Role of 4-1BB:4-1BB ligand in cancer immunotherapy

The activation of T cells plays a central role in antitumor immunity. In order to activate na?ve T cells, two key signals are required. Signal one is provided through the T-cell receptor (TCR) while signal two is that of costimulation. The CD28:B7 molecules are one of the best-studied costimulatory pathways, thought to be the main mechanism through which primary T-cell stimulation occurs. However, a number of molecules have been identified which serve to amplify and diversify the T-cell response, following initial T-cell activation. These include the more recently described 4-1BB:4-1BB ligand (4-1BBL) molecules. 4-1BB:4-1BBL are a member of the TNFR:TNF ligand family, which are expressed on T cells and antigen-presenting cells (APCs), respectively. Therapies utilizing the 4-1BB:4-1BBL signaling pathway have been shown to have antitumor effects in a number of model systems. In this paper, we focus on the 4-1BB:4-1BBL costimulatory molecules. In particular, we will describe the structure and function of the 4-1BB molecule, its receptor and how 4-1BB:4-1BBL costimulation has and may be used for the immunotherapy of cancer.     

Expression of co-stimulatory 4-1BB ligand induces significant tumor regression and protective immunity

Co-stimulatory molecules play an important role in initiating antitumor immune responses. Engineered tumor cells expressing co-stimulatory molecules have been used as cancer vaccines in both experimental tumor models and clinical trials. In this study, we cloned a cDNA gene coding for the mouse co-stimulatory molecule 4-1BBL by RTPCR. The expression vector pCI-4-1BBL was constructed by DNA recombinant technology and further transfected into a moderately immunogenic EL4 and a poorly immunogenic BL6-10 tumor cell line. Expression of the co-stimulatory molecule 4-1BBL is able to induce tumor regression of EL4/4-1BBL but not BL6-10/4-1BBL tumor cell line in syngeneic BALB/c mice. The tumor regression which is mainly mediated by CD8+ T cells further leads to protective immunity against the parental EL4 tumor. Our results thus indicate the potential utility of engineered tumor cells expressing co-stimulatory molecule 4-1BBL, especially in combination with other co-stimulatory molecules such as B7-1 in cancer vaccine.     

4-1BB与肿瘤特异性免疫治疗研究进展

4-1BB(CD137)是新近发现的一种新型共刺激分子,其单独作用(抗4-1BBMoAb或4-1BBL)及联用其他共刺激分子(如B7-1)或细胞因子(如IL-12)显示了强效的抗肿瘤作用,且联用较单用时更显著、持久.现主要从4-1BBL、抗4-1BB-MoAb及联用B7-1、IL-12等方面作一综述.     

4-1BB／4-1BBL介导DC和T细胞共刺激作用的研究进展

4-1BB（CDl37）和4-1BB配体（4—1BBL）属于肿瘤坏死因子／肿瘤坏死因子受体（TNF／TNFR）超家族成员，在树突状细胞（DC）及部分T细胞上表达。4-1BB和4-1BBL相互作用，通过一系列信号转导，激发未成熟DC的分化成熟，并且通过TRAF2和MAPK途径，使未成熟Dc介导初始型T细胞活化进而向Thl型细胞转化，从而产生有效的记忆性CTL和效应性CTL。深人探讨4-1BB和4-1BBL的作用机制，可能为肿瘤等疾病的治疗提供新思路。     

4-1BB在肿瘤治疗中的作用

4-lBB是肿瘤坏死因子受体(TNFR)超家族成员,具有影响T细胞扩增和存活的功能特性.通过4-1BBL或者抗4-1BB单克隆抗体干预4-1BB协同刺激途径可能对肿瘤有治疗作用.最近提出的s4-1BBL即4-1BBL可溶性形式,对恶性血液病的诊断和治疗以及判断预后有一定价值.现综述有关4-lBB在肿瘤治疗中的研究进展.     

