抗CD3人／鼠嵌合抗体的研究

已有的研究表明抗人T细胞CD3抗原的单抗具有激活和抑制人T细胞的双向功能。该单抗在抗肿瘤、抗多种器官移植排斥反应及再生障碍贫血的治疗中已显示出广阔的应用前景。但目前所用的CD3单抗都是鼠源的，大大地限制了它在临床治疗中的应用。为解决这一难题，本研究应用基因工程技术将鼠源CD3单机进行“人化”改造，在国内首次成功地构建并在其核细胞表达了抗CD3人／鼠嵌合抗体。这里综合报道研究结果如下：一、CD3单抗可变区基因cDNA的克隆和鉴定从分泌抗CD3单抗的小鼠杂交瘤HIT3a提取分离RNA，用小鼠Ig轻重涟恒定区保守序列为引物，…     

抗CD71人-鼠嵌合抗体的体外抗肿瘤效应及在肿瘤异种移植模型中的定位研究

Objective To investigate the anti-tumor effects in vitro and in vivo distribution of the human/murine chimeric antibody (D2C).Methods The CD71 positive target cells(K562,CEM and SMMC7721) and the effector cells ,freshly isolated human PBMC,with the ratio of target cells to effector cells 1:50,were incubated in various dilutions of D2C antibody(Ab).Antibody dependent cytotoxicity(AD-CC) was tested by using an LDH-release assay.Instead of effector cells,complement was added to the target cells (CEM,SMMC-7721) with various dilutions of D2C Ab.A method of counting death cells was used in complement dependent cytotoxicity(CDC) assay.Tumor localization and distribution of the chimeric antibody(D2C) were observed by labeling the chimeric Ab with radioiodine(^131I) and injecting in into nude mice(Balb/c nu/nu) transplanted with human hepatocellular carcinoma cells (SMMC-7721).Results A significant ADCC was observed with the increased concentration of the D2C Ab.Cytolysis of CD71-positive target cells by the D2C Ab was found in the presence of fresh rabbit complement.Labeled D2C administered by intraperitoneal as well as tumor regional in-jection,was visualized by SPECT. The distribution of D2C Ab in murine organs and tissues showed that non-specific binding was lower fol-lowing tumor regional administration than when the antibody was administered by an intraperitoneal injection.The human/murine chimeric antibody(D2C) has in vitro anti-tumor effects and can exert its effects in specific tumor localization.Its distribution and local effects in vi-vo can be detected by radioimmunoimaging. Conclusion CD71 human/murine chimeric antibody showed marked killing of tumor cells in vitro,and specific recognition and high affinity binding to tumor tissue in vivo.     

抗人CD3人/鼠嵌合抗体基因的构建

为了降低鼠源抗人CD3单抗的免疫原性．增加其在人体内的生物活性及治疗作用，使该抗体能更广泛更有效地长期多次用于人体治疗肿瘤、器官移植排斥反应及自身免疫性疾病。本文采用PCR技术从分泌抗CD3单抗的杂交瘤细胞HIT3a的mRNA中分离克隆了抗体的轻重链可变区基因的cDNA。以此轻重链可变区cDNA为特异探针从HIT3a基因文库中分离带有调控序列的功能性轻重链可变区基因，并将其插入到含有人k轻链及人71重链恒定区基因的哺乳动物表达载体中成功地构建了抗人CD3人／鼠轻重链嵌合抗体基因，为研制人抗CD3入／鼠嵌合抗体完成了关键性的第一步。     

