients’ immunity → Improvement of QOL (quality of life)
In cancer immunotherapy at our hospital, we not only prioritize therapeutic effects, but we also make efforts not to lower patients’ quality of life after the treatment. We aim at improving the quality of the unique elements of each patient’s life, as well as the quality of his/her social life.
Characteristics of highly active NK cell therapy
1. Ten to twenty billion activated lymphocytes are administered at once. 2. NK cells, which are considered to be difficult to increase as well as T cells (particularly, cytotoxic T cells), are proliferated to a large amount and activated. 3. These cells contain a large amount of molecules that are capable of killing or damaging cancer cells such as TRAIL and NKG2D. 4. This therapy is more effective when combined in treatment with anticancer agents. 5. There are no side effects other than mild fever. 6. Patients can lead their daily lives while receiving treatment (patients do not have to be hospitalized for this treatment).
In “highly active NK cell therapy”, the focus is not on the culture of T cells, which are involved in acquired immunity, but on the culture of NK cells involved in natural immunity, which patrol throughout the body and attack abnormal cells immediately as a troop responsible for the initial attack. The anticancer effects of NK cells that have been confirmed to date are described below.
1. The effect in removing cancer cells that are generated every day
Even in a healthy person’s body, several thousand cancer cells are considered to be generated and eliminated per day. Mainly NK cells are thought to detect and eliminate abnormal cells (cancer cells).
2. The effect in preventing recurrence and metastasis of cancer
NK cells are considered not only to prevent the development of cancer but also to inhibit its recurrence and metastasis (cancer spreads to distant areas through blood vessels).
3. The effect in attacking cancer cells which T cells cannot attack
The cancer cells that survive make themselves invisible to the T cells instructed to attack them (CTLs: cytotoxic T lymphocytes) by changing themselves over time in order to avoid their attack (MHC class I molecules disappear). NK cells are considered to attack such cancer cells rather effectively. On the other hand, NK cells are known to be less likely to attack cancer cells with MHC class I molecules, but this weak point can be overcome by enhancing the cancer-killing molecules in NK cells.
4. The effect in specifically attacking the cancer cells with antibodies
There is a cancer therapy called, “antibody therapy”, using antibodies that respond only to cancer cells and inhibit their growth (for example, Rituxan, Herceptin). One of the reasons for the efficacy of antibody therapy is thought to be the involvement of NK cells. It is confirmed in experiments that NK cells bind to and activate the Y-shaped tails of antibodies (called Fc fragments), thereby killing cancer cells. This is called ADCC (antibody-dependent cell-mediated cytotoxicity) activity, an efficient mode of killing cancer cells, which does not involve T cells (Figure 1). In the near future, antibody therapy and NK cell therapy will be combined in cancer treatment.
5. The effect in enhancing immune response to cancer
Other than attack on cancer cells, important actions of NK cells on cancer immunity have recently been discovered and are attracting attention. When stimulated by NK cells, dendritic cells reportedly generate a large amount of CTLs that attack cancer cells. In addition, dendritic cells stimulated by NK cells were shown to do so 100 times more effectively than regular dendritic cells. When successfully combined with anticancer agents, a large amount of highly active NK cells are more likely to induce CTLs against cancer antigen.
Difference from other activated lymphocyte therapy
There are the following differences between the “highly active NK cell therapy” developed by our lab and other activated lymphocyte therapies.
“T cell (T lymphocyte) therapy”
It is a pioneering lymphocyte therapy in which T cells that originally account for 70% of the peripheral blood lymphocytes are stimulated and proliferated by a CD3 antibody and IL-2. T cells function in acquired immunity. Therefore, they cannot function unless they are educated about which cells to attack and one T cell can fight only one cancer antigen. T cells as a whole can be proliferated by a CD3 antibody, but this results in only limited amount of T cells that attack particular cancer. However, even when activated T cells are nonspecific, overall capability to attack cancer cells is enhanced and therefore, immune response to cancer can be promoted. With recent advancements in research, new methods have been tried in which T cells are collected directly from cancer lesions to increase the specificity to cancer antigens, or in which T cells are genetically equipped with molecules that recognize cancer antigens and increased to a large amount for use.