A functional anti-human 4-1BB ligand monoclonal antibody that enhances proliferation of monocytes by reverse signaling of 4-1BBL

4-1BB Ligand (4-1BBL), a transmembrane molecule, member of the tumor necrosis factor ligand superfamily, is an important costimulatory molecule in the immune response. In this study a functional anti-human 4-1BBL MAb 1F1 was obtained and the specificity of this MAb was verified by flow cytometry and Western blotting. This MAb effectively recognized the 4-1BBL molecule expressed on a series of malignant cell lines as well as on DC and monocytes and it inhibited the proliferation of T lymphocytes, costimulated by soluble 4-1BBL and agonist anti-human CD3 MAb. Furthermore, we demonstrated that MAb 1F1 induced an impressive proliferation of monocytes from peripheral blood by triggering the reverse signal through 4-1BBL. This functional anti-human 4-1BBL MAb provides a valuable tool for further study of biological functions as well as signal transduction of 4-1BBL/4-1BB.     

Polarization effects of 4-1BB during CD28 costimulation in generating tumor-reactive T cells for cancer immunotherapy

Using murine tumor-draining lymph node (TDLN) cells, we investigated the polarization effect of 4-1BB (CD137) during CD28 costimulation in generating antitumor T cells. Costimulation of TDLN cells through the newly induced 4-1BB molecules, CD3, and CD28 using monoclonal antibodies significantly enhanced cell proliferation. The greater cell yield with 4-1BB signaling appeared to be related to the inhibition of activation-induced cell death. Activation of TDLN cells through 4-1BB in addition to CD3/CD28 signaling shifted T-cell responses toward a type 1 cytokine pattern because 4-1BB ligation plus CD3/CD28 stimulation significantly augmented type 1 cytokine (e.g., IFN-gamma) and granulocyte macrophage colony-stimulating factor secretion. By contrast, type 2 cytokine (e.g., interleukin 10) secretion by the activated TDLN cells was significantly reduced. The in vivo antitumor reactivity of TDLN cells activated through 4-1BB in conjunction with CD3/CD28 pathways was examined using an adoptive immunotherapy model. The number of pulmonary metastases was significantly reduced and survival was prolonged after the transfer of anti-CD3/anti-CD28/anti-4-1BB-activated TDLN cells compared with an equivalent number of cells activated without anti-4-1BB. The antitumor effect through 4-1BB involvement during CD28 costimulation was dependent on IFN-gamma production and abrogated after IFN-gamma neutralization. By contrast, interleukin 10 neutralization resulted in significantly enhanced tumor regression. These results indicate that costimulation of TDLN cells through newly induced 4-1BB and CD3/CD28 signaling can significantly increase antitumor reactivity by shifting T-cell responses toward a type 1 cytokine pattern while concomitantly decreasing type 2 responses.     

THEMIS-SHP1 Recruitment by 4-1BB Tunes LCK-Mediated Priming of Chimeric Antigen Receptor-Redirected T Cells

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Anti-4-1BB monoclonal antibody enhances rejection of large tumor burden by promoting survival but not clonal expansion of tumor-specific CD8+ T cells

Anti-4-1BB monoclonal antibody (mAb) has been shown to induce antitumor immunity by a CD4/CD8-dependent mechanism, but its direct effect on tumor-specific CD8+ T cells in tumor rejection is unclear. Here we used transgenic CD8+ T cells against the unmutated tumor rejection antigen P1A to analyze whether this mAb can promote CD8+ T-cell function against large tumors in the absence of CD4+ T-helper cells. RAG-2(-/-) mice were challenged with P1A-expressing plasmacytoma J558. Once tumor size reached a diameter of 0.85-1.75 cm, mice were treated with P1A-specific CD8+ CTL (P1CTL) in conjunction with anti-4-1BB mAb or control IgG. All of the mice showed a partial regression of tumor, but mice treated with anti-4-1BB mAb exhibited markedly enhanced tumor rejection, delayed tumor progression, and prolonged survival. Correspondingly, we observed a substantial increase in the number of P1CTL in anti-4-1BB mAb-treated mice. Surprisingly, anti-4-1BB mAb did not accelerate division of the tumor-specific CD8+ T cells, and the increase in tumor-specific T-cell number was due to reduced activation-induced cell death. These results indicate that anti-4-1BB mAb can promote CD8+ T cell-mediated protection against large tumors in the absence of CD4+ T-cell help by promoting P1CTL survival without increasing initial clonal expansion.     

