TWI278320B - A complex immunity gene medicine composition for inhibiting tumor cell - Google Patents

Abstract

This invention relates to a complex immune gene medicine composition for inhibiting tumor cell. Via activating natural killer cell primarily, it improves immune response. By the merging way of using several cell hormones, for instance, it uses the first helper T cell (T-helper type 1, Th1) and the second helper T cell (T-helper type 2, Th2) type cell hormone. By using the Th2 type cell to inhibit the anti-extension growth factor to the natural killer cell suppression, it relieves the host immune inhibitory state. Furthermore, by using the Th1 type cell to activate vivacious natural killer cell in host, it strengthens the ability of host immune system to resist the growth of tumor cell. Finally, it achieves the goal of removing tumor cell.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for inhibiting tumor cells, and more particularly to a composite immunogenic composition for inhibiting tumor cells, and inhibiting the composition using the pharmaceutical composition A method of tumor cell growth. [Prior Art] Canine transmissible venereal Uim〇r (CTVT) is a naturally occurring, low-differentiated cancerous cell. It is mainly infected by contact with injured skin or mucous membranes by living tumor cells by mating or biting. It can be transmitted between dogs and is similar to allograft. Experimentally inoculated canine infectious sputum tumors will exhibit a predictable growth pattern. Changes in tumor growth can be divided into Progressive (P phase), Stasis (s phase), and Regression (R phage). Infectious canine tumor cells can only express a very small amount of major histocompatibility complex (MHC) during the growth phase, but produce a large number of transforming growth factors during the growth phase and the regression phase. Transf〇rming to (10) let factor-β, TGF-β). The transforming growth factor has the property of rendering the first and second major histocompatibility complexes (MHC I and MHC II) non-expressing and inhibiting the activity of natural killer (NK) cells. In addition, 90% of the cells in the tum〇r infihming lymphoytes (TILs) of the canine infectious tumor are non-T non-B lymphocytes that do not express the antigens of the cells and B cells. Large-sized lymphocytes, which are very similar to natural killer cells, are observed under the type observation 1278320. This non-T non-B lymphocytes should be natural killer cells. The first major histocompatibility complex antigen is a glycoprotein expressed on the cell membrane together with β2 small globulin W2_mi(10)globulin, β2ηι), which is about 44kDa in size, and is also called histocompatibility leukocyte antigens in humans. , HLA) In the host, tumors grow by evading the monitoring of the host immune system by a variety of different mechanisms, and the non-expression or low-expression of major histocompatibility complex molecules is a common mechanism for tumor cells. one. Case S swells include primary breast CarCin〇ma, advanced renal cell CaiXln〇ma, melanoma, prostate cancer, Lung carcinoina, as well as other tumors including various types of tissues such as the skin and the endometrium, have been found to have reduced levels of major histocompatibility complexes, and even some malignant tumors. Will not behave completely. In animal terms, it occurs in avian T-lymphoma caused by Marek's virus, as well as in dogs with canine infectious swells. It is also seen that the main histocompatibility complex has a very low performance. When the amount of major histocompatibility complex in tumor cells is low, the natural killer cells in the host will cause loss of self (missing factor (7) to activating receptors on the cell surface. However, many tumor cells produce a transforming growth factor to inhibit natural killer cells that are not activated by the major histocompatibility complex molecules. Therefore, the immune system cannot perform its function normally, 1278320, so The tumor can grow unscathed in the host. The transforming growth factor is a homodimeric protein of about 25kDa, which has a very strong regulatory effect on the mammalian immune system. By means of in vitro culture of lymphocytes, it is found to inhibit the division of B cells, mature tau cells, thymocytes, natural killer cells, and lymphokine-activated killer cells (LAK). Many tumors produce transforming growth factors, including colorectal cancer (C〇l Rectalcancer, breast tumor, thyroid tumor, hepatocellular carcinoma (10)a, Hcc), and Meth a tumor. In addition to promoting angiogenesis and cell adhesion, the transforming growth factor can help tumor growth. Avoiding the initiation of the host immune system by reducing the expression of major histocompatibility complex molecules and intercellular adhesion factor-1 (Intercellular adhesi〇nqingxin I IC AM-1). Inhibition of the immune system 'If the effect of transforming growth factor is reduced by antagonism or reduced secretion, the immune system in the body can perform functions normally to remove tumor cells. Current immunity against inhibition of transforming growth factor Therapy is mainly based on the application of antibodies against anti-transformation factors and gene therapy of anti-infected oligonuclear acid (oh m 仏 oh oh oh oh oh oh oh oh oh oh oh oh oh oh oh oh oh oh oh oh oh oh oh oh oh oh oh oh oh oh oh oh When the tumors exhibiting major histocompatibility complex molecules, the antigen-specific T lymphocyte cytotoxicity in the immune system cannot be Execution, therefore, the role of natural killer cells that are not toxic to cells that do not express 1278320 major histocompatibility complex molecules is extremely important. Therefore, it is closely related to the differentiation, activation and executive function of natural killer cells. Whether natural killer cells can successfully remove tumors is highly relevant. These cytokines include: γ-interferon (IFN-γ), interleukin-2 (hereinafter referred to as IL-2), Baisu-12 (hereinafter referred to as il-12), interleukin-15 (hereinafter referred to as IL-15), interleukin-18 (hereinafter referred to as il-18), and interleukin-21 (hereinafter referred to as il-2) 1) They have effects on the τ lymphocytes, sputum lymphocytes, natural killer cells, and related immune cells in the immune system. It has been known that several kinds of cytokines have been confirmed to have significant anti-tumor effects in vitro and in specific animal experiments, and this discovery has led to the attention of cytokines in the treatment of human cancers. The cytokines currently used