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.     

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。     

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/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。     

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.     

抗人肝癌血管内皮功能性单抗的筛选与鉴定

目的:筛选、鉴定抗人肝癌血管内皮功能性单抗,为治疗肝癌提供靶向治疗剂,并为分离获得肝癌相关的分子靶标打下基础。方法:采用活细胞荧光、MTT细胞增殖实验、成管实验和动物体内治疗实验,筛选鉴定抑制肝癌内皮细胞的功能性单抗。结果:从能与肝癌内皮细胞膜反应的119株克隆中,筛选出16株单抗显著地抑制肝癌内皮增殖其抑制率达21%~46%,46株能显著抑制肝癌内皮细胞的成管,3株单抗1F9、12B5和1B11能显著抑制肝癌移植瘤的生长抑制率分别为50.8%、48.7%和47.0%。选择1株体内抑瘤效果最好的1B11单抗,通过抗体抗原亲和层析法进行纯化,并对纯化的单抗的相关抗原蛋白进行免疫组织化学实验鉴定,结果显示1B11抗原在肝癌血管组织较高的表达而在正常肝血管组织极少表达。蛋白质印迹法显示其抗原相对分子质量约46×103。结论:采用大容量功能性抗体库技术成功获得了多株具有抑制肝癌内皮恶性生物学行为的功能性单抗,体内外抑制肺癌的生长,具有成为肺癌靶向治疗剂的潜力。其中1株可能是一个靶向治疗肝癌的新靶位。     

Eradication of established renal cell carcinoma by a combination of 5-fluorouracil and anti-4-1BB monoclonal antibody in mice

Renal cell carcinoma (RCC), one of the most incurable malignancies, is highly resistant to chemotherapy and radiotherapy. Cytokine immunotherapy has been the standard approach, but the overall response rate is still very low. Administration of agonistic anti-4-1BB monoclonal antibody (mAb) has been shown to induce regression of several animal tumors but its effect on RCC is unknown. We show here that monotherapy with either anti-4-1BB mAb or the cytotoxic drug, 5-fluorouracil (5-FU), has little effect on established RCC, Renca tumors, but combination therapy with anti-4-1BB mAb and 5-FU eradicates the tumors in more than 70 % of mice. The regressing tumor tissues from mice receiving the combination therapy contained more apoptotic tumor cells and tumor infiltrating lymphocytes than tumor tissues from mice receiving 5-FU or anti-4-1BB mAb monotherapy. The number of lymphocytes in the spleens and tumor- draining lymph nodes (TDLNs) of the combination therapy mice was greatly increased compared to that of control or 5-FU monotherapy mice. Mice that had recovered due to the combination therapy rapidly rejected rechallenge with the tumor, pointing to the establishment of long-lasting tumor-specific memory. Our results indicate that targeting tumors with 5-FU, and immune cells with 4-1BB stimulation, could be a useful strategy for treating incurable RCC.     

Tumor expression of 4-1BB ligand sustains tumor lytic T cells

Inadequate costimulation by solid tumors is generally believed to induce immune tolerance during primary tumor growth. We looked for tumor-specific immunity vs. tolerance in patients with Ewing's sarcoma. Circulating T cells from patients with progressively growing Ewing's tumors displayed MHC restricted tumor-induced proliferation and robust tumor lysis. Tumor-reactive T cells reside within the memory CD3+CD8+ subset and are CD28-/4-1BB+. Autologous Ewing's tumors expressed 4-1BBL, and tumor-induced T cell proliferation and activation required costimulation by 4-1BBL. Stimulation of PBL with anti-CD3/4-1BBL, but not anti-CD3/anti-CD28 induced tumor lytic effectors. Similarly, in a xenograft model, anti-CD3/4-1BBL expanded T cells controlled primary growth and prevented metastasis of autologous tumors while nonactivated and anti-CD3/anti-CD28 activated CD8+ cells did not. These results question prevailing models of tumor induced tolerance accompanying progressive tumor growth; rather, we show coexistence of progressive tumor growth and anti-tumor immunity, with costimulation provided by the tumor itself. They further demonstrate a potential new therapeutic role for 4-1BBL mediated costimulation in expanding tumor reactive CTLs for use in the adoptive immunotherapy of cancer.     