In vivo persistence, tumor localization, and antitumor activity of CAR-engineered T cells is enhanced by costimulatory signaling through CD137 (4-1BB)

Human T cells engineered to express a chimeric antigen receptor (CAR) specific for folate receptor-α (FRα) have shown robust antitumor activity against epithelial cancers in vitro but not in the clinic because of their inability to persist and home to tumor in vivo. In this study, CARs were constructed containing a FRα-specific scFv (MOv19) coupled to the T-cell receptor CD3ζ chain signaling module alone (MOv19-ζ) or in combination with the CD137 (4-1BB) costimulatory motif in tandem (MOv19-BBζ). Primary human T cells transduced to express conventional MOv19-ζ or costimulated MOv19-BBζ CARs secreted various proinflammatory cytokines, and exerted cytotoxic function when cocultured with FRα(+) tumor cells in vitro. However, only transfer of human T cells expressing the costimulated MOv19-BBζ CAR mediated tumor regression in immunodeficient mice bearing large, established FRα(+) human cancer. MOv19-BBζ CAR T-cell infusion mediated tumor regression in models of metastatic intraperitoneal, subcutaneous, and lung-involved human ovarian cancer. Importantly, tumor response was associated with the selective survival and tumor localization of human T cells in vivo and was only observed in mice receiving costimulated MOv19-BBζ CAR T cells. T-cell persistence and antitumor activity were primarily antigen-driven; however, antigen-independent CD137 signaling by CAR improved T-cell persistence but not antitumor activity in vivo. Our results show that anti-FRα CAR outfitted with CD137 costimulatory signaling in tandem overcome issues of T-cell persistence and tumor localization that limit the conventional FRα T-cell targeting strategy to provide potent antitumor activity in vivo.     

4-1BB/4-1BBL在树突状细胞上的表达及其生物学意义

41BB/41BB配体（41BBL）属于肿瘤坏死因子受体/肿瘤坏死因子超家属成员,是机体特异性免疫应答中一对重要的共刺激分子，41BB/41BBL相互作用产生的共刺激信号在维持T细胞的增殖、活化及功能介导中发挥了重要作用。最新研究发现,41BB/41BBL共表达于树突状细胞（dendritic cells , DC），通过激发41BB/41BBL为DC活化提供了新的思路，进而增强DC激发T细胞的能力。因此，调节41BB/41BBL信号在以DC为主介导的肿瘤免疫中具有广阔的应用前景。     

Tumor necrosis factor-related apoptosis-inducing ligand retains its apoptosis-inducing capacity on Bcl-2- or Bcl-xL-overexpressing chemotherapy-resistant tumor cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family and has recently been shown to exert tumoricidal activity in vivo in the absence of any observable toxicity. The signaling pathways triggered by TRAIL stimulation and the mechanisms involved in resistance against TRAIL-mediated apoptosis are still poorly defined. We show here that TRAIL-induced apoptosis involves late dissipation of mitochondrial membrane potential (delta psi(m)) and cytochrome c release. These events follow activation of caspase-8 and caspase-3 and induction of DNA fragmentation. In addition, caspase-8-deficient cells are resistant against TRAIL-induced apoptosis, and inhibition of caspase-8 but not caspase-9 prevents mitochondrial permeability transition and apoptosis. In contrast, various Bcl-2- or Bcl-xL-overexpressing tumor cell lines are sensitive to TRAIL-induced apoptosis; however, they show a delay in TRAIL-induced mitochondrial permeability transition compared with control transfectants. This indicates that TRAIL-induced apoptosis depends on caspase-8 activation rather than on the disruption of mitochondrial integrity. Because most chemotherapeutic drugs used in the treatment of malignancies lead to apoptosis primarily by engagement of the mitochondrial proapoptotic machinery, we tested whether drug-resistant tumor cells retain sensitivity for TRAIL-induced apoptosis. Tumor cells overexpressing Bcl-2 or Bcl-xL become resistant to apoptosis induced by the chemotherapeutic drug etoposide. However, these cells are not protected or are only marginally protected against TRAIL-induced apoptosis. Thus, TRAIL may still kill tumors that have acquired resistance to chemotherapeutic drugs by overexpression of Bcl-2 or Bcl-xL. These data will influence future treatment strategies involving TRAIL.     

