KR20160134989A

KR20160134989A - Pharmaceutical compositions containing human active protein

Info

Publication number: KR20160134989A
Application number: KR1020150067408A
Authority: KR
Inventors: 한만덕; 김용현; 한국일; 정의길; 우구; 백영준; 이성희
Original assignee: 순천향대학교 산학협력단; (주)한국비엠아이
Priority date: 2015-05-14
Filing date: 2015-05-14
Publication date: 2016-11-24

Abstract

The present invention relates to an anticancer adjuvant composition, or a pharmaceutical composition for treating or preventing lymphocytopenia by comprising human intereukin-2 protein. The composition can increase the number of reduced lymphocytes and prevent reduction in lymphocytes, significantly increase differentiation and proliferation of helper T cell (CD4 T cell), cytotoxic T cell (CD8 T cell) and natural killer cell (NK cell), and lower danger of opportunistic infection and especially, make a favorable prognosis of anticancer treatment.

Description

[0001] PHARMACEUTICAL COMPOSITIONS CONTAINING HUMAN ACTIVE PROTEIN [0002]

The present invention relates to a pharmaceutical composition for adjuvant therapy comprising a human interleukin-2 protein and a pharmaceutical composition for the treatment or prevention of lymphopenia.

Lymphocytopenia refers to cases where the number of total lymphocyte sites in the blood is less than 1,000 / mm ^{3 for} adults and less than 3,000 / mm ³ for children less than 2 years of age. These lymphocytopenia, together with neutropenia, cause a decrease in immunity, resulting in opportunistic infection, health deterioration or autoimmune disease. Therefore, if lymphocytopenia occurs, the patient should be treated for immunodeficiency disease or lymphocyte subtype analysis, and the cause should be prescribed.

The number of lymphocytes in the blood of normal persons is 1,000 to 4,800 / mm ³ (μl) for adults and 3,000 to 9,500 / mm ³ for children under two years of age. Lymphocytes, which account for 20 to 40% of total white blood cells, are composed of 75% T lymphocytes (T cells) and 25% B lymphocytes (B cells). Therefore, if there is no difference in the above ratio when the number of cells is examined, it is difficult to determine whether or not the lymphocyte is decreased. 65% of blood T cells CD4 + (Helper T cell) as a most lymphopenia patient represents a T cell, particularly the reduction of CD4 + cells (note; the range of normal human CD4 + cells from 300 to 1,300 / mm ^3, mean 1000 Cells / mm ³ , and the range of CD8 + cells is 100 to 900 cells / mm ³ , an average of 600 cells / mm ³ ).

The occurrence of lymphocytopenia is inhibited by the production of lymphocytes, is destroyed faster than the production, or is caused by the loss of lymphocytes, the cause of which is largely classified as acquired or inherited.

The causes of lymphocytopenia include AIDS, hepatitis, influenza, TB, typhoid fever and sepsis, nutritional deficiencies due to various causes (alcoholism, protein energy deficiency, zinc deficiency, etc.) There are a number of diseases, such as cytotoxic chemotherapy, glucocorticoids (steroid hormones), high-dose psoralen and UV-A, radiation therapy, immunosuppressant medications, and autoimmune diseases such as aplastic anemia, hodgkin lymphoma, myasthenia gravis and renal fail. In addition, surgery and various causes of trauma cause lymphocytopenia. Among them, protein energy deficiency is the most common cause of AIDS, thymus (Thymus) or damage caused by lymphoid organs.

Genetic causes of lymphocytopenia include aplasia lymphopoietic stem cells, ataxia-telangiectasia (capillary dilatation dysfunction), cartilage-hair hyplasia, idiopathic CD4 + T lymphocytopenia, and immunodeficiency with thymoma.

Symptoms of lymphocytopenia include loss of tonsils or lymph nodes, cellular immune deficiency, abnormal skin disease, alopecia, eczema, and scurvy. In addition, lymphocyte deficiency patients have recurrent infections and special pathogen infections, and are especially risk factors for autoimmune diseases and cancer.

The diagnosis of lymphocytopenia is based on the clinical suspicion of immunodeficiency such as virus, fungi, parasitic organisms, etc., abnormal distribution of blood cells, direct lymphocyte subsets, immunoglobulin This is done by measuring the quantity.

In patients with lymphopenia, even if the ratio of lymphocytes is in the usual category, the same symptoms as those of immune deficiency are shown, so it is desirable to carry out an experimental test to confirm the immunodeficiency. Patients who are at risk for lymphocytopenia include AIDS or infected patients, autoimmune patients, hematologic malignancies, hematologic malignancies, and genetic factors. Among those treated with steroids, Phenia belongs to high risk group.

In Korea, in order to enhance the body's immune function after chemotherapy, the extract of Phellinus linteus is prescribed as an anti-malignant tumor agent, and the fundamental purpose of this prescription is to increase the expression of interleukin or interferon in the body. That is, the interleukin and interferon in the body after the chemotherapy activates the immune function according to each functional group to improve the prognosis of the patient, thereby preventing the recurrence of cancer and inhibiting the cancer progression.

Korean Patent No. 10-1041986

It is an object of the present invention to provide an anticancer-aid pharmaceutical composition containing human interleukin-2 protein and a pharmaceutical composition for the treatment or prevention of lymphopenia.

In order to achieve the above object, the pharmaceutical composition for anti-cancer adjuvant according to one embodiment of the present invention comprises a human interleukin-2 protein as an active ingredient to alleviate or treat lymphocytopenia caused by an anticancer agent or radiation therapy.

The anticancer agent may be an alkylating agent, an antimetabolic agent, or a combination thereof, and the radiation therapy may be pelvic radiation therapy.

The anticancer agent is an alkylating agent such as cisplatin, carboplatin, dacarbazine, procarbazine, busulfan, thiotepa, cyclophosphamide, , Or a combination thereof as an active ingredient.

The anticancer agent may be an antimetabolite containing a fluorinated pyrimidine nucleoside derivative as an active ingredient.

The fluorinated pyrimidine nucleoside derivative may be one containing 5-fluorouracil, cytarabine, capecitabin or a combination thereof.

The human interleukin-2 protein is a recombinant human interleukin-2 protein having a modification in which alanine as the first amino acid in the 5 'terminal base sequence is removed from the human interleukin-2 protein and the 125th amino acid cysteine is substituted with serine .

The human interleukin-2 protein may be a recombinant human interleukin-2 protein having the amino acid sequence of SEQ ID NO: 1.

The cancer to which the above chemotherapeutic pharmaceutical composition is applied is a solid cancer and the solid cancer is cancer of the uterus, cervix cancer, ovarian cancer, breast cancer, colon cancer, stomach cancer, liver cancer, colon cancer, rectal cancer, esophagus cancer, pancreatic cancer, lung cancer, Bladder cancer, prostate cancer, head and neck cancer, or testicular cancer.

