Improving Adoptive T Cell Therapy by Targeting and Controlling IL-12 Expression to the Tumor Environment

Interleukin-12 (IL-12) is an important immunostimulatory cytokine, yet its clinical application has been limited by the systemic toxicity associated with its administration. In this work, we developed a strategy to selectively deliver IL-12 to the tumor environment using genetically engineered lymphocytes. However, peripheral blood lymphocytes (PBLs) transduced with a γ-retroviral vector, which constitutively expressed IL-12, failed to expand in culture due to apoptosis. To circumvent this problem, a vector was designed where IL-12 expression was directed by a composite promoter-containing binding motifs for nuclear factor of activated T-cells (NFAT.hIL12.PA2). The NFAT-responsive promoter was activated to drive IL-12 expression upon the recognition of tumor-specific antigen mediated by a T cell receptor (TCR) that was engineered into the same lymphocytes. We tested the efficacy of the inducible IL-12 vector in vivo in a murine melanoma model. Adoptive transfer of pmel-1 T cells genetically engineered with NFAT-murineIL12 (NFAT.mIL12.PA2) significantly enhanced regression of large established B16 melanoma. Notably, this targeted and controlled IL-12 treatment was without toxicity. Taken together, our results suggest that using the NFAT.hIL12.PA2 vector might be a promising approach to enhance adoptive cancer immunotherapy.     

通过FasL通路阻抑肿瘤免疫逃逸的研究

许多白血病和实体瘤细胞通过高表达Fas配体 (FasL)而发生肿瘤的免疫逃逸。为了观察腺病毒载体向肿瘤细胞导入小鼠可溶性Fas(sFas)基因后能否阻抑肿瘤细胞通过FasL发生肿瘤免疫逃逸作用 ,采用AdEasy腺病毒载体系统 ,利用大肠杆菌内质粒间同源重组的方法分别构建携带小鼠sFas基因及增强型绿色荧光蛋白 (EGFP)的重组腺病毒载体 ,扩增纯化后利用空斑试验测定滴度 ,Westernblot检测蛋白的表达。然后 ,分别感染肿瘤细胞 EL4细胞 ,用氚胸腺嘧啶核苷 ( 3 H thymidine,3 H TdR)掺入法检测其诱导靶细胞YAC 1的凋亡率。结果表明 ,获得了重组成功的复制缺陷的腺病毒载体AdsFas和AdEGFP ,密度梯度离心纯化后其滴度达到 10 11pfu/ml,Westernblot分析证实前者能够高效表达出sFas蛋白。转染肿瘤细胞EL4细胞之后与YAC 1细胞混合培养 ,导入sFas组的YAC 1凋亡率在效靶比 (E∶T)为 3∶1,10∶1和 30∶1时分别为 6 %、7%和 9% ,与对照组 (分别为 2 8%、37%和4 5 % )相比明显下降 ,统计学有显著性差异 (P <0 .0 1) ;而导入EGFP组YAC 1凋亡率 (分别为 30 %、36 %和 4 8% )与对照组相似 ,没有统计学差异 (P >0 .0 5 )。结论 :腺病毒介导sFas的导入能够明显抑制肿瘤细胞EL4诱导Fas+细胞 YAC 1的凋亡 ,说明通过腺病毒转移sFas基因     

Prevention of UV radiation-induced immunosuppression by IL-12 is dependent on DNA repair

The immunostimulatory cytokine IL-12 is able to antagonize immunosuppression induced by solar/ultraviolet (UV) radiation via yet unknown mechanisms. IL-12 was recently found to induce deoxyribonucleic acid (DNA) repair. UV-induced DNA damage is an important molecular trigger for UV-mediated immunosuppression. Thus, we initiated studies into immune restoration by IL-12 to discern whether its effects are linked to DNA repair. IL-12 prevented both UV-induced suppression of the induction of contact hypersensitivity and the depletion of Langerhans cells, the primary APC of the skin, in wild-type but not in DNA repair-deficient mice. IL-12 did not prevent the development of UV-induced regulatory T cells in DNA repair-deficient mice. In contrast, IL-12 was able to break established UV-induced tolerance and inhibited the activity of regulatory T cells independent of DNA repair. These data identify a new mechanism by which IL-12 can restore immune responses and also demonstrate a link between DNA repair and the prevention of UV-induced immunosuppression by IL-12.     

