Enhanced antitumor immune responses of IL-2 gene-modified tumor vaccine by combination with IL-1 and low dose cyclophosphamide.

To enhance the antitumor immunity induced by IL-2 gene-modified tumor vaccine, we proposed a combined protocol to treat tumor-bearing mice using IL-2 gene-modified tumor vaccine in combination with IL-1 and low-dose Cyclophosphamide(Cy). After treatment with IL-2 gene-modified B16 melanoma cell vaccine alone, the pulmonary metastases of tumor-bearing mice were reduced and their survival time was prolonged. The anti-metastases effect was improved when the vaccine was used in combination with IL-1 or low-dose Cy. The best therapeutic effect was achieved when the IL-2 gene-modified vaccine was combined with IL-1 and low-dose Cy. The cytotoxicity of the splenic CTL, NK, and the levels of IL-2, TNF secreted by splenocytes increased after tumor-bearing mice were treated with the IL-2 gene-modified tumor vaccine. The above antitumor immune functions were augmented more significantly when IL-1, low-dose Cy were used in combination with IL-2 genemodified tumor vaccine. These results demonstrated that the IL-2 gene modified vaccine could exert more potent anti-metastases effects when it is combined with IL-1 or/and low-dose Cy by activating the specific and non-specific antitumor immune responses more effectively.     

腺病毒介导的IL—2基因及B7—1基因联合转染G422细胞致瘤原性和免疫原性的变化

目的研究联合细胞因子基因转染的D422胶质母细胞瘤细胞体内致瘤原性和免疫原性的变化,为胶质瘤的免疫基因治疗打下基础.方法IL-2基因和B7-1基因转染的G422细胞1×105皮下和脑内接种,观察肿瘤生长速度和荷瘤小鼠的存活期,2周取脾脏,检测NK、LAK和CTL的杀伤活性.结果IL-2和B7-1基因联合转染的G422细胞,皮下接种后肿瘤生长明显减慢,脑内接种动物存活期明显延长,NK、LAK和CTL的杀伤活性增强.结论IL-2基因和B7-1基因联合转染的G422细胞,致瘤原性下降,免疫原性增强,能有效激活机体特异性与非特异性抗肿瘤免疫反应.     

瘤内／瘤周注射巨噬细胞集落刺激因子或（和）干扰素—γ基因转染的 …

目的 观察巨噬细胞集落刺激因子（Ｍ－ＣＳＦ）和干扰素－γ（ＩＦＮ－γ）基因单独或联合转染的巨噬细胞对局部黑色素瘤的治疗效果及相关免疫机理。     

单克隆抗体MGb2和LAK细胞的协同抗胃癌作用

本研究探讨了成纤维细胞介导的G-CSF基因疗法联合应用IL-2基因疗法及时对小鼠I期肾癌的治疗作用。经NIH3T3-IL-2基困疗法．NIH3T3-G-CSF基因疗法单独治疗后的荷瘤小鼠存话期明显延长，而经以上两者联合治疗后的荷癌小鼠存活期的延长更为明显，且有75％荷瘤小鼠长期存活。对经G-CSF基因疗法及IL-2基因疗法联合治疗后的荷瘤小鼠体内抗肿瘤免疫功能的检测表明，经治疗后第14天，荷瘤小鼠睥脏明显增大，睥脏淋巴细胞数量明显增多；肿瘤局部的常规病理检查可见数量较多的嗜酸性粒细胞浸润。荷瘤小鼠睥细胞NK活性、经诱导后的LAK话性及CTL，杀伤活性均明显升高，小鼠腹腔巨噬细胞杀伤活性在NIH3T3-IL-2基因疗法治疗组升高．而在NIH3T3-G-CSF基因疗法组未见明显升高。以上结果表明．联合应用成纤维细胞介导的G-CSF基因疗法与IL-2基因疗法可因对小鼠体内抗肿瘤免疫功能的联合增强作用而取得更佳的抗肿瘤效果。     

NK and CD8+ T cell-mediated eradication of poorly immunogenic B16-F10 melanoma by the combined action of IL-12 gene therapy and 4-1BB costimulation