The anticancer adjuvant pharmaceutical composition may be administered before the anticancer agent or the radiation therapy, or simultaneously with the anticancer agent or the radiation therapy.

The pharmaceutical composition for treating or preventing lymphocytopenia according to another embodiment of the present invention includes human interleukin-2 protein as an active ingredient to alleviate or treat lymphocyte reduction by an anticancer agent or radiation therapy.

Hereinafter, the present invention will be described in more detail.

The anticancer adjuvant pharmaceutical composition according to an embodiment of the present invention includes human interleukin-2 protein as an active ingredient to alleviate or treat lymphocytopenia caused by an anticancer agent or radiation therapy.

The pharmaceutical composition has the effect of preventing or treating lymphocyte reduction and alleviating individual or overall reduction of T, B lymphocytes and NK cells caused by lymphocyte reduction. Accordingly, when the above pharmaceutical composition is administered, it is an adjuvant to chemotherapy, chemotherapy, surgery, radiotherapy, or a combination thereof, which is applied for the purpose of reducing opportunistic infections and improving recurrence rate and survival rate, It is highly utilized.

The radiation therapy may be applied to radiation therapy applied to chemotherapy, but the effect of the chemotherapy-assisted drug composition may be excellent especially when applied to pelvic radiation therapy. In the case of pelvic topical treatment, for example, it is an anti-cancer therapy applied to cervical cancer, cervical cancer, ovarian cancer and the like. When the anti-cancer adjuvant pharmaceutical composition of the present invention is applied together with chemotherapy, Reduction in chance infection rate, recurrence rate, and survival rate.

The anticancer agent may be an alkylating agent, an antimetabolite, or a combination thereof.

The alkylating agent may be selected from the group consisting of cisplatin, carboplatin, dacarbazine, procarbazine, busulfan, thiotepa, cyclophosphamide, or A combination of these may be contained as an active ingredient, and preferably, cisplatin or carboplatin may be applied.

When the anticancer adjuvant pharmaceutical composition of the present invention is applied together with the above alkylating agent, it is possible to reduce the chance of lymphocytopenia caused by the anticancer agent or the condition of the patient, and to decrease the chance infection rate, recurrence rate and survival rate have.

The antimetabolite may include a fluorinated pyrimidine nucleoside derivative as an active ingredient. The fluorinated pyrimidine nucleotide derivative has been widely applied because of its excellent anticancer effect. However, as it has been confirmed in animal studies, the adverse effect of the fluorinated pyrimidine nucleotide derivative is remarkable in the lymphocyte reduction phenomenon after administration of the drug. , Recurrence rate, and survival rate, the anti-cancer adjuvant pharmaceutical composition of the present invention may be administered before or simultaneously with the administration of the anti-cancer drug to alleviate or treat lymphocytopenia and assist in chemotherapy.

The fluorinated pyrimidine nucleoside derivative may be 5-fluorouracil, cytarabine, capecitabin, or a combination thereof.

The cancer to which the above chemotherapeutic pharmaceutical composition is applied may be applied to solid tumors such as cervical cancer, cervical cancer, ovarian cancer, breast cancer, colon cancer, gastric cancer, liver cancer, colon cancer, rectal cancer, esophageal cancer, pancreatic cancer, lung cancer, Laryngeal cancer, bladder cancer, prostate cancer, head and neck cancer, or testicular cancer.

The human interleukin-2 protein is a recombinant human interleukin-2 protein in which the alanine 1 amino acid in the 5'-terminal base sequence of the human interleukin-2 protein is removed (substituted with methionine) and the 125th amino acid cysteine is substituted with serine Human interleukin-2 protein as an active ingredient. In addition to the above modifications, the recombinant human interleukin-2 protein (rhIL-2) has a silent mutation that transforms the gene sequence "GGAGC" into "GGAAC" without changing the base sequence of the 15th amino acid of the IL- The branch may be derived from a structural gene.

The recombinant human interleukin-2 protein may have the amino acid sequence shown in FIG. 11 or the amino acid sequence shown in SEQ ID NO: 1. Specifically, there are three cysteins in the human IL-2 protein. Cysteine, which is the 125th amino acid in FIG. 12 or SEQ ID NO: 2, does not participate in the formation of the secondary structure and therefore does not affect the activity of IL-2 In order to prevent secondary IL-2 protein from forming during the purification process after IL-2 expression in Escherichia coli, undesired disulfide bonds may be formed during the oxidation and reduction processes. Therefore, serine- . For this purpose, point mutation modification is performed so that the 125th amino acid site is substituted with serine in cysteine. The nucleotide sequence of the 15th amino acid, which is the initiation codon of the human IL-2 structural gene (SEQ ID NO: 2) and is the initiation codon and has the methionine amino acid sequence "ATG" , And this sequence has a ribosome binding site (RBS, Ribosome Binding Site). This results in fragmented proteins in which the expressed IL-2 protein is cleaved 22 amino acids at the 5'-end of the 5'-end, and these fragmented proteins are not well removed during the purification process, so that the purification yield may be very low for its removal. Thus, a silent mutation in which the nucleotide sequence of the 15th amino acid of the IL-2 structural gene is not changed and the gene sequence "GGAGC" is changed to "GGAAC" (SEQ ID NO: 3) is induced and the sequence shown in FIG. A recombinant human interleukin-2 protein having the sequence of SEQ ID NO: 1 can be obtained.

The recombinant human interleukin-2 protein was transformed with the recombinant E. coli pBMIL2 (manufactured by Korean Patent No. 10-1041986 (manufactured by Korea Biomedical Co., Ltd., unconventional mass production of human interleukin-2) No. KCTC11284BP). The contents of production and refining are cited and omitted from the above patent.

The administration of the pharmaceutical composition may be carried out by a method generally accepted as a method of administering a pharmaceutical agent having the above-mentioned pharmacological activity, and specifically, a method of administering it by oral, parenteral or local administration, Can be done. The pharmaceutical composition may be administered to a patient by way of intravenous injection in a pharmaceutically acceptable carrier. The pharmaceutical composition may also be formulated as a pharmaceutical composition suitable for parenteral administration to a patient, for example, intravenous, intraarterial, spinal or intraperitoneal administration.

The pharmaceutical compositions may preferably be used in the form of solid, semi-solid or liquid preparations such as tablets, capsules, powders, liquids, suspensions and the like, in unit dosage form suitable for administration in precise dosages. The pharmaceutical composition includes conventional pharmaceutical carriers or excipients, and may further include other pharmaceutical preparations, pharmaceutical preparations, carriers, additives, and the like. Such excipients may, for example, include but are not limited to human serum albumin or other proteins such as plasma proteins.

Preferably, the anti-cancer adjuvant pharmaceutical composition may be administered before the chemotherapeutic agent or the radiotherapy, or concurrently with the chemotherapeutic agent or the radiotherapy.

The pharmaceutical composition or formulation thereof to be administered contains an amount of the active ingredient in an amount effective to achieve a desired effect in the subject being treated and the amount of the active ingredient to be administered to the subject depends on the characteristics of the subject, The manner of administration, the judgment of the physician, etc., but it is preferable to administer the protein at a relatively low concentration.