IFN-gamma enhances the antimyeloma activity of the fully human anti-human leukocyte antigen-DR monoclonal antibody 1D09C3

To investigate the therapeutic activity of the fully human anti-HLA-DR antibody 1D09C3 in multiple myeloma (MM), we reevaluated HLA-DR expression on CD138(+) cells, analyzed the capacity of IFN-gamma to up-regulate HLA-DR expression on MM cell lines, and tested the in vitro and in vivo activity of 1D09C3 alone or in combination with IFN-gamma. CD138(+)HLA-DR(+) cells were detected in 31 of 60 patients, with 15 of 60 patients having >/=20% CD138(+)HLA-DR(+) cells (median, 50%; range, 23-100). Because primary plasma cells cannot be efficiently cultured in vitro, we used a panel of MM cell lines with a dim/negative to bright HLA-DR expression to evaluate 1D09C3-induced cell death. Annexin V/propidium iodide (PI) staining showed that 1D09C3-induced cell death correlated with constitutive HLA-DR expression. Induction of HLA-DR by IFN-gamma restored the sensitivity of HLA-DR dim cell lines to 1D09C3. In vivo, the combined IFN-gamma/1D09C3 treatment significantly increased the median survival of nonobese diabetic/severe combined immunodeficient mice xenografted with KMS-11 cell line, compared with controls (147 versus 48 days, P 相似文献   

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.     

Anti-human FLT3 monoclonal antibody that inhibits proliferation of monocytic leukemia cell line SHI-1

FLT3, a transmembrane molecule, was found on hematopoietic stem/progenitor cells and leukemia cells and determined to be a promising target in leukemia diagnosis and therapy. In this study a functional anti-human FLT3, monoclonal antibody (MAb) 10G6, was obtained and the specificity of this MAb was verified by flow cytometry. This MAb effectively recognized the FLT3 molecule expressed on a series of malignant cell lines. Furthermore, we demonstrated that MAb 10G6 inhibited the proliferation and migration ability and induced the apoptosis of SHI-1 cells that derived from a human monocytic leukemia. This functional anti-human FLT3 MAb provides a valuable tool for further study targeting the FLT3 on leukemia cells.     

Combination therapy with anti-CTL antigen-4 and anti-4-1BB antibodies enhances cancer immunity and reduces autoimmunity

The majority of cancer antigens identified thus far have limited expression in normal tissues. It has been suggested that autoimmune disease is a necessary price for cancer immunity. This notion is supported by a recent clinical trial involving an anti-CTL antigen-4 (CTLA-4) antibody that showed significant clinical responses but severe autoimmune diseases in melanoma patients. To selectively modulate cancer immunity and autoimmunity, we used anti-CTLA-4 and anti-4-1BB antibodies to treat mice with a preexisting cancer, MC38. The combination of the two antibodies led to CD8 T-cell-mediated rejection of large established MC38 tumors and long-lasting immunity to the same tumor cells, although the same regimen was not effective for B16 melanoma. More importantly, whereas individual antibodies induced inflammation and autoimmune manifestations, combination therapy increased cancer immunity while reducing autoimmunity. The reduction of autoimmune effects correlates with an increased function of regulatory T cells. Our results suggest a novel approach to simultaneously enhance cancer immunity and reduce autoimmunity.     

Partial characterization of endothelial FGF receptor functional domain by monoclonal antibody VBS-1

Polypeptide growth factors mediate their cellular responses by binding to and activating specific cell surface receptors. Monoclonal antibody (MAb) VBS-1, produced against native fibroblast growth factor receptor-1 (FGFR-1), inhibited the binding of fibroblast growth factor-2 (FGF-2) to its receptor on coronary venular endothelial cells (CVECs) as determined by 125I-FGF-2 Scatchard analysis and [3H]thymidine uptake assays (ED50 = 80 ng/mL). Enzyme studies demonstrated that MAb VBS-1 binds to a protein epitope. Proteolytic mapping of the CVEC-FGFR established that a 52 kDa doublet contained the FGF binding site and the MAb VBS-1 antigenic epitope. N-glycanase digestion suggested the presence of a 50 kDa core protein for the CVEC-FGFR. Tunicamycin treatment resulted in the loss of expression of the core protein and the mature receptor, indicating the importance of CVEC-FGFR n-linked glycosylation. By Northern blot analysis, it was determined that CVECs express fgfr-1 and not fgfr-2. VBS-1 recognized FGFR-1 (140 kDa) and crossreacted weakly with FGFR-2 (135 kDa). Using a combination of affinity crosslinking, proteolytic mapping and Mab VBS-1 binding studies, we have located the FGF binding site near the NH2-terminal domain of the receptor close to the highly acidic box.     

A murine monoclonal antibody that binds N-terminal extracellular segment of human protease-activated receptor-4

Abstract A monoclonal antibody that recognizes native G protein coupled receptors (GPCR) is generally difficult to obtain. Protease-activated receptor-4 (PAR4) is a GPCR that plays an important role in platelet activation as a low-affinity thrombin receptor. By immunizing peptide corresponding to the N-terminal segment of human PAR4, we obtained a monoclonal antibody that recognizes cell surface expressed PAR4. Epitope mapping using a series of artificial fusion proteins that carry PAR4-derived peptide revealed that the recognition motif is fully contained within the 6-residue portion adjacent to the thrombin cleavage site. The antibody blocked PAR4 peptide cleavage by thrombin, suggesting its utility in the functional study of PAR4 signaling.     