in clinical anti-cancer are mainly divided into two types, one is the first type of helper cell (T-helper type 1, Th1) cytokine, and the response caused by such cytokines is mainly IL- 2 and the production of IFN-γ, and subsequent induced cellular immunity, such as: IL-2, IL-12, IL-15, IL-18, IFN-γ; the other is a second type of helper T cells ( T-helper type 2, Th2) cytokine, which triggers the production of IL-4, IL-5 and IL-6 (all three are cytokines that stimulate the growth and differentiation of beta cells) The immune response is characterized. However, the clinical results of cytokine immunotherapy show that the way of administration, the dose, the type of tumor, whether it is necessary to use other cytokines or drugs, and the possible side effects, etc., each factor is related to this one. The effectiveness and clinical applicability of immunotherapy. Although immunogenic therapy has a certain degree of efficacy for tumors, there are still 1278320 restricted in use. On the other hand, in view of the immunological characteristics of the tumor itself, the concept of combining a plurality of cytokines is emphasized to achieve the effect of tumor regression. SUMMARY OF THE INVENTION The object of the present invention is to provide a composite immunogenic composition for inhibiting tumor cells, which antagonizes the immunosuppressive effect of a transforming growth factor (J'TGFj) to restore the killing ability of natural killer cells. Another object of the present invention is to provide a recombinant immunogenic pharmaceutical composition for inhibiting tumor cells, which activates the killing activity of the cells from the hand cells to the tumor cells. Due to the growth of certain tumors, tumor cells evade specific immune responses (CD8+ τ cells) in the host by reducing the expression of major histocompatibility complex (MAJ〇r hist〇c〇mpatibility MHC) molecules. Attack, while natural killer cells are activated by the lack of expression of histocompatibility complex molecules, but tumors also produce transforming growth factors to inhibit the differentiation and activity of natural killer cells, while causing a decrease in the number and cell killing The inhibition of the ability, the amount of expression of the α-chain of the cell surface IFN_a receptor, the IL-2 receptor, and the amount of secreted ΙΝρ_γ are decreased. The present invention provides a composite immunogenic drug composition for activating the immune response of natural killer cells in response to the above-described mechanism of tumor escaping host system. The composite immunogenic drug composition is a natural killer cell by using a combination of a plurality of cytokines, that is, a combination of sputum and Th2 type cytokines, by using a Th2 type cytokine to antagonize the transformation of the 1278320 long factor to the natural killer cell. The inhibition, the release of the host's immunosuppressive state, and then the Thul type cytokines activate the natural killer cells in the host to enhance the host immune system's ability to resist tumor cell growth, with the goal of finally achieving tumor removal. The aforementioned Th1 type cytokines include: IL_2, IL_12, IL-15, IL-18, and IFN-γ; and the aforementioned Th2 type cytokines include IL_4, IL-5, and IL-6. In order to confirm the inhibitory effect of the composite immunogenic pharmaceutical composition according to the present invention on tumor cells, the present invention uses a canine infectious sputum tumor (hereinafter referred to as CTVT) as a test tumor model because (〇CTVT is a kind) Tumors with low expression of major histocompatibility complex molecules; (2) CTVT produces a large number of transforming growth factors; (3) CTVT tumor infiltrating lymphocytes (TILs), up to 85% of cells do not exhibit CD3 And a non-tau-B lymphocyte of CD21, which should be a killer cell of a dog. According to the composite immunogene drug composition indicated by the present invention, a plastid of "and IL_15" can be constructed by a conventional technique. After the constructed plastid is sequenced and determined to be correct, the cells of the in vitro (10) vzir〇 are transfected, and the supernatant is transfected to test whether the functional IL-6 and IL-15 proteins can be produced. Then, in vitro, when IL-6 and IL-15 were combined and used alone, the recovery effect on the killing ability of the natural killer cells inhibited by the transforming growth factor was further introduced into the living body with IL-6 and il-15 plastids. Internal (BALB/C mice)' measurement of the performance of genes in mice, as well as the distribution of lymphocyte populations in the spleen and the ability of natural killer cells to kill, 278320 solution IL_6 and IL-15 plastids The performance of the produced cytokines after being administered into mice. Finally, the immunodeficient C b_17 scid mice were used as the animal model for tumor inoculation, due to the small gas T and B lymphocytes of this line. The ball is incapable of performing functions, only functional natural killer cells, to evaluate the effect of the composite immunogenic pharmaceutical composition indicated in accordance with the present invention on natural killer cells and its inhibitory effect on CTVT. It is confirmed that the composite immunogenic drug composition according to the present invention can achieve the antagonism of the inhibition of natural killer cells by the transforming growth factor, the immunosuppressive state of the host, and the activation of natural killer cells in the host to enhance host immunity. The purpose of the system against the ability of tumor cells to grow, that is, the fineness of the diced hi and Th2 species as indicated by the present invention The compound immunological gene composition of the compound can target tumors with low expression of histocompatibility complex molecules and produce transforming growth factors, which can not only provide the right medicine, but also exhibit anti-tumor effect, and can be adjacent to other immune reactions. The present invention will be further described with reference to the following embodiments, which are not intended to limit the disclosure of the present invention. Those skilled in the art can make a little The improvement and repair _, but still does not deviate from the scope of the present invention. 1278320 [Embodiment] Since the IL-6 gene of the dog cannot be easily obtained from dogs, and the IL-15 mRNA is rarely contained in normal animal tissues. Therefore, the IL-6 (SEQ ID NO: 1) and IL-15 (SEQ ID NO.