共刺激分子4-1BB与肿瘤治疗的研究进展

近年来,诸如PD-1,PD-L1等免疫检查点为靶点的单克隆抗体成为了肿瘤免疫治疗的热点,其作用机制是阻断T细胞表面的共抑制分子与配体的相互作用,从而激活T细胞进行肿瘤免疫应答.共刺激分子4-1BB(CD137)又名肿瘤坏死因子超家族受体9,多表达于活化的T细胞表面,也可表达于粒细胞或树突状细胞表面.4-1BB在免疫应答...     

4-1BB Protects Dendritic Cells from Prostate Cancer-Induced Apoptosis

It has been shown that human prostate cancer (PCa) cells induced apoptotic death of the most potent antigen-presenting cells, dendritic cells (DCs), which are responsible for the induction of specific antitumor immune responses. Here, we investigated the function of 4-1BB on protecting DCs from prostate cancer-induced apoptosis with an agonistic mAb to 4-1BB. RM-1 cells and DCs were co-incubated for 48 h and DC apoptosis was assessed by Annexin Vassay. TNF-α and IL-12 production were assessed by enzyme-linked immunosorbent assay (ELISA) and Bcl-2 and Bcl-xL on DCs were analyzed by Western blot. We have shown that co-incubation of RM-1 cells with DCs is accompanied by an increased level of DCs apoptosis. Triggering 4-1BB on DCs resulted in increased resistance of DCs to RM-1 cells-induced apoptosis, which was owing to the up-regulated expression of Bcl-2 and Bcl-xL, and increased secretion of TNF-αand IL-12. These results demonstrate that triggering 4-1BB on DCs could increased resistance of DCs to PCa-induced apoptosis.     

Leukemia-specific T-cell reactivity induced by leukemic dendritic cells is augmented by 4-1BB targeting.

PURPOSE: Acute myelogenous leukemia (AML) blasts are able to differentiate into leukemia-derived dendritic cells (AML-DC), thereby enabling efficient presentation of known and unknown leukemic antigens. Advances in culture techniques and AML-DC characterization justify clinical application. However, additional measures are likely needed to potentiate vaccines and overcome the intrinsic tolerant state of the patients' immune system. Engagement of the costimulatory molecule 4-1BB can break immunologic tolerance and increase CTL responses. In this study, we examined the role of the 4-1BB ligand (4-1BBL) on T-cell responses induced by AML-DC. EXPERIMENTAL DESIGN: In allogeneic and autologous cocultures of T cells and AML-DC, the effect of the addition of 4-1BBL on T-cell proliferation, T-cell subpopulations, and T-cell function was determined. RESULTS: Addition of 4-1BBL to cocultures of AML-DC and T cells induced a preferential increase in the proliferation of CD8(+) T cells. Increased differentiation into effector and central memory populations was observed in both CD4(+) and CD8(+) T cells in the presence of 4-1BBL. AML-DC induce a T helper 1 response, characterized by high IFN-gamma production, which is significantly increased by targeting 4-1BB. T cells primed in the presence of 4-1BBL show specificity for the leukemia-associated antigen Wilms' tumor 1, whereas cytotoxicity assays with leukemic blast targets showed the cytolytic potential of T cells primed in the presence of 4-1BBL. CONCLUSION: We conclude that 4-1BBL is an effective adjuvant to enhance T-cell responses elicited by AML-DC.     

4-1BB基因在不同分化胃癌细胞株中的表达

目的：研究共刺激因子4-1BB在人胃高分化腺癌细胞株MKN-28、人胃中分化腺癌细胞株SGC-7901、人胃低分化腺癌细胞株BGC-823、人胃粘液腺癌细胞株MGC-803中的表达及其生物学意义。方法：用RT-PCR法测定胃癌细胞株中4-1BB mRNA表达;免疫细胞化学法测定胃癌细胞株中4-1BB蛋白表达;MTT法测定人淋巴细胞对胃癌细胞的体外杀伤活性;ELISA法检测人淋巴细胞与胃癌细胞共培养上清液中IL-2、IL-10、TNF-α的含量。结果：RT-PCR结果示4-1BB mRNA表达率从高到低依次为：MGC-803（77.30%）、BGC-823（71.68%）、SGC-7901（40.06%）、MKN-28（37.65%）。免疫细胞化学结果显示4-1BB着色程度由深到浅依次为：MGC-803、BGC-823、SGC-7901、MKN-28。MTT结果显示各时间段（24、48、72h）不同效靶比（10∶1,20∶1,40,∶1）下,人淋巴细胞对胃癌细胞的体外杀伤活性由高到低的顺序为：MKN-28、SGC-7901、BGC-823、MGC-803。ELISA结果显示在各效靶细胞比下,人淋巴细胞与胃癌细胞共培养上清液中IL-2、TNF-α的含量由高到低的顺序均为：MKN-28、SGC-7901、BGC-823、MGC-803。而IL-10的含量由高到低的顺序为：MGC-803、BGC-823、SGC-7901、MKN-28。结论：人胃癌细胞株MKN-28、SGC-7901、BGC-823、MGC-803中4-1BB基因的mRNA和蛋白均有表达,且胃癌细胞株的恶性程度越高其4-1BB表达水平越高;淋巴细胞对低表达4-1BB胃癌细胞的杀伤力较高;提示4-1BB可能通过抑制细胞因子TNF-α、IL-2和促进细胞因子IL-10的产生调节肿瘤免疫。     

