Clinical evaluation (Phase I) of a human monoclonal antibody against hepatitis C virus: safety and antiviral activity

BACKGROUND/AIMS: HCV-AB68, a human monoclonal antibody against the envelope protein of hepatitis C virus (HCV), neutralizes HCV in cell-culture and in the HCV-Trimera mouse model. A Phase 1 clinical trial was designed to test safety, tolerability, and antiviral activity of HCV-AB68 in patients with chronic HCV-infection. METHODS/RESULTS: Single doses of HCV-AB68, 0.25-40 mg, administered to 15 patients were well tolerated with no moderate or serious adverse events (SAEs) reported. In six patients, HCV-RNA levels transiently decreased by 2- to 100-fold immediately following infusion and rebound to baseline in 24-48 h. Multiple doses of HCV-AB68, 10-120 mg, were administered to 25 patients. Doses were given weekly for 3 weeks, then 3x a week during the fourth week, after which patients were followed for 3 months. No drug-related SAEs were reported and no specific pattern of adverse events was evident. Eight out of 25 patients had at least a 1-log reduction and 17 had at least a 0.75-log reduction in HCV-RNA levels from baseline at one or more time points following HCV-AB68 infusion. CONCLUSIONS: These data support the investigation of HCV-AB68 in the prevention of recurrent HCV-infection in patients who had received hepatic allografts for end-stage liver disease.     

Use of a monoclonal antibody (GA3) to demonstrate lineage restricted O- glycosylation on leukosialin during terminal erythroid differentiation

A murine monoclonal antibody (GA3) obtained by immunizing mice with cells of the human erythroleukemic cell line K562 is shown to define a 105 kilodalton (kd) membrane antigen on K562 cells that is restricted within the hematopoietic system to the erythroid lineage and to a minor population of CD3, CD4 positive T lymphocytes. Cocapping studies and immunoprecipitation experiments performed with GA3 and L10, an anti- sialophorin monoclonal antibody reacting with leukosialin (Gp 105) on K562 cells, demonstrate that the antigen detected by GA3 on K562 cells is identical to leukosialin. Neuraminidase treatment but not tunicamycin treatment of K562 cells abolishes the expression of the GA3- epitope without affecting the L10-epitope thus providing evidence that terminal sialic acid present on O-linked oligosaccharide chains on Gp 105 is essential for the expression of the GA3-epitope. Further analysis by flow cytometry and immune panning experiments performed on bone marrow cells with GA3 or L10 demonstrate that, in contrast to L10, which reacts with all types of hematopoietic progenitors, the epitope recognized by GA3 is restricted to the erythroid lineage, and appears during erythroid differentiation before glycophorin A on the earliest morphologically recognizable erythroid precursor, the proerythroblast. Our results therefore suggest that O-linked oligosaccharides on leukosialin express lineage restricted and even maturation restricted antigenic structures that might serve as cell lineage specific markers.     

Anti-Kveim monoclonal antibody. New monoclonal antibody reacting to epithelioid cells in sarcoid granulomas

A monoclonal antibody to the sarcoid granulomagenic agent contained in Kveim suspension was prepared by immunizing mice with Kveim suspension. One monoclonal antibody (IHY-1) that reacted with the epithelioid cells in sarcoid granulomas on immunoperoxidase technique was selected. The immunoperoxidase technique was used to compare this monoclonal antibody's binding to sarcoidosis- or tuberculosis-affected lymph nodes. IHY-1 is a monoclonal antibody of IgM class. This antibody did not react to erythrocytes, lymphocytes, monocytes, alveolar macrophages, or the macrophage-derived cell lines such as U-973 and KG-1. It reacted to granuloma epithelioid cells of sarcoidosis-affected lymph nodes. The monoclonal antibody also reacted positively to epithelioid cells in tuberculous granulomas although the reaction was not as strong. Since IHY-1 was found to bind to both types of granulomas, this suggests that the epithelioid cells in sarcoidosis have antigenicity common to the epithelioid cells in tuberculosis.     

Killing of human cytomegalovirus-infected fibroblasts by antiviral antibody and complement

Complement-dependent cytolytic antibodies (CyAb) to cytomegalovirus (CMV)-infected fibroblasts were detectable in acute- and convalescent-phase sera from renal allograft recipients (n = 44) and nonimmunocompromised patients (n = 14) with symptomatic CMV infection but not in sera from control donors (n = 75; P less than .001 by Wilcoxon rank sum test). Renal allograft recipients with secondary CMV infection had the highest levels of CyAb activity. Activity closely correlated with the serum antibody titer to CMV membrane antigens (r = .9106 by linear regression analysis) and was present in both the IgM and IgG fractions of human sera. IgG F(ab)2 fragments were inactive, thus implicating the classical pathway of complement activation. Maximal CMV-specific lysis was obtained with target cells expressing CMV late membrane antigens (greater than or equal to 72 hr after inoculation) irrespective of the CMV strain used. Adsorption and cold target inhibition studies indicated that the target antigens for the CyAb response are specific for the plasma membrane of CMV-infected cells and may only partly be shared by the virion envelope.     

