Preparation and characterization of a new monoclonal antibody against CXCR4 using lentivirus vector

CXCR4 is a member of chemokine receptors and plays a vital role in numerous diseases and cancer processes, which makes the CXCR4/CXCL12 chemotactic axis a potential therapeutic target. In this study, we used lentiviral vectors as a novel technology to produce a monoclonal antibody against CXCR4. Lentivirus vector pLV-CXCR4-Puro was successfully constructed and a hybridoma cell line 1A4 was generated. The CXCR4 monoclonal antibody (MAb) 1A4 had high titer and affinity, and the isotype was identified as IgG1a. The recombinant lentivirus vector could effectively stimulate the production of 39 kDa CXCR4 antibody in vivo after immunization. Western blot analysis showed that the MAb could recognize the CXCR4 antigen expressed on transfected 293T cells as well as various human cancer cell lines. Immunofluorescence assays showed that MAb 1A4 mainly localized and strongly stained on the membrane of transfected 293T cells. Immunohistochemistry assays demonstrated that 1A4 could recognize strong expression of CXCR4 on the hepatocellular carcinoma (HCC). Thus, the method using lentiviral vectors may have application on effective and large-scale production of the CXCR4 monoclonal antibody, which will be a potential tool for the diagnosis and treatment of human cancers.     

Historical development of monoclonal antibody therapeutics

Since the first publication by Kohler and Milstein on the production of mouse monoclonal antibodies (mAbs) by hybridoma technology, mAbs have had a profound impact on medicine by providing an almost limitless source of therapeutic and diagnostic reagents. Therapeutic use of mAbs has become a major part of treatments in various diseases including transplantation, oncology, autoimmune, cardiovascular, and infectious diseases. The limitation of murine mAbs due to immunogenicity was overcome by replacement of the murine sequences with their human counterpart leading to the development of chimeric, humanized, and human therapeutic antibodies. Remarkable progress has also been made following the development of the display technologies, enabling of engineering antibodies with modified properties such as molecular size, affinity, specificity, and valency. Moreover, antibody engineering technologies are constantly advancing to enable further tuning of the effector function and serum half life. Optimal delivery to the target tissue still remains to be addressed to avoid unwanted side effects as a result of systemic treatment while achieving meaningful therapeutic effect.     

Trends in therapeutic monoclonal antibodies of cancer

Therapeutic monoclonal antibodies are increasingly applied in clinical application with great success. A variety of antibody products have been approved by the FDA since 1997. Furthermore, the industries have been paying more attention to and efforts in the field of antibody development than ever, suggesting the grand potential of the market and benefits. At present, many monoclonal antibodies have proven their therapeutic value in combination with established treatment for many diseases, as shown in FDA approved expanded indications. This old-fashioned immunotherapy exerts profound effects in many refractory and formidable diseases, especially cancers. With further understanding of the interaction between immune system and cancer, more target molecules were discovered and more promising therapeutic antibodies with improved effects will be feasible in the future. Regardless of initial development or ultimate approved drug, therapeutic monoclonal antibodies have always been associated with numerous patent applications. This review mainly focuses on potential therapeutic monoclonal antibodies in oncology and related antibody patents, and discusses the trend for antibody development and therapeutic applications in humans.     

Monoclonal Antibodies as Diagnostics; an Appraisal

Ever since the development of Hybridoma Technology in 1975 by Kohler and Milstein, our vision for antibodies as tools for research for prevention, detection and treatment of diseases, vaccine production, antigenic characterization of pathogens and in the study of genetic regulation of immune responses and disease susceptibility has been revolutionized. The monoclonal antibodies being directed against single epitopes are homogeneous, highly specific and can be produced in unlimited quantities. In animal disease diagnosis, they are very useful for identification and antigenic characterization of pathogens. Monoclonal antibodies have tremendous applications in the field of diagnostics, therapeutics and targeted drug delivery systems, not only for infectious diseases caused by bacteria, viruses and protozoa but also for cancer, metabolic and hormonal disorders. They are also used in the diagnosis of lymphoid and myeloid malignancies, tissue typing, enzyme linked immunosorbent assay, radio immunoassay, serotyping of microorganisms, immunological intervention with passive antibody, antiidiotype inhibition, or magic bullet therapy with cytotoxic agents coupled with anti mouse specific antibody. Recombinant deoxyribonucleic acid technology through genetic engineering has successfully led to the possibility of reconstruction of monoclonal antibodies viz. chimeric antibodies, humanized antibodies and complementarily determining region grafted antibodies and their enormous therapeutic use.     