共刺激分子4-1BBL和B7-1在人脑胶质瘤细胞中的表达

背景与目的：4-1BBL和B7-1为诱导和维持T细胞活化提供了重要的共刺激信号,目前被认为是提高抗肿瘤免疫的治疗靶点。本研究探讨4-1BBL和B7-1在7株胶质瘤细胞系表面的表达情况。方法：用流式细胞仪检测7株胶质瘤细胞株表面的共刺激分子4-1BBL和B7-1的表达,同时用MTT法分析胶质瘤细胞系对抗癌药物长春新碱（VCR）敏感性,并分析4-1BBL的表达与耐药性的相关性。结果：发现在所检测的胶质瘤细胞表面有不同程度的表达4-1BBL,但均不表达B7-1。其中T98G和MGR1细胞表面的4-1BBL表达〉30％,对VCR不敏感。UW28、SKMG1、MGR2、SF767、SKMG4细胞表面的4-1BBL表达〈10％。对VCR敏感。结论：本研究所检测的胶质瘤细胞均不表达共刺激分子B7-1。但有不同程度的表达4-1BBL,并且4-1BBL高表达的胶质瘤细胞对长春新碱敏感性差。     

Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia.

To develop a therapy for drug-resistant B-lineage acute lymphoblastic leukemia (ALL), we transduced T lymphocytes with anti-CD19 chimeric receptors, consisting of an anti-CD19 single-chain variable domain (reactive with most ALL cases), the hinge and transmembrane domains of CD8alpha, and the signaling domain of CD3zeta. We compared the antileukemic activity mediated by a novel receptor ('anti-CD19-BB-zeta') containing the signaling domain of 4-1BB (CD137; a crucial molecule for T-cell antitumor activity) to that of a receptor lacking costimulatory molecules. Retroviral transduction produced efficient and durable receptor expression in human T cells. Lymphocytes expressing anti-CD19-BB-zeta receptors exerted powerful and specific cytotoxicity against ALL cells, which was superior to that of lymphocytes with receptors lacking 4-1BB. Anti-CD19-BB-zeta lymphocytes were remarkably effective in cocultures with bone marrow mesenchymal cells, and against leukemic cells from patients with drug-resistant ALL: as few as 1% anti-CD19-BB-zeta-transduced T cells eliminated most ALL cells within 5 days. These cells also expanded and produced interleukin-2 in response to ALL cells at much higher rates than those of lymphocytes expressing equivalent receptors lacking 4-1BB. We conclude that anti-CD19 chimeric receptors containing 4-1BB are a powerful new tool for T-cell therapy of B-lineage ALL and other CD19+ B-lymphoid malignancies.     

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.     

人平足蛋白单抗SZ168抑制人骨肉瘤细胞增殖和迁移作用及机制研究

目的:探究人骨肉瘤细胞株MG63的平足蛋白（podoplanin,PDPN）表达及PDPN对MG63细胞增殖、迁移作用的影响和机制,在此基础上进一步研究抗人PDPN单抗SZ168体外抑制MG63细胞增殖和迁移的作用,判断SZ168靶向治疗骨肉瘤的临床应用潜力。方法:流式细胞术及蛋白免疫印迹实验检测PDPN在MG63中的表达。血小板聚集实验探究MG63细胞诱导血小板聚集作用,研究SZ168抑制血小板聚集作用及机制;CCK8实验检测活化血小板对MG63细胞增殖的影响;细胞划痕实验和Transwell实验探究活化血小板在MG63细胞迁移中的作用及机制。结果:MG63细胞高表达PDPN,该细胞能够诱导血小板聚集,且该作用能被SZ168呈剂量依赖性抑制,最大抑制率为（90.8±3.0）%。5×105个/mL及5×104个/mL活化血小板促进MG63细胞的增殖和迁移,与无血小板组相比差异有显著统计学意义（P＜0.01)。在CCK8实验中,与对照组比较, SZ168显著抑制MG63细胞增殖（P＜0.01）;在Transwell实验及细胞划痕实验中,与对照组比较,SZ168均显著抑制MG63细胞迁移（P＜0.05)。结论:高表达PDPN的MG63细胞与血小板表面的CLEC2受体相互作用使血小板活化,活化血小板进一步促进MG63细胞增殖和迁移。SZ168通过阻断PDPN-CLEC2轴,有效抑制MG63细胞的增殖及迁移,具有靶向治疗骨肉瘤的临床应用潜力。