抗人胃癌3H11人-鼠嵌合抗体的构建及表达

为了降低抗人胃癌鼠单抗3H11的免疫原性,以利于该单抗在临床中的应用。我们构建并表达了3H11的人-鼠嵌合抗体,将3H11的轻、重链可变区基因分别插入到含有人k链及IgGl重链恒定区基因的真核细胞表达载体中,构建了3H11人-鼠嵌合抗体轻、重链表达载体。应用Lipofectin方法先将嵌合轻链表达载体转染到Sp2/0细胞中,经用含霉酚酸的选择培养基筛选及克隆化培养,获得稳定分泌3H11人-鼠嵌合轻链的转染细胞株。再将嵌合重链表达载体转染该细胞系,用含有组氨醇的选择培养基筛选,获得组氨醇抗性细胞株,经亚克隆后得到可稳定分泌人k链和人IgGl的转染细胞系,经ELISA检测该细胞系所分泌的上清含有可与人胃癌细胞系803结合的人IgG抗体活性,RT-PCR结果显示该细胞株有人-鼠嵌合抗体mRNA的转录,证明已获得分泌3H11人-鼠嵌合抗体的细胞系。     

抗人CD3人/鼠嵌合抗体的表达及特性

将从HIT3a基因库分离克隆的功能性抗CD3轻重链可变区基因插入含有人k轻链及γ1重链恒定区基因的哺乳动物表达载体中，构建了抗CD3嵌合抗体基因,用Lipofectin将抗CD3人／鼠嵌合轻重链基因共转染SP2／0细胞表达，获得稳定传代和稳定表达抗CD3嵌合抗体的转染杂交瘤细胞株C-HIT3a，ELISA，免疫荧光和PCR实验证实嵌合抗体与原鼠CD3单抗有相同的特异性和亲和性。体外生物活性的初步分析表明嵌合抗体与鼠CD3单抗对人的PBMC细胞的增殖有相同类型的作用，高剂量抗体抑制人T细胞的增殖，低剂量显示明显的激活增殖作用，当与IL-2联合应用其激活增殖作用增加20倍。细胞毒实验表明嵌合CD3抗体或由该抗体介导的免疫活性细胞(CD3-AK)对多种肿瘤细胞有明显的杀伤活性，较单用IL-2或单用LAK细胞其杀伤活性增加25倍。     

抗人卵巢癌-抗人CD3单链双特异性抗体体外介导的细胞毒作用及其机制

目的了解抗人卵巢癌-抗人CD3（BHL—I）单链双特异性抗体（scBsAb）体外介导的外周血淋巴细胞（PBL）对靶细胞SKOV3的细胞毒作用及可能的机制。方法二苯基溴化四氮唑蓝（MTr）法检测BHL-I体外介导PBL对SKOV3细胞的杀伤作用；逆转录-聚合酶链反应（RT—PCR）检测BHL—I介导的细胞毒作用过程中效应细胞PBL对穿孔素（perforin）、颗粒酶（GrB）mRNA表达的影响；酶联免疫吸附（ELISA）法检测细胞培养上清液中人肿瘤坏死因子-α（hTNF—α）和人干扰素-γ（hIFN-γ）含量的变化。结果BHL-I体外介导PBL对SKOV3细胞的细胞毒作用显著高于对MCF-7细胞的作用（P〈0.01），并且在效靶比为12．5：1、作用时间为36h、BHL-I浓度为25μg/ml时，PBL对靶细胞的细胞毒作用最为显著；BHL—I体外介导的细胞毒作用中，PBL表达perforin、GrBmRNA及混合细胞培养上清液中hTNF—α和hIFN-γ的含量均显著升高，并且白介素2（IL-2）的存在有利于这些因子的表达。结论BHL—I介导PBL对靶细胞的细胞毒作用具有一定的特异性，并且IL-2能增强BHL—I介导PBL的细胞毒作用。     

基因工程抗体的研究

本文综述了本实验室近年来在基因工程抗体方面所做的工作：①从杂交瘤细胞基因组中筛选和鉴定出抗乙型脑炎病毒单克隆抗体的重链、轻链可变区基因，构建成人-鼠嵌舍基因并在骨髓瘤细胞中表达出抗乙型脑炎病毒的人-鼠嵌合抗体；②根据文献中抗CD3单抗的序列，进行了分子设计，设计出改形抗体的分子序列．用合成和PCR补齐的方法构建了改形的单域抗体（Vn)表达载体，并在大肠杆菌中表达；④构建了外分泌型-附着型的表达单链抗体的表达载体；④克隆和测序了抗人肺腺癌．膀胱癌、CD3的单抗重,轻链可变区基因，并正在构建鼠的抗体库。     