In previous reports, systemic administration of a stimulatory monoclonal antibody directed against the 4-1BB receptor had no effect on survival or tumor burden in mice inoculated with the poorly immunogenic B16-F10 melanoma. We combined IL-12 gene transfer with 4-1BB costimulation to explore a previously noted cooperative anti-tumor effect against this model tumor. We hypothesize that the innate immune response mediated by IL-12-activated natural killer (NK) cells initiates the activation of the immune system, leading to the priming of T cells, whereas 4-1BB costimulation enhances the function of primed tumor-specific T cells. The effect of the combination therapy on the growth of subcutaneous (s.c.) tumors and pulmonary metastasis was examined. The combination therapy significantly retarded the growth of subcutaneously-inoculated tumors, and 50% of tumor-bearing mice survived with complete tumor regression. In contrast, neither IL-12 gene transfer nor anti-4-1BB antibody administration alone was as effective. Enhanced CTL activity against both B16-F10 tumor cells and TRP-2-pulsed EL4 syngeneic tumor cells was observed in tumor-bearing animals treated with the combination therapy 2 weeks after treatment and, in long-term survivors from this combination therapy, at >120 days. In a pulmonary metastatic model, only the combination therapy generated significant protection against metastasis. In vivo depletion of NK or CD8(+) but not CD4(+) subsets eliminated the protective immunity. Furthermore, NK cell depletion significantly reduced both tumor-specific CTL activity and the number of tumor-specific IFN-gamma-producing cells, suggesting that this synergistic effect requires the participation of both NK and CD8(+) T cells.     

联合自杀基因与IL-2基因转移疗法的抗肿瘤效果及其抗肿瘤免疫应答的诱导作用

单用自杀基因疗法或单用细胞因子基因疗法抗肿瘤效果不理想,本研究中我们观察了大肠杆菌胞嘧啶脱胺酶(CD)基因与白细胞介素2(IL-2)基因联合转移对荷瘤小鼠的治疗效果及其对抗肿瘤免疫的诱导作用。复制荷瘤小鼠模型后在荷瘤部位注射表达CD基因的重组腺病毒(AdCD)及表达小鼠IL-2基因的重组腺病毒(AdIL2),并连续10天、每天1次腹腔注射5氟胞嘧啶(5FC)对荷瘤小鼠进行治疗。结果表明,AdCD/5FC/AdIL2联合基因治疗能显著抑制荷瘤小鼠皮下肿瘤的生长,并明显延长其生存期(P<0.01)。联合基因治疗组小鼠肿瘤细胞发生明显的坏死,瘤内及瘤周有大量的炎性细胞浸润,瘤内CD4~ 和CD8~ T细胞明显增加,脾细胞NK和CIL杀伤活性明显高于单用AdCD/5FC、对照病毒AdLacZ/5FC或PBS组。实验结果表明,联合应用自杀基因与细胞因子基因治疗可更有效诱导机体的抗肿瘤免疫反应,从而更显著地抑制荷瘤小鼠肿瘤的生长。     

Therapeutic effects of adoptive splenocyte transfer following in situ AdIL-12 gene therapy in a mouse prostate cancer model

We developed a preclinical prostate cancer model to study the feasibility of adoptive immunotherapy for residual tumor following neo-adjuvant in situ adenoviral-vector-mediated interleukin 12 (AdIL-12) gene therapy. Splenocytes were obtained from mice with orthotopic 178-2 BMA metastatic mouse prostate cancers treated previously with AdIL-12, or a vector with the IL-12 genes plus the costimulatory gene B7-1 (AdIL-12/B7), or a control gene (Adbetagal). The splenocytes were subsequently injected intravenously into syngeneic mice bearing orthotopic 178-2 BMA tumors generated 3 days previously. Significant orthotopic tumor growth suppression was achieved with splenocytes derived from mice whose tumors had been injected with AdIL-12 compared to splenocytes from control Adbetagal mice (P = 0.0005) and splenocytes from AdIL-12/B7-treated mice significantly suppressed spontaneous lung metastases compared to splenocytes from control mice (P = 0.0356). Adoptive transfer of splenocytes from either AdIL-12 (P = 0.004) or AdIL-12/B7 (P = 0.009)-treated mice significantly prolonged survival relative to controls. Transfer of NK and tumor-specific CTL activities was detected and depletion of CD4+ and CD8+ T cells by in vitro antibody-mediated complement lysis of the splenocytes prior to injection abrogated the effects. Systemic IL-12 administration delivered by intramuscular AdIL-12 injection enhanced the antitumor effects of adoptive splenocyte transfer and boosted the CTL response. Our data provide evidence that this form of adoptive immunotherapy can enhance the effectiveness of neo-adjuvant in situ IL-12 gene therapy in cases of persistent malignancy.     