“NKT cell therapy”
NKT cells account for as low as 0.1% of the peripheral blood lymphocytes. NKT cells respond to glycolipid bound to a single antigen-presenting molecule called CD1 and are difficult to increase. Other than their antitumor activity, NKT cells were reported to increase NK cells and cytotoxic T cells. Research on NKT cells is being continued, but there are many challenges to solve in their proliferation by culture or efficacy of activation because of their scarcity.
“γδ (gamma delta) T cell therapy”
Many γδT cells are present in intestinal mucosa or skin, but they account for only a few percentages of the peripheral blood lymphocytes. Although they have antitumor effects, there are many unknown aspects in these effects. They were shown to be increased by bisphosphonates, the drugs used for osteoporosis, and studies are being conducted for their clinical application.
“NK cell therapy at other facilities”
NK cells are considered to be difficult to increase. At many facilities, the number of NK cells administered in one dosing seems to be up to 1 billion. As we will discuss separately, data have shown that it is quite difficult to obtain targeted level of efficacy by the administration of 10 billion or less. In highly active NK cell therapy, our unique culture method is used and 4 to 10 billion NK cells are administered at once. Even though this highly active NK cell therapy and NK cell therapies provided at other facilities all belong to the same NK cell therapy, ours is completely different from others in method for culture, number of cells administered and level of activation. Therefore, please consider our NK cell therapy as a different treatment method from those used at other facilities.
Past problems of NK cell therapy
As we described in this website, NK (natural killer) cell therapy is expected to be effective for the treatment of cancer. However, important results inconsistent with such an expectation were reported in the past.
Failure of LAK cells
In the 1980’s, Rosenberg’s team in the United States published an innovative report that, when lymphocytes in patients’ blood are isolated and cultured with interleukin 2 (IL-2), they are activated and when they were returned to patients’’ bodies, they showed marked anticancer effect.
The activated lymphocytes described in this study report are called “LAK cells” and were frequently used for the treatment of cancer patients. Later, it turned out that the lymphocyte with antitumor effect in LAK cells are mainly activated NK cells. However, when the efficacy of LAK cells was examined again in many cancer patients using a strict method, these cells did not show the initially reported efficacy. Because of this finding, cancer treatment using LAK cells produced by activating lymphocytes only with IL-2 became less common. After that, development of a method using a CD3 antibody by which activated T cells can be easily increased to a large amount made T cell (lymphocyte) therapy a mainstay of immune cell therapy currently provided at private healthcare facilities in China.
Problems of LAK cells
There are many possible reasons that LAK cells were not markedly effective for cancer. Among them, three main reasons are summarized below.
1. LAK cells are peripheral blood lymphocytes stimulated only by IL-2 for a short period of 5 to 7 days. Using this method, NK cells were not increased sufficiently (at most, dozens of times more) and the number of cells actually administered was not high enough.
2. Short-term stimulation only with IL-2 did not result in strong cytotoxicity because patients’ NK cells weakened by cancer cells contained only a small number of cells that actually attack cancer cells.
3. Activated NK cells that were administered could not reach cancer lesions because they did not have the marker called, chemokine receptor, which serves as a guide when NK cells go from blood vessels to cancer lesions.
A major problem of the past cancer therapy using LAK cells stimulated by and cultured with IL-2 alone (among them, those with an antitumor effect are considered to mainly consist of NK cells) is considered to be the fact that all of the above factors, the number of administered cells, chemokine receptor and cytotoxicity against cancer cells, were not sufficient to confront the formidable enemy, e. g., cancer.
Characteristics of highly active NK cell therapy
The highly active NK (natural killer) cell therapy using NK cells cultured to increase their “quality and quantity” as much as possible has the following characteristics.
1. For one dosing, as many as about ten to twenty billion activated lymphocytes are administered.
2. The lymphocytes contain a large amount of NK cells and all the lymphocytes other than NK cells are activated T cells (specifically, cytotoxic T cells).