抗人卵巢癌/抗人CD3单链双特异性抗体的生物学活性研究

背景与目的目前,常规疗法已难以提高卵巢癌患者的生存率,已有实验数据及临床前试验结果表明,双特异性抗体能有效介导效应细胞对肿瘤细胞的杀伤作用。本研究旨在通过检测抗人卵巢癌/抗人CD3单链双特异性抗体(BHL-I)的生物学活性,为其临床前试验及应用提供实验依据。方法观察BHL-I介导的外周血淋巴细胞(peripheralbloodlymphocyte,PBL)与靶细胞SKOV3结合、MTT法检测外周血单个核细胞(peripheralbloodmononuclearcells,PBMCs)增殖及PBL对靶细胞SKOV3杀伤效应,ELISA法检测杀伤过程中PBL分泌hIFN-γ、hTNF-α变化。结果BHL-I介导的花环形成率(15.7%)显著高于对照组(11.1%)(P<0.01);在抗原存在下,BHL-I显著促进PBMC增殖和PBL对靶细胞的杀伤(P<0.01),且杀伤率与花环形成率呈正相关(r=0.946);杀伤过程中上清液中hIFN-γ、hTNF-α显著增高(P<0.01)。结论BHL-I能介导PBL和SKOV3结合并活化PBL特异杀伤效应,杀伤可能与其hIFN-γ、hTNF-α表达增高有关。     

双特异抗体介导的CD3AK对人小细胞肺癌细胞株的细胞毒作用

目的：研究在双特异性抗体的介导下ＣＤ３ＡＫ细胞（ＣＤ３单克隆栓激活的杀伤细胞）对人小细胞肺癌细胞株ＬＴＥＰ－ｓｍｌ的细胞毒作用。方法：用＾３１Ｃｒ－Ｎａ２ＣｒＯ４释放试验检测单抗（２Ｄ６、ＵＣＨＴ１）和双特异性抗体（ＷＳＴ－Ｈ７）与ＣＤ３ＡＫ共同对人小细胞肺癌细胞株的杀伤活性。持异性抗体体外能明显增强ＣＤ３ＡＫ细胞对人小细胞肺细胞株ＬＴＥＰ－ｓｍｌ的杀伤活性结论：与单纯ＣＤ３ＡＫ相比,加入双特异性     

双特异性抗体对LAK细胞增殖和细胞毒作用影响的体外研究

LAK细胞过继免疫治疗恶性肿瘤具有一定的疗效，南于不能特异性识别和攻击靶细胞影响了LAK细胞的治疗效果进一步提高。双特异抗体能同时识别效应细胞和靶细胞，使其特异地结合，选择性地将靶细胞杀死，本研究应用化学偶联剂SPDP将鼠源抗CD3与抗HBs连接得到化学嵌合双特异性抗体，     

Feasibility and pharmacokinetic study of a chimeric anti-CD20 monoclonal antibody (IDEC-C2B8, rituximab) in relapsed B-cell lymphoma

Background: In clinical trials in the USA, IDEC-C2B8 (a mouse-humanchimeric anti-CD20 monoclonal antibody) has demonstrated high response rateswith only mild toxic effects in relapsed B-cell lymphoma at a dose of fourweekly 375 mg/m² infusions. The aim of the present trial wasto determine whether or not this dose is practically applicable to Japanesepatients with relapsed B-cell lymphoma with respect to safety,pharmacokinetics and efficacy.Patients and methods: Patients with relapsed CD20+ B-cell lymphomareceived intravenous infusions of IDEC-C2B8 once a week for four weeks. Atotal of 12 patients (four at 250 mg/m² and eight at 375mg/m²) were enrolled.Results: All 11 eligible patients treated with either dose leveltolerated IDEC-C2B8 well. Commonly observed adverse drug reactions weregrades 1 or 2 non-hematologic toxicities during the infusion, consistingmostly of flu-like symptoms and skin reactions. All of the observedhematologic toxicities were of grade 3 or less, and transient. A rapid andsustained B-cell decrease in peripheral blood was observed, but noinfectious episodes were encountered. Human anti-mouse and anti-chimericantibodies were not detected. Of the 11 eligible patients (eight withfollicular, two with diffuse large-cell and one with mantle cell lymphoma),two showed a complete response and five showed a partial response, and allof the seven responders had lymphoma with follicular histology. Apharmacokinetic analysis showed that the elimination half-life (T1/2) ofIDEC-C2B8 was 445 ± 361 hours, and that the serum antibody levelsincreased in parallel with the course of infusions, and in most patients wasstill measurable at three months.Conclusions: The dose of four weekly 375 mg/m² infusionsof IDEC-C2B8 is safe and effective in Japanese patients with relapsed B-celllymphoma. Further studies evaluating IDEC-C2B8 are warranted.     