For example, when the pharmaceutical composition is administered intravenously, the amount of active ingredient per person (based on 50 kg) calculated on the basis of an animal rat is 1 x 10 ⁶ IU / person to 5 x 10 ⁶ IU / person And can be 1.6 x 10 ⁶ IU / person to 3.5 x 10 ⁶ IU / person.

The anticancer agent may be an alkylating agent, an antimetabolite, or a combination thereof. The radiation therapy may be pelvic radiation therapy, and the contents of each anticancer agent and radiation therapy are the same as those described above.

The human interleukin-2 protein comprises a recombinant human interleukin-2 protein having a modification in which alanine as the first amino acid in the 5'-terminal base sequence of the human interleukin-2 protein is removed and the 125th amino acid cysteine is substituted with serine And the detailed description of this recombinant human interleukin-2 protein is the same as that described above, so that the description thereof will be omitted.

The recombinant human interleukin-2 protein is useful as a pharmaceutical composition for the treatment or prevention of lymphopenia. The pharmaceutical composition may be applied for the purpose of alleviating lymphocytopenia and may reduce the serum concentration of IL-6, a cytokine that inhibits lymphocyte function and replication at the initial stage of administration.

In addition, the administration of the pharmaceutical composition containing the protein can increase lymphocyte and eosinophil in the blood, and promote clonal proliferation of T-lymphocytes and T helper lymphocytes. Furthermore, it may induce a decrease in circulating dendritic cells (mature dendritic cells, CD 11c), a decrease in blood IL-12 levels and an increase in angiogenic factor (VEGF).

In particular, the pharmaceutical composition can induce a significant increase in the number of lymphocytes by about 90% compared to a control group administered with saline, without clinical abnormal behavior or death in an animal rat . In addition, the pharmaceutical composition may also result in a significant increase in neutrophils in the blood cells. In the case of lymphocyte subtypes, the pharmaceutical composition also increases the helper T cell (CD4 T cell), cytotoxic T cell (CD8 T cell) and natural killer (NK cell) It can be increased as much as possible.

The lymphocytopenia can be applied without limitations, for example, due to inherited genetic causes or damage due to acquired infections. For example, the lymphocyte is rapidly reduced by chemotherapy or radiation therapy applied in the treatment of diseases It can be applied to lymphocytopenia, which is not recovered immediately.

The anti-cancer pharmaceutical composition and the pharmaceutical composition for the treatment or prevention of lymphocytopenia of the present invention can increase the number of lymphocytes reduced or prevent the decrease of lymphocytes by an anticancer agent or chemotherapy, and can prevent helper T cell (CD4 T cell), cytotoxic T cells (CD8 T cells) and natural killer cells (NK cells), which may lower the risk of opportunistic infections and improve the prognosis in chemotherapy .

FIG. 1 shows the results of confirming the immunological characteristics of the rhIL-2 protein in Example 2 of the present invention.
FIG. 2 is a diagram showing administration design in which a rhIL-2 protein and an anticancer agent (5-FU) are administered to a laboratory rat and a blood sample is collected in Example 3 of the present invention.
Figure 3 is a graph showing the results of experimental rat blood neutrophils in 5) of Example 4 of the present invention (* p <0.05, Dunnett? S t -test).
Figure 4 is a graph showing the results of experimental rat blood lymphocytes in 5) of Example 4 of the present invention (* p <0.05, Dunnett? S t -test).
5 is a flow cytometric analysis result of CD4 T cells among hemocytes in the blood of rhIL-2 and the test group of 14 days after completion of administration of anticancer drug in Example 6 of Example 4 {A; Saline (0.5 mL), B; IL-2 (2.4 x 10 ⁴ IU / kg), C; IL-2 (1.2 x 10 ⁴ IU / kg), D; IL-2 (0.6 x 10 ⁴ IU / kg).
6 is a flow cytometric analysis of CD8 T cells among hemocytes in the blood of rhIL-2 and the test group on the 14th day after completion of administration of the anticancer drug in Example 4 (6) of the present invention {A; Saline (0.5 mL), B; IL-2 (2.4 x 10 ⁴ IU / kg), C; IL-2 (1.2 x 10 ⁴ IU / kg), D; IL-2 (0.6 x 10 ⁴ IU / kg).
FIG. 7 is a graph showing the results of flow cytometry of CD4 / CD8 T cells among hemocytes in the blood of rhIL-2 and the test group on the 14th day after completion of administration of the anti-cancer drug in Example 4 (6) of the present invention (* p < ** p < 0.01).
8 is a flow cytometric analysis result of NK cells among hemocytes in the blood of rhIL-2 and the test group on the 14th day after completion of the administration of the anticancer drug in Example 4 (6) of the present invention {A; Saline (0.5 mL), B; IL-2 (2.4 x 10 ⁴ IU / kg), C; IL-2 (1.2 x 10 ⁴ IU / kg), D; IL-2 (0.6 x 10 ⁴ IU / kg).
9 is a flow cytometric analysis result of CD45R B cell among hemocytes in the blood of rhIL-2 and the test group on the 14th day after completion of the administration of anticancer drug in Example 6 of Example 4 {A; Saline (0.5 mL), B; IL-2 (2.4 x 10 ⁴ IU / kg), C; IL-2 (1.2 x 10 ⁴ IU / kg), D; IL-2 (0.6 x 10 ⁴ IU / kg).
10 is a graph showing the results of flow cytometry of CD45R B cells and NK cells in hemocytes in the blood of rhIL-2 and the test group of 14 days after completion of administration of anticancer drug in Example 4 (6) of the present invention (* p <0.05 , ** p < 0.01).
11 is a view showing the amino acid sequence of the recombinant interleukin-2 protein of SEQ ID NO: 1 of the present invention.
12 is a diagram showing the amino acid sequence of the human interleukin-2 protein of SEQ ID NO: 2 of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Example 1: Preparation of rhIL-2

(Recombinant human interleukin-2 (rhIL-2)) using a strain (Accession No. KCTC11284BP) prepared by the method described in Patent Registration No. 10-1041986 (novel strain for mass production of human interleukin- ). The production of the strain and the expression of rhIL-2 protein will be briefly described below.

1) Production of rhIL-2 producing strain

Cloning of interleukin-2 gene

CDNA containing the interleukin-2 gene was obtained from the human T-lymphocyte cDNA library of Clontech. This gene is characterized by the removal of alanine, the first amino acid of the natural type interleukin-2. Three cysteines exist in the IL-2 protein expressed from the cDNA. Among them, cysteine, which is the 125th amino acid that does not participate in the secondary structure of the protein, is substituted with serine, and the recombinant interleukin- 2 gene. This is to ensure that the expression of the recombinant gene is smoothly oxidized. Then, pET22b protein expression vector containing this IL-2 gene (pET22b IL2, see Korean Patent No. 10-1041986) was prepared.