·3) gene lines used in the present invention are derived from human IL-6 plastids, respectively, and synthetic IL-containing bodies. -2 Human IL-15 gene (IL-2SP/IL-15MP chimeric gene, SEQ ID NO.. 4) of the signal peptide (SEQ ID NO: 2) sequence. The IL-6 and IL-15 genes were cloned by using the commercial assay kit pcDNA3.1/V5-His-TOPO® Expression Kit (Invitrogen, Groningen, Netherlands) to IL_6 gene and IL-2SP/IL. The -15MP chimeric gene was inserted into the pcDNA3.1/V5-His-TOPO (5523 base pair) plastid to complete the construction of IL-6 and IL-2SP/IL-15MP plastids. The constructed plastid is transformed into a commercial E. coli competent cell (probiotic, Taiwan, Republic of China) using a conventional morphological transformation technique. Enzyme chain reaction, restriction enzyme digestion reaction, and sequencing three steps confirmed the correctness of the gene. The plastids with the correct sequence were further multiplied and the plastid DNA was isolated using the Nucleobond AX bulk plastid DNA isolation kit (Macherey-Nagel, Oensingen, Switzerland). It is then further tested to test whether the constructed IL-6 plasmid exhibits a functional IL-6 protein. Here, the cell line TF-1 (ATCC No. CRL-2003) which requires IL-6 as a growth factor is selected, and the cell supernatant obtained after transfection of the plastid by the conventional MTS test contains 12 1278320 IL- 6 activity. The results of the test showed that the supernatant obtained after transfection of IL-6 plastids into Balb/3T3 cells (ATCC No. CCL-163) was effective for cell division and transfection of PCDNA without any gene. The V5-His-T0P0 plastid and the supernatant of the Baib/3T3 cells cultured alone did not have this activity, indicating that the constructed iL_6 plastid did indeed exhibit a functional IL-6 protein. In addition, the same method was used to test whether the IL-15 plastid also produced a functional IL-15 protein, and HT-2 cells (ATCC No. CRL_1841) which were required to undergo cell division in the presence of IL-15 were used. The supernatant of Balb/3T3 cells transfected with IL-15 plastids was cultured in ht/2 cells, and the results showed that the supernatant obtained after transfection of IL-15 plastids with Balb/3T3 cells was effective. HT-2 cells were subjected to cell division, and the supernatant of pcDNA3.1/V5-His-TOP plastids without any gene and the supernatant of Balb/3T3 cells cultured alone did not have this activity, indicating that the constructed IL_15 was completed. The plastid does indeed exhibit a functional IL-15 protein. The effect of IL-6 and IL-15 on the natural killer cell activity inhibited by the transforming growth factor was then tested in vitro. It can be seen from the test results that the combined use of IL-6 and IL-15, and the treatment with IL-6 or IL-15 alone, compared with the ratio of the killing rate of YAC-1 mouse lymphoma cells . This result shows that, in the case of acting alone, if only the presence of IL-6 'releases the inhibitory effect of the transforming growth factor on natural killer cells, but lacks the substance that activates natural killer cells, the effect of poisoning is not awarded. If the opposite is true, only IL-15, which activates natural killer cells, is present, but the growth factor of the natural killer cells is inhibited by 13 1378320 and cannot be completely recovered. Only in the presence of IL-6 and IL-15, IL-6 antagonizes the immunosuppressive effect of transforming growth factor on natural killer cells, and then further activates natural killer cells with IL-1 5 to be effective. Improve the ability of natural killer cells to kill. To further confirm the effect of IL-6 and IL-15 in combination on in vivo immunity, iL-6 and IL-15 plastids were separately or together sent to BALB/C mice by muscle bio-injection. In vivo, to assess its effect on immune cells in the spleen of mice. Muscle in vivo gene electroporation is a very common method for transmitting genes in non-viral vectors in gene therapy in recent years. Its safety is not like a viral vector, which easily causes the host's immune response, and is highly efficient in anti-meat-transfer plastids. The ability of the protein is also strong, and it is easy to operate near the surface of the valley. The results of enzyme-linked immunosorbent assay (ELISA) showed that IL-6 and IL-1 5 plastids could be fully expressed in mice. On the 14th day after the electroporation of the shellfish gene, it was found that the number of natural killer cells in the spleen of the mice treated with the two plastids was compared with that of IL-6 or IL-15 plastid alone. The poisoning ability showed a significant increase. In addition, the IL-15 treatment group had a natural killer cell killing ability at E/Trati〇5〇/1 and 12%, although it was lower than IL_6 and IL_丨5 combined users, but still significantly higher than IL-6 and Vector (Vector) treatment group, this result indicates that IL-1 5 itself can promote the activation of natural killer cells. C.B-17 SCID mouse. An immunodeficient strain caused by a genetic defect caused by artificial selection, which does not have mature functional 14 1278320 T cells and B cells. However, other immune cells, including myeloid cells, antigen-presenting ceiis (APCs), and natural killer cells, all have normal functions. Here, the C.B-17 SCID mice were subjected to xenotransplantation of canine infectious squamous tumors, and then IL-6 and IL-15 plastids were fed by muscle biopsy. The test was divided into two parts. The first part evaluated the effects of both IL-6 and IL-15 on tumor formation; the second part further observed the inhibitory effect of this dicytokine on the formed tumor. The results showed that in the formation of tumors, IL-6 and IL-15 were combined to have the best inhibitory effect on the tumor. IL-15 alone had a slight delay in the growth of canine infectious sputum tumors, but IL-6 tumors alone had no significant effect. However, when tumors have been formed, only mice that use IL-6 and IL-15 plastids can slow the growth rate of canine infectious tumors. Only IL_6 or IL-15 plastids can not be stopped. The continued growth of the tumor. If the anti-asiai GM-1 antibody is administered by intraperitoneal injection and the IL-6 and IL-15 plastids are simultaneously received, the canine infectious sputum tumor continues to grow in the body. This result demonstrates that natural killer cells play a decisive role in this complex gene therapy. Example 1 Acquisition and Synthesis of IL-6 and IL-15 Genes The IL-6 gene line was obtained from the NCBI pubmed (hup://www.ncbi.nlm.nih.gov), GenBank accession number. (Accessi〇n 15 1278320