腺病毒介导的人GM-CSF基因转染瘤苗增强机体免疫功能的实验研究

目的 研究腺病毒介导的人ＧＭ－ＣＳＦ基因转染瘤苗体内抗肿瘤免疫作用极其机理。方法 应用ＧＭ－ＣＳＦ基因转染、的瘤苗对小鼠肝癌模型进行免疫基因治疗,观察该疗法对荷瘤小鼠脾细胞ＮＫ、ＬＡＫ、ＣＴＬ活性的影响、脾淋巴细胞围化反应的影响以及荷瘤习的生存期。结果 经ＧＭ－ＣＳＦ基因转染瘤苗治疗后,插细胞ＣＴＬ活性显著升高,而ＮＫ、ＬＡＫ细胞活性未见明显增强、脾淋巴细胞转化反应显著增强、生存期显著延长。结论     

Correlation between reduced p21(WAF1/CIP1) expression and abnormal p53 expression in esophageal carcinomas

Direct gene transfer into somatic tissue iii vivo is a developing technology with potential application for cancer gene therapy. In this study, recombinant vaccinia virus encoding human IL-2 gene (rVV-IL-2) was used as a candidate vector in mediating iii vivo gene therapy. After rVV-IL-2 was expanded in VERO cells for 72 h, high titer (10(8)-10(10) PFU/ml) rVV-IL-2 were harvested. When 10(6) murine melanoma cells (F16-F10) were infected with rVV-IL-2, about 200 U/ml IL-2 activity was detected in the supernatants at 8 h, and the up-regulation of ICAM-1 and MHC-I expressions on the melanoma cells were observed. The treatment of murine melanoma model by local injection of rVV-IL-2 into the tumor site showed that rVV-IL-2 transfection significantly inhibited the tumor growth and prolonged the survival time of tumor-bearing mice. The splenocytes from rVV-IL-2 treated mice showed higher cytotoxicities of NK, LAK and CTL in comparison with those from the controls. These results suggest that in vivo transfection mediated by rVV-IL-2 has potential effectiveness in enhancing host immunity and would be a useful approach to cancer gene therapy.     

A convenient cancer vaccine therapy with in vivo transfer of interleukin 12 expression plasmid using gene gun technology after priming with irradiated carcinoma cells

We studied interleukin (IL)-12 gene therapy using a gene gun as a new autologous vaccination strategy for cancer. In the first experiment, BALB/c mice were inoculated with syngeneic murine renal cancer cells (Renca) intradermally in the abdomen. This was followed by an injection of IL-12 expression plasmid using the gene gun. About 40% of the mice exhibited rejection of the tumor after the treatment and these mice also acquired immunological resistance against a secondary challenge with Renca cells. Based on these results, we examined whether antitumor activity can be potentiated when mice undergo combination treatment with intradermal inoculation of irradiated Renca cells and transfection with IL-12 gene. Inoculation of irradiated Renca cells alone was partially effective in inducing antitumor immunity, whereas the combined treatment remarkably intensified this effect. Moreover, this combined treatment inhibited tumor establishment and enhanced survival of the mice with tumor infiltration by CD4(+) and CD8(+) T cells, even when the treatment was started after tumor-implantation at a distant site. This antitumor effect was antigen specific and we confirmed the induction of antitumor cytotoxic T cells by this treatment. These results show that local cutaneous transfer of IL-12 expression plasmid using gene gun technology enhances systemic and specific antitumor immunity primed by irradiated tumor cells.     