Methionine aminopeptidase cloning

A DNA containing the methionine aminopeptidase gene of the vector E. coli K12 expressing the methionine aminopeptidase for the first amino acid of the N-terminal of the expressed IL-2 protein in E. coli to remove methionine was obtained. The obtained methionine aminopeptidase gene was digested with restriction enzymes, and the obtained gene fragment was cloned into an expression vector pSTV29 vector to produce an expression vector (pSTV29 MAP, see Korean Patent No. 10-1041986).

Production of double vector strain for the production of interleukin-2

E. coli (Accession No. KCTC11284BP) expressing IL-2 was prepared by transforming the resulting pSTV29 MAP and pET22b IL-2 expression vectors with two vectors in one E. coli .

2) Storage and testing of rhIL-2 producing strains

To use the rhIL-2 expression strain for production, a Master Cell bank and a Working Cell bank were prepared and stored.

The stored strain was restored and the following test was carried out to obtain the stability and safety test of the strain. The stability test was confirmed by the survival rate of the strain, the growth curve of the recovered strain, the expression rate of interleukin - 2 protein during culture, the morphological stability through microscopic observation, and the mutation of the interleukin - 2 structural gene. The safety test of the strain was confirmed through pathogenic microorganism contamination confirmation and bacteriophage contamination confirmation, and all were evaluated as no abnormality.

3) Culture and purification of rhIL-2 producing strain

The above rhIL-2 producing strain was cultivated in LB (Luria Bertani) medium and purified. The final yield of the purified rhIL-2 was 240 mg / L (culture medium) and produced about 200 vials per L of culture medium with high content of rhIL-2 18MIU.

Example 2: Characterization of rhIL-2 protein prepared in Example 1

1) Identification of immunological properties

The purified protein was developed by SDS-PAGE (SDS-polyacylamide gel electrophoresis) method to confirm the immunological characteristics of the rhIL-2 protein prepared in Example 1, and the human interleukin-2 specific antibody (Anti-Hu IL- 2 Goat IgG) was bound and color developed to confirm the antigen-antibody reaction.

The results of the above antigen-antibody reaction are shown in Fig. Referring to FIG. 1, it was confirmed that color development was clearly observed in rHu IL-2 protein at 3, 4 and 5 times, similar to 1 size marker and 2 eldredukin, a control substance.

2) Biological characteristics test

The rHu IL-2 protein prepared in Example 1 above was added to the IL-2 dependent cell line (HT-2), and the degree of differentiation of the cell line upon addition of the interleukin-2 was determined by the intracellular mitochondrial Dehydrogenase The degree of physiological activity of IL-2 in the sample was measured by measuring the phenomenon changing to insoluble formazan crystal. This test has a characteristic that the degree of physiological activity is proportional to the concentration of interleukin-2.

The biological activity of the rHu IL-2 protein prepared in Example 1 was evaluated using NIBSC rHu IL-2 activity standard, and IU (International Unit) per mg protein was determined. As a result, it was confirmed that the activity of 18 × 10 ⁶ IU / mg, which is equivalent to that of prolu- quine, was observed.

Example 3: Evaluation method of activity of rhIL-2

1) Experimental method: Administration of rhIL-2 and anti-cancer drugs (rat experiment)

Preparation of experimental rats

Twenty-four healthy male rats (SD-rats) of 5 weeks of age were purchased from BioLink and purified in a laboratory. Rats were grouped into 4 groups (6 per group) and applied as control (control) and experimental groups 1, 2 and 3, respectively.

Administration of rhIL-2 protein and anticancer drugs to experimental rats

In the control and experimental groups 1 to 3, the drugs were administered as follows.

Control: saline + 5-FU 1 mg / 200 g / 0.5 ml

Experimental group 1: rhIL-2 protein 12 x 10 ³ IU / 0.5 mL + 5-FU 1 mg / 200 g / 0.5 ml

Experimental group 2: rhIL-2 protein 6 x 10 ³ IU / 0.5 mL + 5-FU 1 mg / 200 g / 0.5 ml

Experimental group 3: rhIL-2 protein 3 x 10 ³ IU / 0.5 mL + 5-FU 1 mg / 200 g / 0.5 ml

Specifically, the recombinant interleukin-2 to be administered to the test group was diluted with physiological saline to 24 × 10 ³ , 12 × 10 ³ , and 6 × 10 ³ IU / mL, respectively, 2 < / RTI > In the first 3 days of dosing, the corresponding dose of rhIL-2 protein was administered intravenously to the rat. For the next 5 days, 5-FU (Choongwae Pharmaceutical Co., Ltd.) was intravenously administered at a dose of 1 mg / . The dose of rhIL-2 protein in each group will be 3, 1.5, and 0.75 x 10 ⁶ IU / person, respectively, based on the dose per adult (based on 50 Kg).

Venous blood samples were collected from 1.0 to 1.5 mL in the 1st, 7th, and 14th days after the end of the administration, and lymphocytes and blood cells were counted. The lymphocyte subtypes were analyzed using FACS on the end of the test (day 14) .

2) Experimental method: Analysis of lymphocyte subsets

The lymphocyte subset analysis was performed by flow cytometry in the following manner.

FACS tubes (sterilized microtube 5.0 ml) were prepared and labeled at double the number of samples, and the microtubules were immersed in the following antibodies.

(A) 20 μl of anti-CD3-FITC / anti-CD4-PE / Cy5 / anti-CD8-PE were added to each of the 30 tubes.

(B) 20 μl of anti-CD45R PE (phycoerythrin) was added to the next 30 tubes.

In the next 30 tubes, 20 μl of anti-NK marker PE (phycoerythrin) was added.

In the next 30 tubes, 20 μl of isotype control was added to the target antibody.

Ms IgG3, k-FITC: CD3 isotype control

Ms IgG2a, k-PE-Cy5: CD4 isotype control

Ms IgG1, k-PE: CD8 isotype control

Ms IgG2b, k-FITC: CD45R isotype control

Ms IgG1, k-FITC: CD161 (NK) isotype control

Then, 100 μl of whole blood of rats treated with EDTA was added to the microtube containing the antibody, carefully mixed, and the mixture was reacted for 15 minutes in a cow at room temperature (also wrapped in a foil).

After the reaction, 2 ml of 1 x cell lysis buffer (BD science, Catalog No. 349202) was added and mixed. The solution was left at room temperature for 10 minutes and then centrifuged at 1500 rpm for 5 minutes. After centrifugation, the supernatant was removed, and 2 ml of PBS was added to the well. After centrifugation at 1500 rpm for 5 minutes, the procedure was repeated three times. After the supernatant was discarded, 500 μl of 1% paraformaldehyde was added and the cells were loosened and stored at 4 ° C until measurement.