Engineering and characterisation of chimeric monoclonal antibody 806 (ch806) for targeted immunotherapy of tumours expressing de2-7 EGFR or amplified EGFR

We report the generation of a chimeric monoclonal antibody (ch806) with specificity for an epitope on the epidermal growth factor receptor (EGFR) that is different from that targeted by all other anti-EGFR therapies. Ch806 antibody is reactive to both de2-7 and overexpressed wild-type (wt) EGFR but not native EGFR expressed in normal tissues at physiological levels. Ch806 was stably expressed in CHO (DHFR -/-) cells and purified for subsequent characterisation and validated for use in preliminary immunotherapy investigations. Ch806 retained the antigen binding specificity and affinity of the murine parental antibody. Furthermore, ch806 displayed enhanced antibody-dependent cellular cytotoxicity against target cells expressing the 806 antigen in the presence of human effector cells. Ch806 was successfully radiolabelled with both iodine-125 and indium-111 without loss of antigen binding affinity or specificity. The radioimmunoconjugates were stable in the presence of human serum at 37 degrees C for up to 9 days and displayed a terminal half-life (T(1/2beta)) of approximately 78 h in nude mice. Biodistribution studies undertaken in BALB/c nude mice bearing de2-7 EGFR-expressing or amplified EGFR-expressing xenografts revealed that (125)I-labelled ch806 failed to display any significant tumour retention. However, specific and prolonged tumour localisation of (111)In-labelled ch806 was demonstrated with uptake of 31%ID g(-1) and a tumour to blood ratio of 5 : 1 observed at 7 days postinjection. In vivo therapy studies with ch806 demonstrated significant antitumour effects on established de2-7 EGFR xenografts in BALB/c nude mice compared to control, and both murine 806 and the anti-EGFR 528 antibodies. These results support a potential therapeutic role of ch806 in the treatment of suitable EGFR-expressing tumours, and warrants further investigation of the potential of ch806 as a therapeutic agent.     

The antitumor activity of an anti-CD54 antibody in SCID mice xenografted with human breast, prostate, non-small cell lung, and pancreatic tumor cell lines

We have previously described the development and testing of a monoclonal anti-human CD54 antibody (UV3) in SCID mice xenografted with human multiple myeloma, lymphoma, and melanoma cell lines. In all 3 cases, UV3 was highly effective at slowing the growth of tumors and/or prolonging survival. Since CD54 (ICAM-1) is up-regulated on many different types of cancer cells, we have now investigated the anti-tumor activity of UV3 in several other CD54(+) epithelial tumors. A panel of 16 human breast, prostate, non-small cell (NSC) lung, and pancreatic tumor cell lines was examined for reactivity with UV3, and 13 were positive. A representative CD54(+) cell line from each cancer was grown subcutaneously in SCID mice. Once the tumors were established, UV3 was administered using different dose regimens. UV3 slowed the growth of all 4 tumors, although it was not curative. When UV3 or gemcitabine were administered to SCID mice xenografted with a NSC lung tumor cell line or a pancreatic tumor cell line, UV3 was as effective as the chemotherapy alone. When gemcitabine and UV3 were administered together, the best anti-tumor responses were observed. UV3 has been chimerized (cUV3) and both toxicology studies and clinical trials are planned to assess the safety and activity of cUV3 in patients with one or more of these tumors.     