No.) is the mRNA sequence of human IL-6 of NM-000600, and the IL-6 gene (63th to 701th base pair, SEQ ID NO.·1) having a total length of 636 base pairs is amplified. Referring to the design of Kazuhiro et al. (2001), the human IL-2 signal peptide gene (IL-2SP, 60 base pairs, the gene database accession number V00564 and U14407 mRNA sequences were synthesized by artificial synthesis. 48 to 107 base pairs, SEQ ID NO: 2) the entire peptide gene of human IL-1 5 gene (IL -1 5MP, 342 base pairs, 461 to 802 base pairs) , before SEQ ID NO: 3), constitutes a chimeric gene sequence (IL-2SP/IL-15MP chimeric gene, SEQ ID NO: 4), which is designed to overcome IL-1 5 during translation and transcription. A number of adjustment points must pass, resulting in a very low production of IL-15 protein. In the case of increasing the amplitude, in the case of the sense strand, the gene sequence to be combined is expressed in units of 30 bases, and the sequence of 402 bases in total is written from the 5' end into a primer (a total of 26 primers). ), and the antisense strand is designed from the 15th base of the IL-2SP gene. Each primer of the reverse strand has exactly 15 bases with the sequence of the two forward strands. The overlap of the bases serves as a bridge for the connection. Next, the above pcDNA3 plastid (occurring from Taitung Toxicology Institute) in which the human IL-6 gene is embedded is amplified by a conventional polymerase chain reaction technique and purified to obtain a desired DNA product. As for the human IL-2SP/IL-15MP chimeric gene, the aforementioned 26 primers are placed together, and amplified by a conventional polymerase chain reaction technique and purified to obtain a desired DNA product. Because the designed primer has a reverse 16 1278320 strands and 15 strands of the forward strands of the front and rear sequences, these primers will bind to each other under the action of DNA hydratase, and the bound genes form the original. The template, the polymerase chain reaction can be used to obtain the IL-2SP/IL-15MP chimeric gene. Example 2 In vitro spleen natural killer cell killing test in vitro The BALB/c mice (taken from the National Taiwan University Animal Center and the National Animal Center) at 6-8 weeks of age were sacrificed, and the spleen was removed and placed in RPMI-1640 medium. Prepare a single cell suspension in a cell grinder, remove the supernatant by centrifugation (丨, 5〇·〇 rpm, 4C'10 minutes), then add 5ml of IX ACK lysis buffer (10 X ACK lysis buffer· : 0.15 M NH4C1, 1.0 mM KHC03, 〇·1 mM Na2EDTA, dissolved in double distilled water), allowed to stand at room temperature for 5 minutes to dissolve red blood cells. Join again! The rPMI_丨64〇 culture solution of 〇mi was centrifuged at 1,500 rpm for 10 minutes at 4 C. After removing the supernatant, wash it again with RPMM 640 medium. After cell counting, 'diluted in LAK medium [RPMI -1 0 containing 2-mecaptoethanol: 50μΜ and IL-2: 500U/lxl06 living cells], in a 24-well culture plate, put 2ml/2xl〇6 live in each well. The cells were cultured for 4 to 6 days at 37 ° C in a 5% CO 2 incubator, and IL-1 : 5 〇〇υ / ΐχΐ〇 6 viable cells were added to the third day of culture, and cultured for 4 to 6 days. To evaluate the effects of IL-6, IL-15 and transforming growth factor on the natural killer cell cytotoxicity of mouse spleen cells, spleen cells are cultured with lymphokines to activate killer cells, but the added cytokines are IL- 6, IL-15 and transforming growth factor replacement. 17 1278320 YAC-1 mouse lymphoma cells (ATCC No. TIB-160) were washed twice with RPMI-1640 medium (1,500 rpm, 4 ° C, 10 minutes) and counted for cells [cone blue exclusion method ( Trypan blue exclusion test)], suspended in 5x105 viable cells/ml in RPMI-10 [cell number depending on the effector cells (Effector cells)]. Thereafter, 10 pl 3,3'-dioctadecyloxacarbocyanine (DIOC 18, Sigma, MO, USA) fluorescent dye was added to each 5 x 105 viable cells, and reacted at 37 ° C in a 5% CO 2 incubator for 16 hours. YAC-1 cells were then washed twice (1,500 rpm, 4 ° C, 10 minutes) in RPMI-10. The treated spleen cells were mixed with YAC-1 cells in different ratios (Effector/Target ratio, E/T ratio = 50/1, 25/1, 12.5/1, 6.25/1, 3.125/1) at the bottom. In a 96-well plate (up to 200 μl per well), briefly centrifuge at 1,100 rpm for 4 minutes at 4 °C. It was allowed to act for 4 hours in a dark room at 37 ° C in a 5% CO 2 incubator. The cells were stowed, and then mixed with a cell volume of 1/100 of propidium iodide (PI) (2500 pg/ml) (Sigma, MO, USA), and finally a flow cytometer (FACSCalibur flow cytometer). , Becton Dickinson, NJ? USA) Tested and analyzed with CellQuest software. After stimulation of spleen cells of B ALB/c mice with different doses of IL-6, it was found that the death rate of YAC-1 mouse lymphoma cell line did not increase with the increase of IL-6 dose, and was above 800 U. Under the action of IL-6, the killing ability of natural killer cells began to decline (first figure). In the IL-15 aspect, activation of spleen cells with 400 U of IL-15 resulted in a natural killer cell killing ability and 2000 U of IL-2 activated phase 18 1278320 near; when IL-2 or IL-15 and When the transforming growth factor is added together to culture the cells, it can be seen that the transforming growth factor completely inhibits the killing ability of the lymphocyte-activated killer cell cells activated by the IL-2 group, and only partially inhibits the nature of the IL-15 group. The killing ability of killer cells, this result shows that IL-15 itself is superior to IL-2 in the ability to activate natural killer cells and to restore the natural killer cell cytotoxic activity inhibited by transforming growth factors. In addition, when IL_6 and the transforming growth factor were cultured together with IL-15 and the transforming growth factor and the mouse spleen cells, IL-6 was found here and compared with the treatment group in which only the transforming growth factor was added. It does not contribute to the increase in the killing activity of natural killer cells, while IL-15 can slow & enhance the natural killer cell killing ability inhibited by the transforming growth factor. However, if IL-6 and IL-15 are combined, the improvement effect will be very significant. The results of the poisoning of the transforming growth factor group and the IL_6 + transforming growth factor group were consistent with those of the natural killer cells, and the antagonistic effect of IL-6 on the transforming growth factor could not be seen in the results (Fig. 3). Example 3 Effects of Electron Reperfusion (EP) on IL-6 and IL-15 Genes in BALB/c Mice on Natural Killer Cell Toxicity The BALB/c mice were divided into four groups, respectively (l pcDNA3.1/V5/His/TOPO (Mock) vector lOOpg, (2) IL-6 plastid lOOpg, (3) IL-15 plastid 1 ton, and (4) IL-6 and IL-15 The plastids were combined using each l〇〇pg, with 6 mice per group. The IL-6 and IL-15 plastids were adjusted to a concentration of 1 mg/ml in physiological saline before electroporation. 19 1278320 After anesthetizing BALB/C mice, extract the plastids of ι〇〇μι, and inject 50μ of the muscles on both sides of the mice for 2 minutes to spread the gene evenly in the muscles. Using Electron Poroscope (Electro Square Porator) , BTX ECM 830) Perform muscle electrical impulses (electrode insertion depth 〇 5 cm, loo volts, times, 50 ms each). Blood was collected from Dijon, 3, 8, 12, 14, 15, 20, 25, 27, and 30 days from the date of electroporation, and the obtained blood samples were centrifuged and stored in -20. (:. In order to understand whether IL-6 and IL-15 proteins in the living body can express IL-6 and IL-15 proteins normally, blood collection is performed at a fixed time point after electroporation and electroporation. Serum, linked to the Epidemic Adsorption Assay Kit by Commercial Enzyme (IL-6: Endogen, MA, USA; IL-15:

Bi〇s〇Urce, CA, USA) detects changes in the serum levels of _6 and IL-15 by conventional techniques. The results of the test showed that the two plastids produced protein negative in mice. IL-6 in serum can be measured in each experimental group, but the concentration is high and the performance time is different. The concentration of _15 in serum was only detected in the IL 1 5 and IL-6 + IL-15 treatment groups. (Test results not shown) On the 14th day after muscle electroporation in 4 groups of mice, each group was spleenly collected to collect spleen cells to test lymphocyte population B and natural killer cells in the spleen and natural killer cells. Whether the poisoning ability has occurred. The spleen of the mouse after electroporation of the muscle living body is taken out, and the spleen cells are ground to obtain spleen cells containing lxl〇7 living cells/ml 20 1278320 1 00 μb and various monoclonal antibodies of the following binding fluorescent substances Contains: rat anti mouse CD3-FITC (Serotec, Oxford, UK) ^ rat anti mouse CD19-FITC (Serotec, Oxford, UK) ^ rat anti mouse NK1.1-FITC (PharMingen), and its isotype. Each was added to ΙΟΟμί spleen cells at lpg, and washed at 4 ° C for 45 minutes in the dark in 1X PBS, followed by 500 pg / ml mothidine to identify the cell survival. Finally, the assay was performed by a flow cytometer (FACSCalibur flow cytometer, Becton Dickinson, NJ, USA). The results of analysis by flow cytometry showed that the ratio of T cells and B cells in spleen cells in the 4 treatment groups were very similar, and there was no significant difference between them (Fig. 8 and B); In terms of natural killer cells, it was apparent that the ratio of the treatment groups using IL_6 and IL_15 was significantly increased (fourth panel). At the same time 'natural killer cell poisoning in natural killer cells can be in the other 3 groups; IL-15 killing ability' IL · 6 + il-15 treatment group force regardless of any E / D ratio, are the excellent group Then the E/T ratio = 50 / θ 19 ς / 1 # # _ hand 50 / and 12.5 / 1 is significantly higher than the M 〇 ck and IL _ plastid groups, and at 3 12S / 1 η 士 』 廿 α. There was no significant difference between the two groups in the sputum and the other two groups; the IL- and Mock-treated groups had no significant difference in the killing power of the natural killer cells under the ^ ^ 1 than the sheep. (Picture 5) The combined use of IL_6 and 1L-15 has little inhibitory effect on the inoculation of C.B_17Scid's stained canine tumors - the use of surgical non-surgical methods to adopt human q species on dogs 21 1278320 Infectious flowering Tumors, ground and ground, and the tumor tissue was made into a single cell suspension through a double stainless steel mesh (N〇. 25 stainless steel mesh) and the canine infectious cannabis was isolated using 42% Percoll (Amersham pharmacia biotech, NJ, USA). Tumor cells were determined by viable stain (cone staining method). The lxlO8 live tumor cell suspensions were each inoculated into the subcutaneous back of CB-17 SCID mice using a 18-inch syringe. At the time of inoculation, the tumor growth state was measured twice a week using a vernier scale (Aer〇space, China). The formula for calculating the size is V=tcxLxWxH/4 [V: volume (cm3), L: length (cm), W : Visibility (cm), Η: thickness (cm)] - On the 7th day of CB-17 SCID mice vaccinated with canine infectious sputum tumors (when the tumor has not yet formed), the muscle living gene Rushing, used to observe IL-6 The effect of IL-15 on the formation of canine infectious tumors. It was found that CB-17 SCID mice with IL-6 and IL15 plastids began to grow into visible flat tumors on day 14 The mass (2~3mm in diameter), and its tumor volume was significantly smaller than other treatment groups during the observation period. However, the mice with IL_1 5 plastid alone had a smaller tumor volume than the IL-6 and vehicle treatment groups. However, there was no statistically significant difference (p>〇.〇5). The growth rate of canine infectious lymphoid tumors in the IL_6 plastid group was similar to that in the vehicle-treated group, and there was no statistically significant difference. -asialo GM-1 antibody (Wak〇, Osaka, Japan) was reconstituted with 1 ml of water for injection, and each mouse was intraperitoneally injected with 3 μl twice a week to block the action of natural killer cells and then apply IL. -6 and 22 1278320 gene electroporation. The results showed that the tumor growth rate was significantly accelerated, and the tumor volume was also significantly larger than other treatment groups (Fig. 6). In addition, CB-17 sciD small breast was inoculated with canine infectious sputum tumor. Growing to the diameter of the tumor When the skin was 5 mm, the electrophysical electrofusion was started. The results showed that the treatment group with IL_6 and IL-15 combined had a significantly slower tumor growth rate after electroporation, and there were 3 tumors (N=6, each The mice inoculated on both sides of the back) completely resolved and no longer grew. The average tumor volume of this group during the observation period was significantly lower than that of the other three groups (carrier plastid group, !" and IL_15 plastid group). Compared with the combined use, the inhibition effect was not obvious when IL-6 and IL_15 were used alone, and the volume of the negative control group of the Mock plastid was similar (seventh figure). To further understand whether the tumor suppressive effects exhibited by the combined use of IL_6 and IL_15 are related to natural killer cells, so the design was additionally set to *C.B-17 SCID mice were injected with Anti_asial〇 gm" antibody to block the function of natural killer cells. The results showed that mice administered with gm_丄 antibody did not suppress the growth of tumors even if they were given the same amount of IL_6 and IL_15 plastids, and their mortality increased significantly (Fig. 8 and Fig. ). 23 1278320 [Simple description of the diagram] The effect of IL-6 on the killing ability of natural killer cells. The ratio in the figure is 13/1, and each treatment group performs three repetitions (Ν==3). The χ axis represents the different IL-6 dose; the Υ axis is the percentage of YAcq cells that are sterilized. The second panel compares the reversal effect of IL-15 and IL-2 on the natural killer cell killing ability inhibited by transforming growth factor (TGF_p). In the figure, the Ε/τ ratio is 13/1, and each treatment group performs three repetitions (N==3). χ