IFN-γ and Fas/FasL are required for the antitumor and antiangiogenic effects of IL-12/pulse IL-2 therapy

Systemic administration of IL-12 and intermittent doses of IL-2 induce complete regression of metastatic murine renal carcinoma. Here, we show that overt tumor regression induced by IL-12/pulse IL-2 is preceded by recruitment of CD8(+) T cells, vascular injury, disrupted tumor neovascularization, and apoptosis of both endothelial and tumor cells. The IL-12/IL-2 combination synergistically enhances cell surface FasL expression on CD8(+) T lymphocytes in vitro and induces Fas and FasL expression within tumors via an IFN-gamma-dependent mechanism in vivo. This therapy also inhibits tumor neovascularization and induces tumor regression by mechanisms that depend critically on endogenous IFN-gamma production and an intact Fas/FasL pathway. The ability of IL-12/pulse IL-2 to induce rapid destruction of tumor-associated endothelial cells and regression of established metastatic tumors is ablated in mice with a dysregulated Fas/FasL pathway. The common, critical role for endogenous IFN-gamma and the Fas/FasL pathway in early antiangiogenic effects and in antitumor responses suggests that early, cytokine-driven innate immune mechanisms and CD8(+) T cell-mediated responses are interdependent. Definition of critical early molecular events engaged by IL-12/IL-2 may provide new perspective into optimal therapeutic engagement of a productive host-antitumor immune response.     

Induction of interleukin 12 responsiveness is impaired in anergic T lymphocytes

The cytokine, interleukin 12 (IL-12), stimulates both natural killer cells and T cells to proliferate and to secrete interferon gamma (IFN- gamma). The T cell proliferative response to IL-12 must be induced and is evident after T cell receptor-mediated stimulation. As reported here, tolerant CD4+ T cells and clones, that are anergic for IL-2 production, are also anergic for induction of the proliferative response to IL-12. Murine T helper 1 clones tolerized in vitro, as well as anergic CD4+ T cells isolated from mice tolerized to the Mls-1a antigen (Ag) in vivo, demonstrated defective induction of proliferation to IL-12 upon restimulation with Ag. IL-12-enhanced production of IFN- gamma was observed in both control and anergic cells after Ag/antigen- presenting cell (APC) activation, although total IFN-gamma secretion by anergic cells was less than that produced by control cells, even in the presence of IL-12. These data indicate that T cell clonal anergy results in profound inhibition of proliferative responses, since the autocrine growth factor, IL-2, is not produced, and the APC-derived cytokine, IL-12, is not an effective stimulus for anergic T cell proliferation.     

Antitumor Effects of Chimeric Receptor Engineered Human T Cells Directed to Tumor Stroma

Cancer-associated fibroblasts (CAFs), the principle component of the tumor-associated stroma, form a highly protumorigenic and immunosuppressive microenvironment that mediates therapeutic resistance. Co-targeting CAFs in addition to cancer cells may therefore augment the antitumor response. Fibroblast activation protein-α (FAP), a type 2 dipeptidyl peptidase, is expressed on CAFs in a majority of solid tumors making it an attractive immunotherapeutic target. To target FAP-positive CAFs in the tumor-associated stroma, we genetically modified T cells to express a FAP-specific chimeric antigen receptor (CAR). The resulting FAP-specific T cells recognized and killed FAP-positive target cells as determined by proinflammatory cytokine release and target cell lysis. In an established A549 lung cancer model, adoptive transfer of FAP-specific T cells significantly reduced FAP-positive stromal cells, with a concomitant decrease in tumor growth. Combining these FAP-specific T cells with T cells that targeted the EphA2 antigen on the A549 cancer cells themselves significantly enhanced overall antitumor activity and conferred a survival advantage compared to either alone. Our study underscores the value of co-targeting both CAFs and cancer cells to increase the benefits of T-cell immunotherapy for solid tumors.     