Inhibition of human B-cell lymphoma by an anti-CD20 antibody and its chimeric F(ab')2 fragment via induction of apoptosis

Monoclonal antibodies (mAb) directed against CD20, either unmodified or in radiolabeled forms, have been successfully used in clinic as effective therapeutic agents in the management of non-Hodgkin's B-cell lymphoma. Despite all clinical success the exact mechanisms of action of various anti-CD20 antibodies remains mostly unclear. Several mechanisms have been proposed to be responsible for the therapeutic activity of anti-CD20 antibodies, including antibody-dependent cell-mediated cytotoxicity, complement-mediated cytotoxicity, and direct inhibition of tumor growth via induction of apoptosis. We previously produced an anti-CD20 mAb, HI47, and showed that the antibody effectively blocked human B-cell proliferation in vitro and inhibited xenografted B-cell lymphoma in nude mice. In this study, we engineered the chimeric versions of both the Fab and F(ab)'2 fragments of HI47 and produced the fragments in E. coli. Both fragments competed efficiently with HI47 for binding to CD20+ B cells, and inhibited proliferation of B-lymphoma cells in a dose-dependent manner. Mechanistic studies revealed that both antibody fragments induced significant degree of B-cell apoptosis that is independent of any cross-linking agents. Further, both the F(ab)'2 and Fab fragments when administered in vivo significantly inhibited the growth of human B-cell lymphoma xenografts in nude mice. The bivalent F(ab)'2 fragment showed consistently better efficacy compared to its monovalent Fab counterpart in inducing apoptosis and inhibiting B-cell lymphoma growth both in vitro and in vivo. Taken together, these observations suggest that HI47 and its fragments most likely exert their antitumor activity through induction of cell apoptosis, and cross-linking/dimerization of CD20 molecules on B- cell surface is an important, but not essential, process for therapeutic efficacy of HI47 and its fragments.     

In vitro and in vivo stability and anti-tumour efficacy of an anti-EGFR/anti-CD3 F(ab')2 bispecific monoclonal antibody.

The in vitro and in vivo stability and anti-tumour efficacy of the anti-EGFR/anti-CD3 bispecific monoclonal antibody (biMAb), M26.1, were analysed. The interaction of the intact biMAb with Fc receptor I (Fc gamma RI) present on human leucocytes was not observed when the antibody was used as an F(ab'')2 fragment. A CD8+ T-cell clone coated with M26.1 F(ab'')2 was as effective as the intact biMAb in inducing IGROV1 target cell lysis when tested in a 51Cr-release assay. Variable levels of reduction of F(ab'')2 to monovalent F(ab'') were observed upon incubation with human ovarian cancer ascitic fluid (OCAF) or with human glioblastoma cavity fluid (GCF), but not with mouse or human sera. Activated lymphocytes coated with F(ab'')2 and incubated in vitro with GCF or OCAF for 24 and 48 h respectively maintained their targeting. Thus, the F(ab'')2, when present as a soluble molecule, but not when bound to T cells, might lose some functional activity as a consequence of partial reduction to F(ab''). In normal mice, M26.1 F(ab'')2 retained full cytotoxic activity in the circulation, and clearance values were similar to those obtained with parental and other MAb F(ab'')2. Treatment of IGROV1 tumour-bearing mice with activated human lymphocytes coated with the M26.1 F(ab'')2 significantly prolonged survival of the animals compared with tumour-bearing untreated and control mice treated with lymphocytes or F(ab'')2 alone. Together, these results suggest the clinical usefulness of bispecific M26.1 F(ab'')2 as a targeting agent for local treatment of tumours such as glioma and ovarian cancers that express variable levels of epidermal growth factor receptor (EGFR).     