The axis table has different treatment groups; the axis is the percentage of YAC-i cells that are poisoned. The third panel combines the effects of IL-6 and IL-15 on the natural killer cell activity inhibited by the transforming growth factor. In the figure, the E/butyl ratio is 13/1, and each treatment group is subjected to three repetitions (Ν==3). The x-axis table was different from the treatment group, and the x-axis was the percentage of YAC-1 cells that were poisoned. The fourth figure shows that the muscle living organisms are electrically injected into the IL_6 and IL_15 plastids.

The distribution of spleen lymphocyte populations in B ALB/c mice. (N=3) A · The percentage of CD3+ cells in each treatment group. B: Percentage of CD 19+ B cells in each treatment group. C · The percentage of NK1 · 1+ NK cells in each treatment group. Figure 5 shows the test of the natural killer cytotoxicity of spleen cells after the introduction of IL_6 and IL-15 plastids into BALB/c mice. (N=4) ▲: Table Mock plastid group; □: Table IL-6 plastid group; 24 1278320 Figure 6: Table IL-15 plastid group; ♦: Table IL-6+IL-15 plastid The effect of group immuno-gene therapy on the formation of canine infectious tumors. Each group of treatment groups contained 5 mice (N=5). ▲ ·· Table Mock plastid group; □: Table IL-6 plastid group; ◊ • Table IL-15 plastid group; ♦: Table IL-6+IL-15 plastid group; _ : Table applied Anti- The asialo GM1 antibody was also subjected to the IL-6+IL-15 plastid group; Figure 7 X: Table normalized rabbit sera received the IL-6+IL-1 5 plastid group. A graph of the growth curve of infectious canine tumors in dogs treated with compound immunogenic therapy. There were 6 mice per treatment group (N==6). - ▲: Table Mock plastid group; □: Table IL-6 plastid group; Figure 8 ◊ • Table IL-1 5 plastid group; ♦: Table IL-6+IL-1 5 plastid group. The effect of combined immunotherapy on survival rate of mice (N==5~6). ▲: Table Mock plastid group; □: Table il-6 plastid group; ◊: Table IL-15 plastid group; ♦: IL-6+IL-15 plastid group· _ : Table applied Anti-asialo GM1 The antibody received the IL-6+IL-15 plastid group. Figure IX Effect of blocking natural killer cells on the growth of canine infectious squamous tumors in composite immunogenic therapy (n=:6). + : Anti-asialo GM1 antibody group; - 25 1278320 Sequence Listing <110> National Taiwan University<120>-complex immunogenic drug composition for inhibiting tumor cells <160>4 <210><211>636<212> mRNA and translation PRT < 213 > human IL-6 gene <300><308> NCBI pubmed Genbank; Accession No. : NM 000600 <400>1 63 78 93 108