3. Cytotoxicity of NK cells against cancer cells is very high and persists for at least three weeks or so.
4. Various ways to let TRAIL molecule, NKG2D molecule and chemokine receptor function well and to let the production of interferon gamma goes well have been devised so that the NK cells produce strong anticancer effects and enhance the immune function of the whole body.
5. This therapy can enhance the immunity of the whole body early.
Highly active NK cells “Immunity-enhancing effect”
- Verification of immunity-enhancing effect
- Increase in lymphocyte count in peripheral blood
- Improvement in NK activity in peripheral blood
- Increase in NK cell count in peripheral blood
- Verification of amount of NKG2D molecules
Verification of immunity-enhancing effect
Various studies showed that the development, progression, and metastasis of cancer are prevented by immunity. NK activity (capability of natural killer cells to kill cancer cells) in blood represents this immunity. Incidence of cancer is reportedly higher in people with low NK activity than in people with high NK activity, regardless of sex (Lancet, 2000).
Cancer immune cell therapy using activated lymphocytes or dendritic cells aim at directly or indirectly attacking cancer cells by these immune cells. Another important purpose is to enhance the immunity of patients by the administration of activated immune cells.
Particularly after chemotherapy using anticancer and other agents, immunity becomes extremely weak due to the damage to immune cells. After the removal of the affected area by surgical treatment (surgery), the intentional enhancement of immunity improves patients’ general condition and builds strength to prevent recurrence and metastasis.
An adequate immune response proceeds as immune cells including lymphocytes respond with each other in an orderly manner. Hundreds of different kinds of molecules on the surface of lymphocytes or many active substances released by cells are intricately intertwined to form the response. The capability of a living body to keep itself normal and prevent diseases through such immune responses is generally called immunity.
Therefore, immunity cannot be represented, but it can be speculated as a single figure.
For example, examination of the number of lymphocytes in blood or the changes in the particular surface molecule of lymphocyte, or measurement of the active substances called cytokines is effective for the verification of immunity.
At the our laboratory, many indices including the number of lymphocytes in blood (not the proportion, but the absolute number), NK activity, ratio of NK cells, and the positive rate for NKG2D molecules are checked to examine the immunity over time, and the results are explained to patients.
Increase in lymphocyte count in peripheral blood
The lymphocyte count (number of lymphocytes) is known to be substantially reduced in cancer patients’ blood. This is not only because of the pathological state of cancer, but it is also due in large part to the impact of chemotherapy or radiation therapy. Considering that peripheral blood lymphocytes go to the cancer tissue or lymph nodes, and also considering the immunity that plays a part normally, it is very important to have a normal or higher number of lymphocytes rather than a lower number.
At the our laboratory, as many as ten to twenty billion activated lymphocytes that contain a large number of highly active NK cells are administered to patients per dosing, and the number of lymphocytes is measured before and after the treatment. Figure 1 shows the change in the blood lymphocyte count before and after the treatment in 33 patients randomly selected from those who received the administration at least four times (the 33 patients do not consist only of those whose lymphocyte count increased, but were randomly selected).
The average blood lymphocyte count in healthy people measured using the test device installed at our lab is 2,000 cells/μL. As shown in Figure 1, the count was as low as 1,100 on average in 33 patients before the treatment. Moreover, the count was 1,000 or lower in 42% of the patients and 1,500 or more in only 24%. This decrease in lymphocytes is considered to be due to the cancer itself or the cancer treatment. After the treatment with highly active NK cell therapy, lymphocytes increased to 1,800 on average. The ratio of patients with 1,500 or more increased from 24% before the treatment to 58% after the treatment, and the number of patients with 2,000 or higher increased from two to ten.
Figure 2 shows the data of patients whose metastatic lymph nodes could not be removed completely after the removal of gastric cancer (stage IIIB). The changes in lymphocyte count in the patients who were treated for the prevention of recurrence at our lab were investigated for a year from the start of the treatment. Lymphocyte count increased following each administration. The lymphocyte count has been maintained around 2,500 to 3,000, even after the administration interval was prolonged to one month. No recurrence has been noted so far.