Immunotherapy of cancer by IL-12-based cytokine combinations

Cancer is a multi-faceted disease comprising complex interactions between neoplastic and normal cells. Over the past decade, there has been considerable progress in defining the molecular, cellular and environmental contributions to the pathophysiology of tumor development. Despite these advances, the conventional treatment of patients still generally involves surgery, radiotherapy and/or chemotherapy, and the clinical outcome for many of these efforts remains unsatisfactory. Recent studies have highlighted the feasibility of using immunotherapeutic approaches that seek to enhance host immune responses to developing tumors. These strategies include immunomodulatory cytokines, with TNF-alpha, type I or type II IFNs, IL-2, IL-12, IL-15 and IL-18 being among the most potent inducers of anti-tumor activity in a variety of preclinical studies. More recently, some exciting new cytokines have been characterized, such as IL-21, IL-23, IL-27 and their immunomodulatory and antitumor effects in vitro and in vivo suggest that they may have considerable promise for future immunotherapy protocols. The promise of cytokine therapy does indeed derive from the identification of these novel cytokines but even more fundamentally, the field is greatly benefiting from the ever-expanding amount of preclinical data that convincingly demonstrate synergistic and/or novel biologic effects, which may be achieved through the use of several combinations of cytokines with complementary immune-stimulating capabilities. One cytokine in particular, IL-12, holds considerable promise by virtue of the fact that it plays a central role in regulating both innate and adaptive immune responses, can by itself induce potent anticancer effects, and synergizes with several other cytokines for increased immunoregulatory and antitumor activities. This review discusses the antitumor activity of IL-12, with a special emphasis on its ability to synergize with other cytokines for enhancement of immune effector cell populations and regulation of host-tumor cell interactions and the overall tumor microenvironment.     

Immunotherapy of cancer by IL-12-based cytokine combinations

Cancer is a multi-faceted disease comprising complex interactions between neoplastic and normal cells. Over the past decade, there has been considerable progress in defining the molecular, cellular and environmental contributions to the pathophysiology of tumor development. Despite these advances, the conventional treatment of patients still generally involves surgery, radiotherapy and/or chemotherapy, and the clinical outcome for many of these efforts remains unsatisfactory. Recent studies have highlighted the feasibility of using immunotherapeutic approaches that seek to enhance host immune responses to developing tumors. These strategies include immunomodulatory cytokines, with TNF-α, type I or type II IFNs, IL-2, IL-12, IL-15 and IL-18 being among the most potent inducers of anti-tumor activity in a variety of preclinical studies. More recently, some exciting new cytokines have been characterized, such as IL-21, IL-23, IL-27 and their immunomodulatory and antitumor effects in vitro and in vivo suggest that they may have considerable promise for future immunotherapy protocols. The promise of cytokine therapy does indeed derive from the identification of these novel cytokines but even more fundamentally, the field is greatly benefiting from the ever-expanding amount of preclinical data that convincingly demonstrate synergistic and/or novel biologic effects, which may be achieved through the use of several combinations of cytokines with complementary immune-stimulating capabilities. One cytokine in particular, IL-12, holds considerable promise by virtue of the fact that it plays a central role in regulating both innate and adaptive immune responses, can by itself induce potent anticancer effects, and synergizes with several other cytokines for increased immunoregulatory and antitumor activities. This review discusses the antitumor activity of IL-12, with a special emphasis on its ability to synergize with other cytokines for enhancement of immune effector cell populations and regulation of host–tumor cell interactions and the overall tumor microenvironment.     

重组腺病毒载体介导IL-12基因在间充质干细胞中的表达

目的将大鼠骨髓间充质干细胞(MSC),经重组腺病毒载体Ad-IL-12转染,构建Ad-IL-12-MSC。方法 Percoll法分离与培养大鼠MSC,流式细胞仪测定细胞表面标志CD29,CD44,CD34。以重组腺病毒载体Ad-IL-12转染MSC,RT-PCR、Western Blott检测转染后IL-12基因mRNA和蛋白的表达。结果分离所得的大鼠MSCs细胞组成比较均一。流式细胞仪检测3种MSCs的表面抗原标志,CD29和CD44,阳性率分别达97.1%和98.2%,而CD34的阳性率仅为4.4%。转染后,Ad-IL-12-MSC细胞IL-12基因在mRNA水平和蛋白质水均有表达。结论 Percoll法分离与培养MSC较为可靠且简单,构建的Ad-IL-12-MSC能成功表达IL-12。     