ATG AAC TCC TTC TCC ACA AGC GCC TTC GGT CCA GTT GCC TCC TCC CTG GGG CTG CTC CTG 60 MNSFSTSAFGPVAFSLGLLL 123 138 153 GTG TTG CCT GCT GCC TTC CCT GCC CCA GTA CCC CCA GGA GAA GAT TCC AAA GAT GTA GCC 120 VLPAAFPAPPGPGEDSKDVA 183 198 213 228 GCC CCA CAC AGA CAG CCA CTC ACC TCT TCA GAA CGA ATT GAC AAA CAA ATT CGG TAC ATC 180 APHRQPLTSSERIDKQIRYI 243 258 273 288 CTC GAC GGC ATC TCC GCC CTG AGA AAG GAG ACA TGT AAC AAG AGT AAC ATG TGT GAA AGC 240 LDGISALRKETCNKSNMCES 303 318 333 348 AGC AAA GAG GCA CTG GCA GAA AAC AAC CTG AAC CTT CCA AAG ATG GCT GAA AAA GAT GGA 300 S KEALAENNLNLPKMAE KDG 363 378 393 408 TGC TTC CM TCT GGA TTC AAT GAG GAG ACT TGC CTG GTG AAA ATC ATC ACT GGT CTT TTG 360 CFQSGFNEETCLVKIITGLL 423 438 453 468 GAG TTT GAG GTA TAC CTA CAG TAC CTC CAG AAC AGA TTT GAG A GT AGT GAG GAA CAA GCC 420 EFEVYLEYLQNRFESSEEQA 483 498 513 528 AGA GCT GTG CAG ATG AGT ACA AAA GTC CTG ATC CAG TTC CTG CAG AAA AAG GCA AAG AAT 480 RAVQMSTKVLIQFLQKKAKN 543 558 573 588 CTA GAT GCA ATA ACC ACC CCT GAC CCA ACC ACA AAT GCC AGC CTG CTG ACG AAG CTG CAG 540 LDAITTPDPTTNASLLTKLQ 603 618 633 648 GCA CAG AAC CAG TGG CTG CAG GAC ATG ACA ACT CAT CTC ATT CTG CGC AGC TTT AAG GAG 600 AQNQWLQDMTTHLILRSFKE 1 1278320 663 678 693