Example 4: Results of rat experiment for evaluating the activity of rhIL-2

1) Common symptoms of experimental rats

In all experimental rats, there were no abnormal behavior or deaths due to chemotherapy and rhIL-2 administration during the study period. In addition, the body weight changes of the experimental group rats (control group and rhIL-2 + anticancer drug group) during the entire test period are shown in Table 1 below, and no statistically significant weight change was observed during the entire test period as compared with the control group.

days
rhIL-2 (IU / kg) 0 3 6 9 12 15 18 21 Control group
(Saline) 184.37
± 5.3 207.8
± 7.4 221.72
± 9.0 239.4
± 8.8 254.56
± 10.7 275.38
± 10.7 288.95
± 14.2 301.7
± 17.5 Experiment 1
(2.4 x 10 ⁴ ) 183.07
± 7.3 207.05
± 8.1 221.99
± 10.6 246.43
± 14.7 262.58
± 17.9 287.36
± 21.0 301.16
± 26.3 317.73
± 26.0 Experiment 2
(1.2 x 10 ⁴ ) 183.5
± 6.5 208.77
± 6.1 221.72
± 5.4 241.9
± 6.0 257.71
± 8.9 281.92
± 11.2 295.1
± 13.0 312.45
± 15.5 Experiment group 3
(0.6 x 10 ⁴ ) 183.19
± 7.4 205.4
± 4.8 219.54
± 4.8 238.91
± 5.1 255.31
± 5.2 277.47
± 8.0 291.24
± 9.7 304.44
± 10.4

2) Biochemical analysis of blood of experimental rats

Blood samples were taken at days 1, 7, and 14 after completion of rhIL-2 and anticancer drug administration, and biochemical tests were performed on serum using a blood biochemical analyzer (7080, HITACHI, Japan) (Day 1), Table 3 (day 7) and Table 4 (day 14).

Biochemical parameters
after one day Recombinant IL-2 (IU / kg) Control group
(Saline) Experiment 1
(2.4 x 10 ⁴ ) Experiment 2
(1.2 x 10 ⁴ ) Experiment group 3
(0.6 x 10 ⁴ ) Glucose (mg / dL) 145.0 ± 20.0
153.0 ± 37.0 163.0 + - 34.0 161.0 ± 18.0 Total protein (g / dL) 5.9 ± 0.2 6.0 ± 0.3 60.0 ± 0.2 6.1 ± 0.2 Creatinine (mg / dL) 0.46 + 0.07
0.45 ± 0.05 0.47 + 0.07 0.5 ± 0.07 BUN (mg / dL) 22.8 ± 1.1
21.7 ± 1.9 22.5 ± 1.5 20.1 ± 2.5 Triglycerides (mg / dL) 78.0 ± 36.0 67.0 ± 18.0 69.0 + - 16.0 65.0 + - 21.0
Total cholesterol (mg / dL) 93.0 + - 7.0
90.0 + - 10.0 88.0 + - 5.0 94.0 ± 7.0 ALT (U / L) 44.4 ± 5.8
49.0 ± 104 57.6 ± 17.8 48.3 ± 9.1 AST (U / L) 153.4 ± 18.2
140.7 ± 25.1 149.5 ± 18.5 117.2 ± 16.4 LDH (U / L) 1784.0 + - 406.0
1062.0 ± 700.0 1099.0 ± 595.0 693.0 + - 272.0 ALP (U / L) 1675.8 ± 200.7
1498.2 ± 277.5 1639.9 ± 218.4 1666.1 + - 274.0 GGT (U / L) 0.77 ± 0.3
0.86 ± 0.4 0.75 + - 0.3 0.92 + - 0.35 CK (U / L) 1720 + - 503
1272 + 416 1430 ± 588 1200 ± 423

Biochemical parameters
after 7 days Recombinant IL-2 (IU / kg) Control group
(Saline) Experiment 1
(2.4 x 10 ⁴ ) Experiment 2
(1.2 x 10 ⁴ ) Experiment group 3
(0.6 x 10 ⁴ ) Glucose (mg / dL)
135.0 + - 21.0 134.0 + - 17.0 139.0 + - 14.0 142.0 + - 11.0 Total protein (g / dL) 6.0 ± 0.2 5.9 ± 0.1 6.1 ± 0.1 6.2 ± 0.2 Creatinine (mg / dL)
0.45 ± 0.08 0.46 ± 0.05 0.48 ± 0.05 0.51 + 0.07 BUN (mg / dL)
21.1 ± 1.8 20.9 ± 1.9 21.0 ± 2.4 19.6 ± 2.8 Triglycerides (mg / dL) 87.0 + - 31.0 89.0 ± 32.0 101.0 ± 35.0 98.0 ± 37.0 Total cholesterol (mg / dL) 98.0 + - 10.0 88.0 8.0 94.0 ± 6.0 102.0 + - 11.0 ALT (U / L)
48.7 ± 5.3 41.1 ± 4.2 48.1 ± 7.8 46.9 ± 9.9 AST (U / L)
157.6 ± 22.6 156.7 ± 43.2 146.3 ± 20.5 111.9 ± 11.3 LDH (U / L)
1764.0 ± 1004.0 2158.0 占 1186.0 1827.0 占 685.0 748.0 + - 315.0 ALP (U / L)
1464.8 ± 225.6 1311.0 + 171.5 1464.3 ± 250.2 1501.2 + - 462.2 GGT (U / L)
0.92 + 0.25 0.45 + 0.24 0.56 + - 0.43 1.03 + - 0.39 CK (U / L)
1546.0 + - 468.0 1640.0 + - 727.0 1452.0 ± 489.0 1013.0 + - 707.0

Biochemical parameters
after 14 days Recombinant IL-2 (IU / kg) Control group
(Saline) Experiment 1
(2.4 x 10 ⁴ ) Experiment 2
(1.2 x 10 ⁴ ) Experiment group 3
(0.6 x 10 ⁴ ) Glucose (mg / dL)
122.0 ± 19.0 136.0 + - 10.0 141.0 ± 17.0
160.0 + - 40.0 Total protein (g / dL) 6.2 ± 0.2 6.2 ± 0.2 6.2 ± 0.2 6.2 ± 0.4 Creatinine (mg / dL)
0.49 + 0.11 0.46 ± 0.08 0.53 + - 0.11 0.50 0.07 BUN (mg / dL)
23.3 ± 1.6 22.1 ± 2.1 21.9 ± 2.1 21.2 ± 2.8 Triglycerides (mg / dL) 93.0 + - 24.0 123.0 + - 43.0 99.0 + - 23.0 89.0 + - 34.0 Total cholesterol (mg / dL) 100.0 + - 12.0 87.0 8.0 90.0 ± 6.0 93.0 + - 11.0 ALT (U / L)
49.2 ± 4.8 47.0 ± 8.6 54.1 ± 12.2 53.7 ± 10.2 AST (U / L)
176.8 ± 23.6 165.4 ± 15.4 149.7 ± 29.2 149.1 ± 17.6 LDH (U / L)
2536.0 + - 862.0 2341.0 + - 406.0 1355.0 + - 794.0 1298.0 占 744.0 ALP (U / L)
1450.0 + - 136.9 1286.0 ± 240.3 1441.9 ± 238.7 1308.4 ± 283.4 GGT (U / L)
0.95 + - 0.98 0.62 ± 0.19 0.46 0.22 0.75 + 0.31 CK (U / L)
1752.0 占 570.0 1688.0 + - 235.0 1167.0 + - 483.0 1071.0 + - 327.0

Referring to Tables 2 to 4, biochemical analysis showed no significant blood-phase biochemical changes in all groups administered with IL-2 as compared with the control group.