重组白介素-12治疗小鼠Lewis肺癌的实验研究

背景与目的:白介素-12(interleukin-12,IL-12)是具有抗瘤活性的细胞因子,本研究建立稳定表达小鼠IL-12(murineinterleukin-12,mIL-12)的小鼠Lewis肺癌(Lewislungcarcinoma,LLC)细胞系LLC/mIL-12,评估mIL-12重组质粒及LLC/mIL-12瘤苗治疗小鼠Lewis肺癌移植瘤的疗效。方法:脂质体转染LLC获得LLC/mIL-12瘤苗。于C57BL/6小鼠皮下接种LLC细胞2×106个,成瘤后将小鼠随机分成4组(n=10),第1、4、7天瘤内注射质粒或瘤苗,第14天处死小鼠,观察肿瘤生长曲线、脾细胞中CTL细胞和NK细胞活性以及肿瘤浸润淋巴细胞。结果:LLC/mIL-12瘤苗组和pcDNA3.1( )-mIL-12质粒组肿瘤体积显著缩小,mIL-12能增强NK细胞和CTL细胞活性,两者均以LLC/mIL-12治疗组更显著,LLC/mIL-12和pcDNA3.1( )-mIL-12治疗组有大量CD4 、CD8 淋巴细胞浸润。结论:mIL-12基因能增强NK细胞和CTL活性,LLC/mIL-12及pcDNA3.1( )-mIL-12均能产生抗瘤免疫反应,且前者作用更强。     

The synergistic antitumor effect of recombinant interleukin-1 and low-dose of cyclophosphamide in tumor-bearing mice

Intraperitoneal (i. p.) treatment of MOPC104E ascitic tumor-bearing BALB/c mice with interleukin-1 (IL-1) followed by a low dose of cyclophosphamide (CPA) resulted in synergistic prolongation of their survival time. This antitumor effect was abolished when administration of CPA preceded that of IL-1. The combined i. p. therapy also eradicated subcutaneous (s. c.) tumors, indicating a systemically operating antitumor mechanism. In Winn assay, splenocytes from MOPC104E-bearing mice treated with the combined therapy completely suppressed the growth of MOPC104E cells, but not that of another syngeneic tumor cell line, RL ♀ -8 cells. This tumor-neutralizing activity was completely abrogated by treatment with anti-asialo-GM1 or anti-Thy 1.2 and complement, and reduced by treatment with anti-Lyt2.2 and complement. Treatment of splenocytes with 1-leucine methyl ester (LeuOMe), which depletes natural killer (NK) cells and macrophages in vitro, did not affect the neutralizing activity. © 1994 Wiley-Liss, Inc.     

IL-2和IL-12基因联合治疗小鼠头颈鳞癌的实验研究

目的观察白细胞介素-2(IL-2)基因与白细胞介素-12(IL-12)基因联合治疗小鼠头颈鳞癌的疗效. 方法建立小鼠头颈鳞癌动物模型,在荷瘤部位将脂质体包裹的IL-2基因和IL-12基因直接注入肿瘤中,观察肿瘤大小变化,并检测此两种基因在肿瘤细胞中的蛋白表达情况、小鼠脾脏自然杀伤细胞(NK)和细胞毒T淋巴细胞(CTL)活性. 结果 IL-2基因和IL-12基因联合治疗组,肿瘤生长明显受抑制,疗效显著优于单独治疗组和对照组(P<0.01).在注射有IL-2、IL-12基因的肿瘤组织中,其相对应的IL-2、IL- 12蛋白水平明显升高,小鼠脾细胞NK活性和CTL杀伤活性增强. 结论 IL-2、IL-12基因治疗可抑制小鼠头颈鳞癌生长,提高机体的抗肿瘤免疫应答.二者联合应用,可产生协同效应并加强其抗肿瘤效果.     

Induction of potent antitumor immunity by intratumoral injection of interleukin 23-transduced dendritic cells

Dendritic cells (DCs) are potent antigen-presenting cells that play a critical role in priming immune responses to tumor. Interleukin (IL)-23 can act directly on DC to promote immunogenic presentation of tumor peptide in vitro. Here, we evaluated the combination of bone marrow-derived DC and IL-23 on the induction of antitumor immunity in a mouse intracranial glioma model. DCs can be transduced by an adenoviral vector coding single-chain mouse IL-23 to express high levels of bioactive IL-23. Intratumoral implantation of IL-23-expressing DCs produced a protective effect on intracranial tumor-bearing mice. The mice consequently gained systemic immunity against the same tumor rechallenge. The protective effect of IL-23-expressing DCs was comparable with or even better than that of IL-12-expressing DCs. IL-23-transduced DC (DC-IL-23) treatment resulted in robust intratumoral CD8(+) and CD4(+) T-cell infiltration and induced a specific TH1-type response to the tumor in regional lymph nodes and spleen at levels greater than those of nontransduced DCs. Moreover, splenocytes from animals treated with DC-IL-23 showed heightened levels of specific CTL activity. In vivo lymphocyte depletion experiments showed that the antitumor immunity induced by DC-IL-23 was mainly dependent on CD8(+) T cells and that CD4(+) T cells and natural killer cells were also involved. In summary, i.t. injection of DC-IL-23 resulted in significant and effective systemic antitumor immunity in intracranial tumor-bearing mice. These findings suggest a new approach to induce potent tumor-specific immunity to intracranial tumors. This approach may have therapeutic potential for treating human glioma.     