Interleukin 12 (IL-12) induces tyrosine phosphorylation of JAK2 and TYK2: differential use of Janus family tyrosine kinases by IL-2 and IL- 12

Interleukin (IL-12) has many effects on the function of natural killer and T cells, and is important in the control of cell-mediated immunity. IL-2 and IL-12 display many similar activities, yet each also induces a distinct set of responses. A human IL-12 receptor subunit has recently been cloned and, like the IL-2R beta and IL-2R gamma, is a member of the hematopoietic receptor superfamily; however, the molecular mechanisms of IL-12 action are unknown. In this report we show that IL- 12 and IL-2 induce tyrosine phosphorylation of distinct members of the Janus (JAK) family of protein tyrosine kinases in human T lymphocytes. IL-12, but not IL-2, stimulates the tyrosine phosphorylation of TYK2 and JAK2, whereas JAK1 and JAK3, which are phosphorylated in response to IL-2, are not phosphorylated after IL-12 treatment. The use of distinct but related JAK family tyrosine kinases by IL-12 and IL-2 may provide a biochemical basis for their different biological activities.     

X-linked susceptibility to mycobacteria is caused by mutations in NEMO impairing CD40-dependent IL-12 production

Germline mutations in five autosomal genes involved in interleukin (IL)-12-dependent, interferon (IFN)-gamma-mediated immunity cause Mendelian susceptibility to mycobacterial diseases (MSMD). The molecular basis of X-linked recessive (XR)-MSMD remains unknown. We report here mutations in the leucine zipper (LZ) domain of the NF-kappaB essential modulator (NEMO) gene in three unrelated kindreds with XR-MSMD. The mutant proteins were produced in normal amounts in blood and fibroblastic cells. However, the patients' monocytes presented an intrinsic defect in T cell-dependent IL-12 production, resulting in defective IFN-gamma secretion by T cells. IL-12 production was also impaired as the result of a specific defect in NEMO- and NF-kappaB/c-Rel-mediated CD40 signaling after the stimulation of monocytes and dendritic cells by CD40L-expressing T cells and fibroblasts, respectively. However, the CD40-dependent up-regulation of costimulatory molecules of dendritic cells and the proliferation and immunoglobulin class switch of B cells were normal. Moreover, the patients' blood and fibroblastic cells responded to other NF-kappaB activators, such as tumor necrosis factor-alpha, IL-1beta, and lipopolysaccharide. These two mutations in the NEMO LZ domain provide the first genetic etiology of XR-MSMD. They also demonstrate the importance of the T cell- and CD40L-triggered, CD40-, and NEMO/NF-kappaB/c-Rel-mediated induction of IL-12 by monocyte-derived cells for protective immunity to mycobacteria in humans.     

Discovery of a Linear Peptide for Improving Tumor Targeting of Gene Products and Treatment of Distal Tumors by IL-12 Gene Therapy

Like many effective therapeutics, interleukin-12 (IL-12) therapy often causes side effects. Tumor targeted delivery may improve the efficacy and decrease the toxicity of systemic IL-12 treatments. In this study, a novel targeting approach was investigated. A secreted alkaline phosphatase (SEAP) reporter gene-based screening process was used to identify a mini-peptide which can be produced in vivo to target gene products to tumors. The coding region for the best peptide was inserted into an IL-12 gene to determine the antitumor efficacy. Affinity chromatography, mass spectrometry analysis, and binding studies were used to identify a receptor for this peptide. We discovered that the linear peptide VNTANST increased the tumor accumulation of the reporter gene products in five independent tumor models including one human xenogeneic model. The product from VNTANST-IL-12 fusion gene therapy increased accumulation of IL-12 in the tumor environment, and in three tumor models, VNTANST-IL-12 gene therapy inhibited distal tumor growth. In a spontaneous lung metastasis model, inhibition of metastatic tumor growth was improved compared to wild-type IL-12 gene therapy, and in a squamous cell carcinoma model, toxic liver lesions were reduced. The receptor for VNTANST was identified as vimentin. These results show the promise of using VNTANST to improve IL-12 treatments.     

