Introduction to cancer immunotherapy using highly active NK cells

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Highly active NK cells “Immunity-enhancing effect”

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 New City Osaki Clinic, 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.

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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 New City Osaki Clinic, 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).

Figure 1

The average blood lymphocyte count in healthy people measured using the test device installed at our clinic 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 clinic 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.

Figure 2

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.

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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%).

Figure 3

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.

Figure 4

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.

Figure 5

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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.

Figure 6

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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).

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.

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 New City Osaki Clinic in its “highly active NK cell therapy”.

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Cancer immunotherapy

Immune system | Cancer immunotherapy | Highly active NK cell therapy | Problems of highly active NK cells | Characteristics of highly active NK cells | “immunity-enhancing effect” of highly active NK cells | Recommendation of highly active NK cell therapy