局部注射小鼠白细胞介素12逆转录病毒包装细胞对大鼠肝癌的治疗

目的:研究低剂量环磷酸胺(Cy)联合MHC Ⅰ类限制性肿瘤抗原多肽Mutl致敏、白细胞介素2(IL-2)基因修饰的树突状细胞(DCs)对转移性肺癌小鼠的治疗作用及其免疫学机理.方法:制备小鼠骨髓来源的DCs,用转移性Lewis肺癌特异性多肽Mutl预激经IL-2基因修饰的DCs联合低剂量Cy治疗转移性肺癌小鼠.通过FACS分析其脾细胞内T淋巴细胞比例的变化,~51Cr释放法检测CTL和NK细胞杀伤活性.结果:肿瘤抗原多肽致敏、IL-2基因修饰的DCs与小剂量Cy联合后,能比单用DCs更有效地治疗转移性肺癌,小鼠脾细胞中CD8~ T细胞和NK1.1~ 细胞明显比例升高,联合治疗组诱导出的CTL杀伤活性最高.结论:以肿瘤抗原多肽冲击致敏的IL-2基因修饰的DCs联合小剂量Cy能更有效地促进荷瘤宿主免疫应答,具有显著地体内抑制肺癌转移的效果.     

低剂量环磷酰胺增强MHCⅠ类限制性肿瘤抗原多肽冲击致敏的树突状细胞的抗肿瘤转移效果

目的:研究低剂量环磷酸胺(Cy)联合MHC Ⅰ类限制性肿瘤抗原多肽Mutl致敏、白细胞介素2(IL-2)基因修饰的树突状细胞(DCs)对转移性肺癌小鼠的治疗作用及其免疫学机理.方法:制备小鼠骨髓来源的DCs,用转移性Lewis肺癌特异性多肽Mutl预激经IL-2基因修饰的DCs联合低剂量Cy治疗转移性肺癌小鼠.通过FACS分析其脾细胞内T淋巴细胞比例的变化,~51Cr释放法检测CTL和NK细胞杀伤活性.结果:肿瘤抗原多肽致敏、IL-2基因修饰的DCs与小剂量Cy联合后,能比单用DCs更有效地治疗转移性肺癌,小鼠脾细胞中CD8~+T细胞和NK1.1~+细胞明显比例升高,联合治疗组诱导出的CTL杀伤活性最高.结论:以肿瘤抗原多肽冲击致敏的IL-2基因修饰的DCs联合小剂量Cy能更有效地促进荷瘤宿主免疫应答,具有显著地体内抑制肺癌转移的效果.     

COMBINED IL-2/IL-3 GENE THERAPY FOR G422 MOUSE GLIOBLASTOMA BY INTRATUMORAL INJECTION OF RECOMBINANT ADENOVIRUSES

ＣＯＭＢＩＮＥＤＩＬ２／ＩＬ３ＧＥＮＥＴＨＥＲＡＰＹＦＯＲＧ４２２ＭＯＵＳＥＧＬＩＯＢＬＡＳＴＯＭＡＢＹＩＮＴＲＡＴＵＭＯＲＡＬＩＮＪＥＣＴＩＯＮＯＦＲＥＣＯＭＢＩＮＡＮＴＡＤＥＮＯＶＩＲＵＳＥＳ１ＨｏｎｇＢｏ２洪波ＣａｏＸｕｅｔａｏ３曹雪涛Ｙｕ...     