Migraine Triggered by Rubbing the Eyes

A patient with recurrent exertional headaches was able, on multiple occasions, to trigger his typical headaches within 30 minutes of rubbing his eyes gently and inducing bilateral photopsias. No intracranial or orbital lesions were identified. His EEG, obtained while rubbing his eyes and inducing photopsias, did not show epileptic discharges or background changes. His ophthalmologic examination, including visual field testing, was normal. Ocular pressure and massage of the carotid sinuses failed to cause bradycardia to imply vagal mediation of his physically induced headaches. It is hypothesized that in this patient mechanical displacement of the eyes precipitated retinal auras leading to migraines.     

IL-12及IL-12P40对NK抗肿瘤功能的免疫调节

目的　通过对正常人及肿瘤患者血清中IL 12以及IL 12P4 0含量的检测 ,观察其分子对NK细胞抗肿瘤细胞细胞毒效应的影响 ,探讨IL 12、IL 12P4 0分子在NK细胞抗肿瘤功能上的免疫调节作用。方法　ELISA酶联免疫法检测IL 12、IL 12P4 0分泌水平以及MTT释放法检测NK细胞毒活性。结果　①肿瘤患者外周血PBMC中CD3 、CD5 6 细胞阳性率低于对照组 (P <0 0 5 )。②肿瘤患者NK细胞对NK敏感细胞K5 6 2以及NK不敏感细胞Raji和Ddaudi的杀伤活性均低于正常对照组 (P <0 0 5 )。③正常人外周血NK细胞经含肿瘤患者血清培养后 ,NK细胞杀伤活性明显下降 (P <0 0 5 )。④在肿瘤患者血清中 ,IL 12分泌水平为 14 3 31± 0 93pg ml,高于对照组 84 97± 3 7pg ml(P <0 0 5 ) ;IL 12P4 0含量为 2 2 4 90± 0 7pg ml,高于对照组 32 5 6± 0 7pg ml(P <0 0 5 )。⑤正常人NK细胞经不同浓度 ( 1μg ml、5 μg ml以及 10 μg ml)IL 12诱导后 ,在 5 μg ml、10 μg ml浓度NK细胞杀伤率均高于对照组 ;在肿瘤患者中 ,仅高剂量( 10g ml)IL 12组的杀伤率高于对照组。⑥这种诱导增高的NK细胞杀伤率可被外源性IL 12P4 0的加入而下调。结论　肿瘤患者血清中IL 12P4 0含量的增高可能与肿瘤密切相关的NK细胞功能下降有关。     

白细胞介素12单独或联合白细胞介素2对人脐血单个核细胞抗肿瘤活性的研究

目的　探讨白细胞介素 12 (IL 12 )单独或联合白细胞介素 2 (IL 2 )对人脐血单个核细胞(CBMC)抗肿瘤作用的影响。方法　采用3 H TdR释放法测定经IL 12和 (或 )IL 2刺激的CBMC和人外周血单个核细胞 (PBMC)的抗肿瘤活性 ,及在电镜下观察激活的CBMC所杀伤的K5 6 2细胞的形态特征。结果　① 10IU/mlIL 12激活的CBMC即产生较强的杀伤活性 ,对K5 6 2及Raji细胞的杀伤率分别为 (2 7.2 3± 4.92 ) %和 (2 9.12± 3.46 ) % ;10IU/ml的IL 12、IL 2共用能产生明显的协同作用 ,对两靶细胞的杀伤率分别达 (47.6 0± 4.6 0 ) %和 (38.6 9± 4.86 ) %。②CBMC经IL 12和IL 2刺激不同时间 ,对K5 6 2及Raji细胞的杀伤率不同 ,短时间培养增强对K5 6 2细胞的杀伤活性 ,较长时间则增强对Raji细胞的杀伤活性。③未经细胞因子刺激的CBMC无明显的NK活性 ;经 10IU/mlIL 12刺激后 ,其NK活性与经相同剂量IL 12刺激的PBMC相当 ;两细胞因子合用均能协同增强CBMC和PBMC的NK活性 ,但前者低于后者。④激活的CBMC与K5 6 2细胞作用后 ,后者呈现明显凋亡特征。结论　IL 12激活的CBMC具有显著的抗肿瘤活性 ,与IL 2合用则效果更佳。