3) Electrolyte analysis of experimental rat blood

each of 1 day of rhIL-2 and chemotherapy ends, 7 days, by taking the experimental rat blood 14 days hematology analyzer ^{(ADVIA ® 120 Hematology System, germany} ) and an electrolyte analyzer electrolyte analyzed using (AVL9181, Roche, Germany) And the results are shown in Tables 5 to 7, respectively.

Electrolytes
after one day Recombinant IL-2 (IU / kg) Control group
(Saline) Experiment 1
(2.4 x 10 ⁴ ) Experiment 2
(1.2 x 10 ⁴ ) Experiment group 3
(0.6 x 10 ⁴ ) Chloride (mmol / L)
98.0 ± 2.0 99.0 ± 2.0 101.0 ± 2.0 102.0 ± 3.0 Sodium (mmol / L)
139.0 ± 1.0 140.0 ± 2.0 141.0 ± 1.0 144.0 ± 2.0 Potassium (mmol / L)
6.1 ± 0.6 5.9 ± 0.8 5.6 ± 0.3 5.9 ± 0.3 Calcium (mg / dL)
10.6 ± 0.3 11.3 ± 0.3 11.2 ± 0.5 11.4 ± 0.3 Phosphorus (mg / dL)
8.79 ± 0.7 9.04 ± 0.7 8.61 ± 0.8 9.06 ± 0.7

Electrolytes
after 7 day Recombinant IL-2 (IU / kg) Control group
(Saline) Experiment 1
(2.4 x 10 ⁴ ) Experiment 2
(1.2 x 10 ⁴ ) Experiment group 3
(0.6 x 10 ⁴ ) Chloride (mmol / L)
101.0 ± 2.0 101.0 ± 2.0 105.0 ± 3.0 104.0 ± 1.0 Sodium (mmol / L)
143.0 ± 1.0 144.0 ± 2.0 147.0 ± 1.0 147.0 ± 1.0 Potassium (mmol / L)
6.1 ± 0.2 5.7 ± 0.4 6.3 ± 0.2 6.7 ± 0.5 Calcium (mg / dL)
10.6 ± 0.4 10.9 ± 0.4 11.2 ± 0.1 11.4 ± 0.6 Phosphorus (mg / dL)
8.54 ± 0.5 8.60 ± 0.59 8.38 ± 0.63 8.85 ± 0.64

Electrolytes
after 14 days Recombinant IL-2 (IU / kg) Control group
(Saline) Experiment 1
(2.4 x 10 ⁴ ) Experiment 2
(1.2 x 10 ⁴ ) Experiment group 3
(0.6 x 10 ⁴ ) Chloride (mmol / L)
99.0 ± 2.0 102.0 ± 2.0 105.0 ± 2.0
106.0 ± 3.0 Sodium (mmol / L)
140.0 ± 1.0 145.0 ± 2.0 149.0 ± 3.0
150.0 + - 3.0 Potassium (mmol / L)
6.6 ± 0.5 6.4 ± 0.5 6.1 ± 0.5 5.9 ± 0.4 Calcium (mg / dL)
10.6 ± 0.3 10.9 ± 0.1 11.7 ± 0.7 11.5 ± 0.8 Phosphorus (mg / dL)
8.22 + 0.44 7.99 + - 0.40 9.48 ± 1.44 9.10 ± 0.98

Referring to Tables 5 to 7, there was no significant change in chloride, sodium, potassium, calcium, and phosphorus in all groups during the test period.

4) Analysis of blood characteristics of experimental rats

after rhIL-2 and chemotherapy end to a respective day, 7 days, and by collecting the experimental rat blood of 14 days using a hematology analyzer ^{(ADVIA ® 120 Hematology System, germany} ) and the blood composition analysis result table 8 to 10.

Hematological parameters
after one day Recombinant IL-2 (IU / kg) Control group
(Saline) Experiment 1
(2.4 x 10 ⁴ ) Experiment 2
(1.2 x 10 ⁴ ) Experiment group 3
(0.6 x 10 ⁴ ) RBC
(× 10 ⁶ / μl) 7.86 ± 0.17 7.87 ± 0.59 7.88 0.31 7.91 + - 0.38 HGB
(g / dl) 15.43 + - 0.62 15.97 ± 0.77 16.05 + - 0.33 15.88 + - 0.56 HCT
(%) 47.57 ± 2.21 48.22 + - 2.27 47.95 ± 1.52 48.28 ± 1.76 RBC Index MCV (fL) 60.52 + - 2.22 61.47 ± 2.33 60.95 + - 2.44 61.13 + - 2.52 MCH (pg) 19.68 ± 0.71 20.32 + - 0.68 20.37 + - 0.61 20.1 ± 0.68 MCHC (g / dL) 32.5 ± 0.76 33.07 ± 0.26 33.43 + - 0.58 32.92 + 0.64 PLT
(× 10 ³ / μl) 868.67 ± 296.87 753.83 + - 421.92 651.33 + - 227.66 970.33 + - 398.21

Hematological parameters
after 7 day Recombinant IL-2 (IU / kg) Control group
(Saline) Experiment 1
(2.4 x 10 ⁴ ) Experiment 2
(1.2 x 10 ⁴ ) Experiment group 3
(0.6 x 10 ⁴ ) RBC
(× 10 ⁶ / μl) 7.69 0.35 7.35 ± 0.54 6.14 ± 2.79 7.42 + - 0.42 HGB
(g / dl) 15.53 + - 0.84 15.34 ± 0.59 11.68 ± 6.39 15.12 ± 0.53 HCT
(%) 46.43 + - 2.56 45.44 + 1.66 36.08 + - 16.03 45.18 ± 1.25 RBC Index MCV (fL) 60.35 + 1.03 62.02 + - 2.47 59.23 1.52 60.98 + - 3.04 MCH (pg) 20.23 + - 0.34 20.94 + - 0.85 17.43 + - 4.69 20.4 ± 0.72 MCHC (g / dL) 33.5 ± 0.08 33.74 + - 0.38 29.58 8.39 33.5 ± 0.69 PLT
(× 10 ³ / μl) 1107.75 ± 123.96 1203.8 + 236.32 945.25 + 472.37 1169.6 ± 127.42