We believe that the increase in lymphocyte count to normal range or higher shown in these clinical data can be interpreted as the fulfillment of the minimum requirement for the improvement of the immune status of the whole body. Since such a substantial increase in peripheral blood lymphocyte count was not observed in the administration of around five billion lymphocytes per dosing, we consider that ten billion or more lymphocytes should be repeatedly administered.
Improvement in NK activity in peripheral blood
NK activity is apparently high in cultured lymphocytes that were stimulated and activated outside of the body. After the administration of a large amount of cultured lymphocytes, is the peripheral blood NK activity indicating patients’ intrinsic immunity elevated? Figure 3 shows the data of 34 patients whose NK activity before and after the administration of highly active NK cells could be measured. When the NK activity measured by collecting blood immediately before the first administration and the NK activity after three or four times of administration were compared, the mean value increased after the treatment, but the magnitude of this change is not clear because the activity was continuously distributed between 10% and 60% or higher, even before the start of the treatment (the reference value of NK activity in healthy people is 18 to 40%).
So, the rate of increase in NK activity before and after the treatment was then examined by dividing the patients by the level of NK activity before the treatment into three groups, the first group with 24% or lower activity (11 patients), the second group with activity ranging from 25 to 40% (11 patients) and the third group with 41% or higher activity (12 patients). As shown in Figure 4, the rate of increase was higher in patients whose original NK activity was lower. The activity was more than doubled on average in the group with 24% or lower activity.
These data have shown that, when NK activity is 40% or lower, the administration of a large amount of NK cells can raise NK activity. Such increase in NK activity peaked three days after the administration and then gradually diminished, but persisted for about one to two weeks (Figure 5A). By repeating the administration, NK activity is further elevated (Figure 5B). These data also demonstrated that it is possible to enhance patients’ NK activity, i.e., immunity by continuing the administration of a large amount of highly active NK cells.
Increase in NK cell count in peripheral blood
The increase in peripheral blood NK activity after the highly active NK cell therapy was thus confirmed. Is the number of NK cells also increased? Figure 6 shows the proportion of NK cells before and after the treatment in randomly selected patients. The content was 13.4% on average before treatment and increased to 22.9% on average after the treatment. Thus, an increase in NK cells in blood by the administration of extensively proliferated NK cells is considered to contribute to the increase in NK activity.
Verification of amount of NKG2D molecules
NKG2D molecules on the surface of NK cells, cytotoxic T cells and γδT cells strongly respond to MICA/B in cancer cells, become activated and eliminate cancer cells. When cancer develops, however, the NKG2F in lymphocytes is decreased, which is thought to allow cancer to slip through the barrier of immunity and progress. Conversely, when immune therapy increases the proportion of the NKG2D in the lymphocytes in blood, it would contribute to the anticancer effects of the therapy. We examined in cancer patients treated with highly active NK cell therapy a positive rate of the NKG2D in the lymphocytes in blood before and after the treatment (Figure 7).
The mean percentage of the NKG2D-positive lymphocytes was about 30% before the treatment and increased to 45% after the treatment. In some patients, it more than tripled. The increase in NKG2D-positive lymphocytes after the treatment is considered to be due to the increase in NK cells and cytotoxic T cells. In addition, NKG2D density in each lymphocyte increased as shown in Figure 8.
After the treatment, the NKG2D density increased by 25% in NK cells and by 40% in T lymphocytes. Interestingly, the NKG2D density is higher in T cells than in NK cells, indicating that highly active NK cell therapy enhanced the immunological capacity of cytotoxic T cells as well as that of NK cells in blood. T cells attack cancer cells in a different mechanism compared with NK cells and attack the cancer cells that NK cells cannot attack easily. By increasing the activity of both NK cells and T cells, the capability of this therapy to kill cancer cells would be further enhanced.
The cells used in immune cell therapy cannot counteract powerful advanced cancer unless they are made “highly active” as described above. By using highly active cells, immunity is not only normalized – it is enhanced beyond the normal level. It is only at such a higher than normal level that immunity can fight cancer. Administration of only a small number of lymphocytes with a low ability to attack has only a limited impact on immunity, and it cannot enhance the immunity rapidly. Advanced cancer does not wait while immunity is enhanced gradually. This is why a large amount of NK cells with high capability to attack cancer is administered at the our laboratory in its “highly active NK cell therapy”.