Immunotherapy of spontaneous metastatic lung cancer with tumor antigen-pulsed,interleukin-12 gene-modified dendritic cells

Interleukin-12 (IL-12), with the ability of inducing production of interferon-gamma and enhancing of NK activity and Th1 response, has potent antitumor role and has been used in treatment of tumors[1-7]. Dendritic cells (DC) are the uniquely potent APCs involved in the initiation of immune responses. As adjuvants for Ag delivery, DC pick up Ags in the periphery and carry them to T cells area in lymphoid organs to prime the immune responses. With the development of the methods for propaga…     

Immunomodulatory gene therapy with interleukin 12 and 4-1BB ligand: long- term remission of liver metastases in a mouse model

BACKGROUND: The success of immunomodulatory cancer therapy is frequently hampered by the transient nature of the antitumor immune response. We have shown previously in a mouse model that interleukin 12 (IL-12) generates a strong natural killer (NK) cell-mediated antitumor response and reduces liver metastases induced by a colon carcinoma cell line. However, only a small percentage of the treated animals developed the cytotoxic T-lymphocytic response required for a long-term systemic antitumor immunity. 4-1BB is a co-stimulatory molecule expressed on the surface of activated T cells. Interaction of 4-1BB with its natural ligand (4-1BBL) has been shown to amplify T-cell (especially CD8+)-mediated immunity. In this study, we investigated the effects of adenovirus-mediated gene therapy delivering both IL-12 and 4-1BBL genes on mice with hepatic metastases induced by colon cancer cells. METHODS: Syngeneic BALB/c mice received intrahepatic injection of poorly immunogenic MCA26 colon cancer cells. Various combinations of replication-defective adenoviruses expressing IL-12 and 4-1BBL genes were injected into the established liver tumors. Changes in tumor size and animal survival were then monitored. All statistical tests were two-sided. RESULTS: The long-term survival rate of mice treated with the combination of IL-12 and 4-1BBL was significantly improved over that of animals in the control group (P =.0001). In vivo depletion of NK cells or CD8+ T cells completely abolished the long-term survival advantage of the IL-12 plus 4-1BBL-treated animals (P<.002). Moreover, the systemic immunity induced by this combination treatment protected these animals against a subcutaneous challenge with parental MCA26 cells. CONCLUSION: Adenovirus-mediated transfer of IL-12 and 4-1BBL genes directly into liver tumors resulted in tumor regression that required both NK and CD8+ T cells and generated a potent, long-lasting antitumor immunity.     

Adenovirus-mediated interleukin-18 mutant in vivo gene transfer inhibits tumor growth through the induction of T cell immunity and activation of natural killer cell cytotoxicity

We report here that gene transfer using recombinant adenoviruses encoding interleukin (IL)-18 mutants induces potent antitumor activity in vivo. The precursor form of IL-18 (ProIL-18) is processed by caspase-1 to produce bioactive IL-18, but its cleavage by caspase-3 (CPP32) produces an inactive form. To prepare IL-18 molecules with an effective antitumor activity, a murine IL-18 mutant with the signal sequence of murine granulocyte-macrophage (GM)- colony stimulating factor (CSF) at the 5'-end of mature IL-18 cDNA (GMmIL-18) and human IL-18 mutant with the prepro leader sequence of trypsin (PPT), which is not cleaved by caspase-3 (PPThIL-18CPP32-), respectively, were constructed. Adenovirus vectors carrying GMmIL-18 or PPThIL-18CPP32- produced bioactive IL-18. Ad.GMmIL-18 had a more potent antitumor effect than Ad.mProIL-18 encoding immature IL-18 in renal cell adenocarcinoma (Renca) tumor-bearing mice. Tumor-specific cytotoxic T lymphocytes, the induction of Th1 cytokines, and an augmented natural killer (NK) cell activity were detected in Renca tumor-bearing mice treated with Ad.GMmIL-18. An immunohistological analysis revealed that CD4+ and CD8+ T cells abundantly infiltrated into tumors of mice treated with Ad.GMmIL-18. Huh-7 human hepatoma tumor growth in nude mice with a defect of T cell function was significantly inhibited by Ad.PPThIL-18CPP32- compared with Ad.hProIL-18 encoding immature IL-18. Nude mice treated with Ad.PPThIL-18CPP32- contained NK cells with increased cytotoxicity. The results suggest that the release of mature IL-18 in tumors is required for achieving an antitumor effect including tumor-specific cellular immunity and augmented NK cell-mediated cytotoxicity. These optimally designed IL-18 mutants could be useful for improving the antitumor effectiveness of wild-type IL-18.