4-1BB/4-1BBL在胃癌组织及其外周血中的表达及临床意义

目的探讨胃癌局部免疫与全身免疫状态。方法应用流式细胞术,测定胃癌患者癌组织及外周血中T淋巴细胞亚群、NK细胞水平,同时测定癌组织、癌周正常胃黏膜、淋巴结及外周血单个核细胞中4-1BB、4-1BBL含量。结果胃癌患者外周血中CD3^＋、CD4^＋、CD4^＋/CD8^＋比值和NK细胞表达水平均显著高于胃癌组织（P〈0.05）,CD8^＋细胞计数反之（P〈0.05）。4-1BB表达量由低到高的顺序为外周血单个核细胞、正常胃黏膜、淋巴结、癌组织、癌组织中单个核细胞;其中癌组织明显高于正常黏膜（P〈0.05）,有统计学意义;外周血单个核细胞中4-1BB表达量明显低于其他组织,有统计学意义（P〈0.01）。癌组织中4-1BBL表达量低于正常黏膜,有统计学意义（P〈0.05）。癌组织中4-1BB表达量与CD4^＋/CD8^＋比值、NK细胞表达水平呈负相关关系,4-1BBL与CD4^＋/CD8^＋比值、NK细胞表达水平呈正相关关系,有统计学意义（P〈0.05）。结论胃癌组织内免疫力低下,处于免疫抑制状态,胃癌组织的低免疫状态与4-1BBL缺乏有关;4-1BB与胃癌患者局部和全身的免疫状态亦有密切关系。总之,胃癌组织中4-1BB/4-1BBL的表达失衡与胃癌的免疫逃逸、低免疫状态有关。     

Intratumoral T cell subset ratios and Fas ligand expression on brain tumor endothelium

Summary Introduction: T cell presence in TIL, and the ratio of CD8⁺ and CD4⁺ T cell subsets in particular, can correlate with tumor prognosis in some tumors, although the significance of such infiltration into glioma is controversial. However, gliomas represent a lower extreme in their extent of T cell infiltration, and are thus useful in assessing factors that can decrease T cell presence within tumor tissue. Fas ligand, a pro-apoptotic cell surface protein, may play a key role in reduction of T cells in tumor tissue. Objective: To assess the level of FasL expression on brain tumor endothelium and to correlate this with relative levels of CD4⁺ and CD8⁺ T cell subsets in TIL from brain tumors. Methods: CD3⁺, CD4⁺, and CD8⁺ cells were quantified in fresh TIL by flow cytometry. Paraffin embedded sections of tumors, including meningiomas and gliomas as well as extracranial malignancies, underwent immunohistochemical staining for FasL and Von-Willebrand’s factor (Factor VIII) to determine expression levels of endothelial FasL. Results: FasL expression was high in aggressive intracranial malignancies compared to more indolent neoplasms, and correlated inversely with CD8⁺/CD4⁺ TIL ratios in all tumor classes combined (ANOVA,p<0.05). Conclusion: Low levels of T cells within TIL, as well as low CD8⁺/CD4⁺ TIL ratios appear to be a property of parenchymal tumor presence. Together with the inverse correlation seen between FasL expression and CD8⁺/CD4⁺ TIL ratios, the high levels of endothelial FasL expression in gliomas suggests that FasL decreases T cell presence in brain tumors in a subset-selective manner, thus contributing to glioma immune privilege.     

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.