氨基端前B型钠尿肽单克隆抗体的制备及其鉴定

目的通过经典单克隆抗体制备技术制备氨基端前B型钠尿肽(NT-pro BNP)单克隆抗体,将单克隆抗体进行特异性和亲和力鉴定,并用于免疫学检测试剂盒的开发。方法用带His-Trx-标签蛋白的NT-pro BNP免疫Balb/C小鼠制备单克隆抗体。使用PEG1500化学融合法将小鼠脾细胞与骨髓瘤细胞SP2/0进行融合。用不带标签的NT-pro BNP对获取的杂交瘤细胞进行反复多次克隆筛选,将稳定分泌抗体的杂交瘤细胞株经腹腔注射Balb/C小鼠诱导产生腹水,经蛋白G亲和纯化后获得NT-pro BNP单克隆抗体并对其进行亲和力和特异性进行鉴定。结果成功进行细胞融合并获得4株稳定分泌单克隆抗体的杂交瘤细胞株,抗体分泌亚型为Ig G1,可特异性识别NT-pro BNP。经腹腔体内诱生法和蛋白G亲和纯化,获得高质量NT-pro BNP单克隆抗体,经ELISA检测抗体效价在1∶10~5以上。结论通过传统经典单克隆抗体制备技术成功制备NT-pro BNP单克隆抗体,为NT-pro BNP检测试剂盒的研发奠定了基础。     

肝细胞DNA损伤诱导蛋白DDI2的单克隆抗体制备及鉴定

目的制备鼠抗DDI2蛋白单克隆抗体,并对其在细胞和组织中的定位进行初步研究。方法利用体外原核表达的DDI2蛋白,免疫BLAB/c小鼠,制备单克隆抗体。利用免疫组织化学技术明确该蛋白在组织中的定位。结果利用杂交瘤细胞技术,成功制备了两株DDI2单克隆抗体细胞系2H3、2B2。表达纯化的单克隆抗体具有较高的免疫活性。与正常对照组相比,肝损伤组织中该蛋白表达增强,且高表达于门脉周围肝实质细胞,并主要表达于细胞核。结论 DDI2的表达与四氯化碳诱导的肝损伤有关,损伤肝细胞主要表达于肝实质细胞的细胞核。     

Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques

Filovirus infections can cause a severe and often fatal disease in humans and nonhuman primates, including great apes. Here, three anti-Ebola virus mouse/human chimeric mAbs (c13C6, h-13F6, and c6D8) were produced in Chinese hamster ovary and in whole plant (Nicotiana benthamiana) cells. In pilot experiments testing a mixture of the three mAbs (MB-003), we found that MB-003 produced in both manufacturing systems protected rhesus macaques from lethal challenge when administered 1 h postinfection. In a pivotal follow-up experiment, we found significant protection (P < 0.05) when MB-003 treatment began 24 or 48 h postinfection (four of six survived vs. zero of two controls). In all experiments, surviving animals that received MB-003 experienced little to no viremia and had few, if any, of the clinical symptoms observed in the controls. The results represent successful postexposure in vivo efficacy by a mAb mixture and suggest that this immunoprotectant should be further pursued as a postexposure and potential therapeutic for Ebola virus exposure.     

A monoclonal antibody to hemoglobin F

Summary A monoclonal antibody BMU7-17 which recognizes an antigenic site unique to the cyanogen bromide fragment, CB2 (residues 56 through 133) of the -chain of human hemoglobin has been produced using cell hybridization techniques. The antibody does not crossreact with hemoglobin A, the isolated - or -chains, or with hemoglobin of various mammals. Affinity chromatography on Staphylococcal protein A-Sepharose 4B and radial immuno-diffusion revealed the antibody to be of the mouse IgG 1 class. BMU7-17 identifies F cells in adult and in cord blood specimens, as demonstrated by an indirect immunofluorescence assay. The dissociation constant of the antibody as determined using tritiated hemoglobin F with a double antibody radioimmunoassay has a K_d=1.2×10^–9 moles by Scatchard plot analysis.     