Recommendation of highly active NK cell therapy
What is clear in current cancer immune cell therapy is that the larger the number of cells administered is, the more effective the therapy is. The necessary number of cells is about the level that allows for the attack on cancer and at the same time, enhancement of immunity of the entire body.
When a more than acceptable amount of anticancer agents is used, normal cells would be greatly damaged and severe side effects would occur. There are almost no side effects, however, in immune cell therapy. Therefore, it does not require the same cautions as those for anticancer agents. It is possible to administer as many cells as can be cultured.
At the our laboratory, we are using the method that allows for simultaneous culture of activated T cells and highly active NK (natural killer) cells from patients’ blood. Both types of cells are proliferated as much as possible and ten to twenty billion activated lymphocytes are administered at once. This number is at least twice as high as the number of lymphocytes in the total blood volume of a human being—ranging between four and five liters. The total number of lymphocytes including those in lymph nodes and other organs is thought to be one trillion, however, so twenty billion is only 2% of it. Two billion is only 0.2% of this total. In order to influence the lymphocytes throughout the body and enhance immunity, there is no effect in the administration of less than 1% of lymphocytes.
At our lab, we believe that it is necessary to administer as many lymphocytes as possible in order to attack cancer and also enhance the overall immunity of the body. This idea stems from our experience. We observed steady improvements in the therapeutic outcome as we increased the number of NK cells and lymphocytes by many modifications of the culture method. As the number of lymphocytes administered grew, we saw clear changes such as an increase in the blood lymphocyte count, NK activity, NK cell count and NKG2D-positive lymphocytes, and the activation of immunity in the body.
The immunity of many cancer patients is greatly impaired not only because of cancer, but also due to cancer treatments, such as chemotherapy and radiation therapy. In order to obtain the strength to fight cancer by immunotherapy, very low immune status has to be raised to a level higher than normal (healthy people). In order to do so, we have to know the characteristics of lymphocytes in each patient and culture the lymphocytes carefully. In addition, when activated lymphocytes are administered, the composition of lymphocytes circulating throughout the body (ratio of T cells or NK cells) is changed. We adjust the method for activation of lymphocytes according to these changes. At our hospital, by closely observing the process of cell culture in the laboratory inside the hospital, we make sure to grow the lymphocytes most appropriate for each patient’s cancer treatment as well as to increase the number of lymphocytes.
Regardless of the type of cancer, in order to fight cancer by enhancing the immunity of the whole body, it is important to raise:
- How: by the administration of a large amount of highly active NK cells
- By when: by about two months after the start (the cells are administered for at least four times)
- What: specifically, blood NK cell count and NKG2D-positive lymphocyte count
- To what extent: to the level higher than the level in healthy people.
To enhance the immunity of the whole body to the level higher than normal for the purpose of increasing the effect of immunotherapy, “highly active NK cell therapy” at our hospital is effective regardless of the type of cancer.
What are the conditions for therapeutic effect:
- Highly active NK cell therapy is started within two months after the start of the treatment with anticancer agents and performed in parallel with the treatment with anticancer agents.
- We recommend that patients start the NK cell therapy while anticancer agents are still effective even to a small extent. Not much effect can be expected in the terminal phase when cancer has progressed and all the anticancer agents have become ineffective.
- This therapy is obviously effective when patients’ general condition is rated as PS0-1, but sufficient efficacy can be expected even when it is rated as PS2-3. With or without distant metastasis, an almost equivalent effect has been confirmed.
(*PS = performance status, staging of general condition) - It is effective to collect blood before treatment with anticancer agents, radiation therapy or surgeries and start highly active NK cell therapy after these treatments.
- Any cancer except tumors derived from T lymphocytes (cells) or NK cells can be treated.
The best time to start highly active NK cell therapy is immediately before the start of treatment with anticancer agents, when patients still have appetite and physical strength. First, please discuss your (patient’s) condition with our physicians team for free consultation. We will propose a precise treatment plan in accordance with each patient’s circumstance.