Hematological parameters
after 14 days Recombinant IL-2 (IU / kg) Control group
(Saline) Experiment 1
(2.4 x 10 ⁴ ) Experiment 2
(1.2 x 10 ⁴ ) Experiment group 3
(0.6 x 10 ⁴ ) RBC
(× 10 ⁶ / μl) 7.4 ± 0.27 7.77 + - 0.68 7.73 ± 0.19 7.37 + - 0.46 HGB
(g / dl) 14.75 ± 0.72 15.63 + - 0.83 15.68 + - 0.41 14.9 ± 0.68 HCT
(%) 44.73 + - 2.43 47.43 + - 1.45 48.27 ± 1.35 45.08 ± 2.06 RBC Index MCV
(fL) 60.43 + - 2.33 61.23 + - 3.50 62.5 ± 2.29 61.3 ± 4.00 MCH
(pg) 19.9 + 0.47 20.1 ± 0.78 20.3 ± 0.83 20.24 0.88 MCHC
(g / dl) 32.92 + 0.71 32.9 ± 0.87 32.5 ± 0.89 33.06 + - 0.75 PLT
(× 10 ³ / μl) 1238.5 ± 109.17 1399.67 + - 42.36 1217.5 ± 193.60 1039.6 + 347.60

Referring to Tables 8-10, there were no significant hematological changes in the IL-2 administration group compared to the control group during the test period.

5) Hematological analysis of blood of experimental rat

Blood samples were taken at 1, 7, and 14 days after completion of rhIL-2 and anticancer drug administration. Hematological analysis of peripheral blood cells was performed by the ADVIA ^® 120 Hematology System (germany) The results are shown in Table 11 (day 1), Table 12 (day 7), and Table 13 (day 14).

WBC Differential Counting
after one day Recombinant IL-2 (IU / kg) Control group
(Saline) Experiment 1
(2.4 x 10 ⁴ ) Experiment 2
(1.2 x 10 ⁴ ) Experiment group 3
(0.6 x 10 ⁴ ) WBC (x 10 ³ / μl) 10.46 ± 0.68 11.2 ± 1.98 9.13 ± 1.10 9.39 ± 0.68 WBC Differential Counting (%) NEU 7.4 ± 1.04 12.07 * 0.85 8.13 *? 2.40 4.54 ± 0.73 LYM 86.93 + 1.61 92.63 * 1.34 87.23 + - 1.57 88.13 + 1.55 MONO 1.16 ± 0.27 1.84 0.63 1.5 ± 0.24 1.37 + - 0.36 EOS 1.6 ± 1.10 1.38 + - 0.63 1.87 ± 1.07 1.43 ± 1.38 BASO 0.33 + - 0.10 0.38 + 0.10 0.37 + - 0.10 0.47 + 0.10 LUC 0.93 + - 0.27 0.93 + - 0.23 0.9 ± 0.18 0.85 ± 0.26

WBC Differential Counting
after 7 day Recombinant IL-2 (IU / kg) Control group
(Saline) Experiment 1
(2.4 x 10 ⁴ ) Experiment 2
(1.2 x 10 ⁴ ) Experiment group 3
(0.6 x 10 ⁴ ) WBC (x 10 ³ / μl) 8.78 ± 0.83 10.99 ± 1.26 9.73 ± 1.06 10.97 ± 1.91 WBC Differential Counting (%) NEU 9.6 ± 0.26 10.8 ± 5.77 9.8 ± 5.56 6.3 ± 1.49 LYM 84.38 ± 2.68 90.35 *? 2.99 88.57 * 1.81 89.53 ± 1.20 MONO 2.15 ± 0.58 1.58 0.35 2.05 ± 0.94 1.8 ± 0.43 EOS 1.6 ± 0.89 1.34 + 0.62 1.7 ± 1.48 0.78 + 0.13 BASO 0.35 + 0.06 0.3 ± 0.07 0.4 ± 0.16 0.44 + 0.15 LUC 0.7 ± 0.14 0.6 ± 0.12 0.73 + 0.30 0.66 + 0.17

WBC Differential Counting
after 14 days Recombinant IL-2 (IU / kg) Control group
(Saline) Experiment 1
(2.4 x 10 ⁴ ) Experiment 2
(1.2 x 10 ⁴ ) Experiment group 3
(0.6 x 10 ⁴ ) WBC (x 10 ³ / μl) 7.92 ± 1.87 8.76 ± 0.03 5.37 + 1.53 6.75 ± 1.73 WBC Differential Counting (%) NEU 8.08 ± 2.50 7.3 ± 4.10 9.27 ± 1.71 7.85 ± 1.20 LYM 86.45 + 1.24 90.20 * + - 3.74 87.8 ± 2.08 86.55 7.35 MONO 1.68 + 0.47 0.97 + - 0.12 1.22 ± 0.29 1.42 ± 0.20 EOS 1.58 ± 1.05 0.93 + - 0.49 0.9 ± 0.59 1.56 ± 1.15 BASO 0.3 ± 0.15 0.4 ± 0.17 0.38 + 0.15 0.34 0.11 LUC 1.58 ± 2.22 0.8 ± 0.10 0.6 ± 0.24 0.52 + 0.15

In addition, among the measurement results are given in Table 11 to 13 above, the results for neutrophils (neutrophil) in Figure 3, showed the results for lymphocytes (Lymphocyte) in Figure 4, the statistical processing is a Dunnett? S t -test And * p <0.05, respectively.

3 and 4, neutrophil in the whole white blood cell (WBC) was administered at 2.4 x 10 ⁴ IU / kg on the first day after completion of rhIL-2 and 5-FU administration ( P <0.05) compared with the control group (saline + 5-FU) in the group treated with 12.07% and 1.2 × 10 ⁴ IU / kg in one group. Lymphocytes also showed a significant increase of 92.63% in the group administered with 2.4 × 10 ⁴ IU / kg ( p <0.05). That is, the total WBC and neutrophil values did not change at 7 days and 14 days after administration, but lymphocyte was increased in the 2.4 × 10 ⁴ and 1.2 × 10 ⁴ IU / kg administration groups compared with the control group (P < 0.05). &Lt; / RTI >

6) Analysis of T-cell Subset using FACS

The results analyzed according to the experimental method 2) of Example 3 were as follows.

a. CD4 / CD8 assay results

Analysis was performed by flow cytometry (Cytomics, FC500, Beckman Coulter) in order to confirm the effect of differentiation on CD4 and CD8 using FACS in the blood of test group on day 14 after completion of rhIL-2 and anti-cancer drug administration And the results are shown in Table 14 and Figs. 5 to 7, respectively.