抗弓形虫单克隆抗体的制备鉴定及应用

目的制备抗弓形虫全抗原、天然P30、重组P30的单克隆抗体（McAb），为抗原提纯、弓形虫病诊断及亚单位疫苗的研制奠定基础。方法用弓形虫全抗原、天然P30、重组P30分别免疫BALB／c小鼠，取脾细胞与骨髓瘤细胞融合，筛选稳定分泌高滴度McAb的杂交瘤细胞株。用ELISA法测定McAb亚类和效价；通过SDS—PAGE和Western blot进行特异性鉴定;Giemsa染色观察杂交瘤细胞的染色体数目；利用电镜及IFAT观察McAb对弓形虫的杀伤作用及其作用位点。结果筛选出3株（4810、283、1H6）特异性较好的杂交瘤细胞株。Western blot结果显示，283、1H6产生的MeAb与弓形虫全抗原的阳性反应带均在30kDa处，4810反应带主要在22kDa处；283，1H6，4810MeAb的效价（ELISA）分别为：1：102400、1：51200、1：12800；283、1H6为IgM，4810为IgG2b；3株细胞的染色体数均在100条以上。电镜观察到McAb作用后的弓形虫虫体聚集、肿胀，表面出现缺口和空洞，虫体变形破碎、膜变厚。抗弓形虫全抗原及天然P30的McAb能准确识别重组体pET-30a—ROP2、pET-30a-P30的表达蛋白。结论抗弓形虫全抗原、P30抗原的McAb均对弓形虫具有明显的杀伤作用，能准确识别P30抗原，可应用于弓形虫病诊断、抗原鉴定及亚单位疫苗的研制。     

Hepatic immunohistochemical staining with a monoclonal antibody against HCV-E2 to evaluate antiviral therapy and reinfection of liver grafts in hepatitis C viral infection

BACKGROUND/AIMS: A simple and reproducible hepatic immunohistochemical staining (IHS) for hepatitis C virus (HCV) is not available. We aimed to validate hepatic IHS with monoclonal antibody (Mab) IG222, directed against the HCV-envelope 2 (E2) protein. METHODS: A three-step indirect immunoperoxidase method was used for frozen sections and a two-step indirect EnVision technique was used for paraffin-embedded sections. RESULTS: Naturally or in vitro HCV infected primary human hepatocytes were immunoreactive to HCV-E2. In the patient study (n=253), IHS had a sensitivity of 96% and a specificity of 91%. Six patients who showed positivity in the liver with Mab IG222, but remained anti-HCV and HCV-RNA negative, had hepatitis C-like changes in their liver biopsy. In one patient HCV-RNA could be detected in the liver biopsy. We confirmed early graft reinfection in patients transplanted for HCV-related disease (34 patients with serial biopsies). Treatment for acute cellular rejection with steroids was associated with an increase in staining intensity. In nine patients with clearance of HCV-RNA during antiviral therapy, seven achieved negativation of immunoreactivity and two a marked reduction. CONCLUSIONS: The IHS with Mab IG222 is an accurate tool for diagnosis and clinical management of chronic hepatitis C.     

High-risk AML evidenced by a monoclonal antibody

A monoclonal antibody has been raised against a surface membrane antigen present on leukemic myeloblasts. In 52 consecutive patients with acute myeloid leukemia treated in a standardized fashion with intensive chemotherapy the immunologic subclass with respect to this antigen was correlated to the clinical outcome. We found the expression of this antigen to predict a poor prognosis, when measured as survival of CR-patients and as survival after 1st relapse.     

A monoclonal antibody reactive with human eosinophils

EO-1, an IgGl murine monoclonal antibody raised against human eosinophilic leukemia cells, reacts with eosinophils, basophils, platelets, and a few (2%) mononuclear cells but not with neutrophils. In the bone marrow, mature and immature eosinophils and basophils express the EO-1 antigen, whereas immature myeloid cells do not. The distribution of EO-1 antigen on leukemic cells is concordant with this finding, ie, typical myeloid lines (HL-60, KG-1, and ML-1) and fresh acute myelogenous leukemia cells are all unreactive with EO-1. Immunoprecipitation of an extract from surface-131I-labeled platelets with EO-1 and sodium dodecyl sulfate polyacrylamide gel electrophoresis, under reducing or nonreducing conditions, yielded a specific band of molecular weight 23,000. Previously described monoclonal antibodies reacting with eosinophils also recognize neutrophils. EO-1 is a unique antibody with specificity restricted to eosinophils, basophils, and platelets and might therefore be a valuable reagent for the study of their function and differentiation.     

A monoclonal antibody reactive with human hepatocytes

A monoclonal antibody, RL23/36, reacting preferentially with a determinant expressed on normal human hepatocytes is described. Use of an immunohistochemical technique on frozen sections from a range of 75 human liver biopsy specimens revealed that the determinant detected by RL23/36 was not expressed on hepatocytes from a number of patients with biopsy-proven liver disease. Although a normal staining pattern was observed in 28 of 29 biopsy specimens from patients with no evidence of liver disease, the antibody did not bind to hepatocytes in some cases of chronic active hepatitis (2/13), alcoholic liver disease (2/9), haemochromatosis (1/1), cirrhosis (1/2) and liver metastases (2/8). Furthermore, as in a previous study undertaken in the rat, the antibody failed to bind to tumour cells in the single human hepatoma observed in this study. These results suggest that further studies using RL23/36 may shed light on the pathogenesis of a number of liver diseases, including primary hepatocellular carcinoma.