HC CTG CAG TCC AGC CTG AGG GCT CIT OGG CAA ATG 636 FLQSSLRALRQM <210>2 <211>60 <212> mRNA and translation PRT <213>human IL-2 signal peptide <300><308>NCBI pubmed Genbank ; Accession No. : V00564 <400>2

ATG TAC AGG ATG CAA CTC CTG TCT TGC ATT GCA CTA AGT CTT GCA CTT GTC ACA AAC AGT MYRMQLLSCIALSLALVTNS <210>3 <211>342

<212> mRNA and translation PRT <213> Human IL-15 translatable partial gene of protein <300><308> NCBI pubmed Genbank; Accession No. : U14407 <400>3

AAC TGG GTG AAT GTA ATA AGT GAT TTG AAA AAA ATT GAA GAT CTT ATT CAA TCT ATG CAT

N W V N V I SDLKK I EDL I Q SMH

ATT GAT GCT ACT m TAT ACG GAA AGT GAT GH CAC CCC AGT TGC AAA GTA ACA GCA ATG

IDATLYTESDVHPSCKVTAM

MG TGC TTT CTC TTG GAG TTA CM GTT ATT TCA CTT GAG TCC GGA GAT GCA AGT ATT CAT KCFLLELQVISLESGDASIH

GAT ACA GTA GAA AAT CTG ATC ATC CTA GCA AAC AAC AGT TTG TCT TCT AAT GGG AAT GTA DTVENLI ILANNSLSSNGNV

ACA GAA TCT GGA TGC AAA GAA TGT GAG GAA CTG GAG GAA GAA AAA AAT ATT AAA GAA TTT TTG TESGCKECEELEEKNIKEFL

CAG AGT TIT GTA CAT ATT GTC CAA ATG Ή0 ATC AAC ACT TCT 342 QSFVHIVQMFINTS 2 1278320 <21〇>4 <211>402 <212>mRNA and translation PRT <213>IL-2SP/IL-15MP Artificial Sequence <220> <223> Human IL_2 signal peptide binds to human IL-15 to translate partial gene of protein <300><308>NCBI pubmed Genbank; IL-2 Accession No. : V00564 IL-15 Accession No . : U14407 <400>4

ATG TAC AGG ATG CM CTC CTG TCT TGC ATT GCA CTA AGT CTT GCA CTT GTC ACA AAC AGT MY RMOLLSCIALSLA LVTNS IL-2 Signal Peptide Gene '

AAC TGG GTG AAT GTA ATA AGT GAT TKj AAA AAA ATT GAA GAT CIT ATT CM TCT ATG CAT NWVNVISDLK KIEDLIQSMH

ATT GAT GCT ACT m TAT ACG GAA AGT GAT GTT CAC CCC AGT TGC AAA GTA ACA GCA ATG ID ATLYTESDVHPSCKVT AM

AAG TGC TIT CTC m GAG TTA CM GTT ATT TCA CTT GAG TCC GGA GAT GCA AGT Απ CAT KCFLLEL Q VISLESGDASIH

GAT ACA GTA GAA AAT CTG ATC ATC CTA GCA AAC AAC AGT TTG TCT TCT AAT GGG AAT GTA DTVENLI ILANNSLSS NGNV

ACA GAA TCT GGA TGC AAA GAA TGT GAG GAA CTG GAG GAA GAA AAA AAT ATT AAA GAA ΤΤΓ Ήΰ TESGCKECEELEEKNIKEFL

CAG AGT TTT GTA CAT ATT GTC CAA ATG TTC ATC AAC ACT TCT 402 QSFVHIVQMFINTS 3

Claims (1)

1278320---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- The plastid and an effective dose of the gene fragment of Inter-6 (Interleukin-6, IL-6, SEQ ID NO: 1) can express Interleukin-15 (IL-15, SEQ ID NO: 3) Gene fragment The plastid, the tumor cell has a characteristic of a major histocompatibility complex (MHC) molecule that is low-performance or non-expressed and produces a transforming growth factor-β (TGF-β). 2. The composite immunogenic pharmaceutical composition according to claim 1, wherein the composite immunogenic pharmaceutical composition antagonizes the transforming growth factor to the host body naturally by the interleukin, -6 The killer cell is inhibited to release the immunosuppressive state of the host, and the natural killer cell is activated by the interleukin-i 5 to enhance the host immune system and achieve the purpose of inhibiting the growth of the tumor cell. The composite immunogenic pharmaceutical composition as described in claim 1, wherein the tumor cell line is a canine infectious tumor cell. 4 · A pharmaceutical composition for inhibiting tumor cells, the pharmaceutical composition being at least An effective amount of interleukin-6 (SEQ ID NO: 1) expressing protein and an effective amount of interleukin-15 (SEQ ID NO: 3) expressing protein having a histocompatibility complex (major) Histocompatibility complex (MHC) is characterized by low or no expression of molecules, and the production of transforming growth factor-β (TGF-β). A pharmaceutical composition for treating tumor cells, the pharmaceutical composition comprising at least an effective amount of a carrier containing interleukin-6 (SEQ ID NO: 1) and capable of causing its expression and an effective dose of interleukin-15 ( SEQ ID NO. 3) and a vector capable of expressing the tumor cells having a low or no expression of a major histocompatibility complex (MHC) molecule, and a transforming growth factor -β, TGF-β) characteristics. 27