Effect of an anti-CD54 (ICAM-1) monoclonal antibody (UV3) on the growth of human uveal melanoma cells transplanted heterotopically and orthotopically in SCID mice

We have shown that administration of a novel anti-CD54 monoclonal antibody (UV3) results in long-term survival of SCID mice bearing human myeloma xenografts. Previous studies have demonstrated a link between the expression of CD54 and the progression of uveal melanoma. Our study assessed the expression of CD54 on 7 human uveal melanoma cell lines and 3 cell lines established from uveal melanoma metastases. In vivo studies examined the efficacy of systemic and local administration of UV3 antibody on the progression of uveal melanoma cells transplanted either heterotopically or orthotopically into SCID mice. Five of the 7 primary uveal melanoma cell lines and all 3 of the metastases cell lines expressed CD54. Intraperitoneal injection of either IgG or F(ab')2 fragments of UV3 significantly inhibited the growth of subcutaneous and intraocular melanomas. Subconjunctival injection of either IgG or F(ab')2 fragments of UV3 produced a significant reduction in the growth of intraocular melanomas, even if the antibody was administered after the appearance of intraocular tumors. The results indicate that both primary and metastatic human uveal melanoma cells express CD54. The marked inhibition of intraocular and subcutaneous uveal melanoma progression suggests that UV3 antibody is a promising therapeutic agent for further evaluation in patients with uveal melanoma. This is especially noteworthy, as no existing therapeutic modality prevents metastasis of uveal melanoma or prolongs the survival of patients with uveal melanoma.     

计算机模建和点突变分析抗人CD40单克隆抗体5C11识别的抗原表位

目的:通过计算机模拟与点突变实验初步探讨本课题组前期研制的抗人CD40激发型单克隆抗体5C11识别的抗原表位。方法:利用InsightⅡ软件分别模拟抗原、抗体结构,构建抗原抗体复合物模型,通过计算推测5C11单抗所识别的抗原表位。构建人野生型CD40(wtCD40)及其第70位苏氨酸突变型(70muCD40)和第114位谷氨酸突变型(114muCD40)的重组真核表达载体pIRES2-EGFP/wtCD40、pIRES2-EGFP/70muCD40和pIRES2-EGFP/114muCD40,脂质体转染法将重组载体导入HEK293细胞,筛选稳定转染细胞株(即HEK293/wtCD40、HEK293/70muCD40和HEK293/114muCD40细胞)。流式细胞术和Western blotting检测5C11单抗与HEK293/wtCD40、HEK293/70muCD40和HEK293/114muCD40细胞的结合能力。结果:成功构建pIRES2-EG-FP/wtCD40、pIRES2-EGFP/70muCD40和pIRES2-EGFP/114muCD40真核表达载体和相应稳定转染细胞株。5C11单抗与HEK293/70muCD40和HEK293/114muCD40细胞结合能力较HEK293/wtCD40细胞明显减弱;Western blotting检测结果表明,5C11单抗仅识别HEK293/wtCD40细胞,不识别HEK293/70muCD40和HEK293/114muCD40细胞。结论:人CD40氨基酸序列的第70位苏氨酸和第114位谷氨酸是其单抗5C11识别的抗原表位,对构建人源化CD40抗体具有潜在的临床意义。     

Expression of connective tissue growth factor (CTGF/CCN2) in a mouse model of rhabdomyosarcomagenesis

Connective tissue growth factor (CTGF/CCN2) is a cysteine-rich matricellular protein that belongs to the CCN (CYR61, CTGF, NOV) protein family. It is highly expressed by human rhabdomyosarcoma cells and sustains their survival. In this study we investigated CCN2 expression in a mouse model of spontaneous rhabdomyosarcomagenesis that combines HER-2/neu oncogene activation and p53 oncosuppressor gene inactivation (BALB-p53neu mice). Murine rhabdomyosarcoma cells showed a 4-26 fold increase in CCN2 mRNA expression regarding to normal thigh muscle. Moreover, they expressed CCN2 protein at levels comparable to human rhabdomyosarcoma cells. Therefore BALBp53neu mice might be useful for the evaluation of the role played by CCN2 in rhabdomyosarcoma in vivo.