Dose (IU / kg) Peripheral blood lymphocyte CD3 ⁺ / CD4 ⁺ CD3 ⁺ / CD8 ⁺ The control (Saline) 35.36 + - 2.72 16.55 ± 3.22 Experiment 1
(2.4 x 10 ⁴ ) 44.28 ** +/- 7.67 20.07 *? 1.63 Experiment 1
(1.2 x 10 ⁴ ) 38.57 ** +/- 3.53 17.24 * + - 4.10 Experiment 1
(0.6 x 10 ⁴ ) 35.79 ± 5.30 15.34 *? 2.66

Referring to Table 14 and FIG. 5 to FIG. 7, the distribution of CD4 was significantly (p <0.05) higher in the experimental group administered with 2.4 and 1.2 × 10 ⁴ IU / kg than the control (A; ), Respectively. In addition, CD8 analysis showed that the distribution of CD8 was significantly (p <0.05) higher than that of the control (Saline + 5-FU) in the group administered with 2.4 and 1.2 x 10 ⁴ IU / .

b. NK / CD45 analysis results

In order to confirm the differentiation effect of NK cells and CD45R on the blood of test group 14 after completion of rhIL-2 and anti-cancer drug administration by FACS, analysis was carried out using a flow cytometer (Cytomics, FC500, Beckman Coulter) And the results are shown in Figs. 8 to 10, respectively.

8, the distribution of NK cells in the test group (B, C in FIG. 8) administered at a concentration of 2.4, 1.2 x 10 ⁴ IU / kg was higher than that of the control group (A, Saline + 5-FU in FIG. 8) (P < 0.05). &Lt; / RTI > In addition, referring to FIG. 9, CD45R was analyzed as a B cell marker of rat, and it was confirmed that there was no significant increase or decrease between all groups.

7) Summary of experimental results

No deaths or clinical behavior was observed for rhIL-2 and 5-FU during the trial using Rat.

The neutrophil count was 12.7% at the dose of 2.4 × 10 ⁴ IU / kg IL-2 and 8.13% at the 1.2 × 10 ⁴ IU / kg concentration on the first day after the end of rhIL-2 and 5-FU administration (P < 0.05). &Lt; tb >< TABLE > On the other hand, lymphocyte was significantly increased (92.63% on day 1, 90.35% on day 7 and 90.20% on day 14) at a dose of 2.4 x 10 ⁴ IU / kg (p <0.05).

T cell subset analysis using FACS showed that CD4 cells were significantly increased in the groups administered with 2.4 and 1.2 × 10 ⁴ IU / kg compared with the control group (35.36%) (44.28 and 38.57%, p <0.05). In addition, CD8 cells were significantly increased in the group administered with 2.4 and 1.2 × 10 ⁴ IU / kg, respectively (20.7 and 17.24%, respectively) than the control group (16.55%). NK cells were significantly increased at 2.4 and 1.2 × 10 ⁴ IU / kg, respectively, compared to the control (2.24%) (3.20 and 2.72%, p <0.05).

Therefore, the effect of IL-2 on lymphocytes after administration of anticancer drugs was significantly higher in the group administered with IL-2 (2.4 × 10 ⁴ IU / kg) compared to the group administered only with anti-cancer drugs (p < 0.05), respectively. In addition, administration of IL-2 inhibited the expression of helper T cells (CD4 T cells), cytotoxic T cells (CD8 T cells) and natural killer cells (IL-2) (P <0.05, p <0.01) in the differentiation and proliferation of NK cells.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, Of the right.

<110> Soonchunhyang University Industry Academy Cooperation Foundation BMI Korea co., Ltd <120> PHARMACEUTICAL COMPOSITIONS CONTAINING HUMAN ACTIVE PROTEIN <130> DP20150140 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> modified human IL-2 <400> 1 Met Pro Thr Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro As Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 2 <211> 133 <212> PRT <213> Human Interleukin-2 <400> 2 Ala Pro Thr Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro As Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 3 <211> 411 <212> DNA <213> Artificial Sequence <220> <223> modified human IL-2 structural gene <400> 3 catatgccta cttcaagttc tacaaagaaa acacagctac aactggaaca tttactgctg 60 gatttacaga tgattttgaa tggaattaat aattacaaga atcccaaact caccaggatg 120 ctcacattta agttttacat gcccaagaag gccacagaac tgaaacatct tcagtgtcta 180 gaagaagaac tcaaacctct ggaggaagtg ctaaatttag ctcaaagcaa aaactttcac 240 ttaagaccca gggacttaat cagcaatatc aacgtaatag ttctggaact aaagggatct 300 gaaacaacat tcatgtgtga atatgctgat gagacagcaa ccattgtaga atttctgaac 360 agatggatta ccttttctca aagcatcatc tcaacactaa cttgaggatc c 411

Claims

A pharmaceutical composition comprising a human interleukin-2 protein as an active ingredient for relieving or treating lymphocytopenia caused by an anticancer agent or radiation therapy.

The method according to claim 1,
Wherein the anticancer agent is an alkylating agent, an antimetabolite, or a combination thereof, and the radiation therapy is pelvic radiation therapy.

The method according to claim 1,
The anticancer agent is an alkylating agent such as cisplatin, carboplatin, dacarbazine, procarbazine, busulfan, thiotepa, cyclophosphamide, , Or a combination thereof as an active ingredient.

The method according to claim 1,
Wherein the anticancer agent is an antimetabolite and comprises a fluorinated pyrimidine nucleoside derivative as an active ingredient.

The method according to claim 1,
Wherein the anticancer agent is a fluorinated pyrimidine nucleoside derivative, which comprises 5-fluorouracil, cytarabine, capecitabin or a combination thereof. Composition.

The method according to claim 1,
The human interleukin-2 protein is a recombinant human interleukin-2 protein having a modification in which alanine as the first amino acid in the 5 'terminal base sequence is removed from the human interleukin-2 protein and the 125th amino acid cysteine is substituted with serine , &Lt; / RTI >

The method according to claim 1,
Wherein said human interleukin-2 protein is a recombinant human interleukin-2 protein having an amino acid sequence of SEQ ID NO: 1.

The method according to claim 1,
The cancer to which the chemotherapeutic adjuvant composition is applied may be one or more selected from the group consisting of cervical cancer, cervical cancer, ovarian cancer, breast cancer, colon cancer, stomach cancer, liver cancer, colon cancer, rectal cancer, esophageal cancer, pancreatic cancer, lung cancer, Head and neck cancer, or testicular cancer.

The method according to claim 1,
Wherein the anticancer adjuvant pharmaceutical composition is administered before the anticancer agent or the radiation therapy, or simultaneously with the anticancer agent or the radiotherapy.

A pharmaceutical composition for the treatment or prevention of lymphocytopenia, which comprises a human interleukin-2 protein as an active ingredient and alleviates or treats lymphocyte reduction by an anticancer agent or radiation therapy.

11. The method of claim 10,
The human interleukin-2 protein is a recombinant human interleukin-2 protein having a modification in which alanine as the first amino acid in the 5 'terminal base sequence is removed from the human interleukin-2 protein and the 125th amino acid cysteine is substituted with serine , A pharmaceutical composition for treating or preventing lymphocytopenia.

11. The method of claim 10,
Wherein the human interleukin-2 protein is a recombinant human interleukin-2 protein having the amino acid sequence of SEQ ID NO: 1.