Recombinant antibodies: a novel approach to cancer diagnosis and therapy

Recombinant antibodies and their fragments currently represent over 30% of all biological proteins undergoing clinical trials for diagnosis and therapy. These reagents dominate the cancer-targeting field, as highlighted by the recent approval of the first engineered therapeutic antibodies by the Food and Drugs Administration (FDA). Last year, important advances have been made in the design, selection and production of recombinant antibodies. The natural immune repertoire and somatic cell affinity maturation has been superseded by large antibody display libraries and rapid molecular evolution strategies. These novel libraries and selection methods have enabled the rapid isolation of high-affinity cancer targeting and antiviral antibodies, the latter capable of redirecting viruses for gene therapy applications. In alternative strategies for cancer diagnosis and therapy, recombinant antibody fragments have been fused to radioisotopes, drugs, toxins, enzymes and biosensor surfaces. Antibody-directed cancer pre-targeting followed by prodrug activation (ADEPT) has proved a most promising therapeutic strategy. Multi-specific antibodies have been effective for cytotoxic T-cell recruitment and antibody-fusion proteins have delivered enhanced immunotherapeutic and vaccination strategies. The new millennium is indeed an exciting time for the design, selection and formulation of a range of new antibody-based products for cancer diagnosis and therapy.     

Recombinant antibodies: a novel approach to cancer diagnosis and therapy

Recombinant antibodies and their fragments currently represent over 30% of all biological proteins undergoing clinical trials for diagnosis and therapy. These reagents dominate the cancer-targeting field, as highlighted by the recent approval of the first engineered therapeutic antibodies by the Food and Drugs Administration (FDA). Last year, important advances have been made in the design, selection and production of recombinant antibodies. The natural immune repertoire and somatic cell affinity maturation has been superseded by large antibody display libraries and rapid molecular evolution strategies. These novel libraries and selection methods have enabled the rapid isolation of high-affinity cancer targeting and antiviral antibodies, the latter capable of redirecting viruses for gene therapy applications. In alternative strategies for cancer diagnosis and therapy, recombinant antibody fragments have been fused to radioisotopes, drugs, toxins, enzymes and biosensor surfaces. Antibody-directed cancer pre-targeting followed by prodrug activation (ADEPT) has proved a most promising therapeutic strategy. Multi-specific antibodies have been effective for cytotoxic T-cell recruitment and antibody-fusion proteins have delivered enhanced immunotherapeutic and vaccination strategies. The new millennium is indeed an exciting time for the design, selection and formulation of a range of new antibody-based products for cancer diagnosis and therapy.     

人绒促性素单克隆抗体的制备与分离纯化

目的制备人绒促性素(hCG)单克隆抗体。方法hCG抗原免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0按5∶1融合,间接ELISA法筛选阳性孔,有限稀释法进行克隆化培养;制备腹水抗体;间接ELISA法测定抗体效价;采用HiTrap rProtein A FF亲和色谱柱纯化抗体。结果得到2株能稳定分泌单克隆抗体的杂交瘤细胞株,细胞培养上清中抗体效价达10-3以上,腹水抗体效价达10-7以上,纯化后的单抗纯度达98%以上,回收率达75%。结论成功获得两株能稳定分泌单克隆抗体的杂交瘤细胞,可用于早孕、肿瘤等诊断的研究。     

Recombinant antibodies in infectious disease

Antibody-based therapies have a proven record of efficacy against many diseases. However, passive immunotherapy is presently underdeveloped in the field of infectious diseases. The emergence of new recombinant technologies for the generation of therapeutic antibodies provides significant opportunities for the development of effective antibody-based drugs. Obstacles to the broader use of antibodies as anti-infectives include a continuous emphasis in the field on the use of microbicidal chemotherapy, the fact that antibodies are usually pathogen-specific drugs with relatively small markets, expense of production, the necessity for early and accurate diagnosis prior to use and the complex logistics necessary for therapeutic use. Nevertheless, many opportunities for developing antibody-based drugs now exist in areas where the available antimicrobial therapies are inadequate. As reflected in patents published in the years 2000 – 2003, activity in this field is increasingly focused on novel antibody drug candidates and it is likely that, in the near future, several antibody reagents will be developed for clinical anti-infective use.     

丙型肝炎病毒单克隆抗体的制备及其鉴定

目的：以基因工程表达的丙型肝炎病毒抗原蛋白（HCAg）为抗原，建立分泌单克隆抗体（McAb）的杂交瘤细胞系．并对其分泌的McAh进行鉴定。方法：用纯化HCV基因工程表达抗原（HCAg）免疫Balb／c小鼠，取脾细胞及骨髓瘤细胞按常规方法融合、筛选、克隆化及腹水注射制备McAb。采用ELISA及分片断鉴定技术进行鉴定。结果：筛选出5株能稳定分泌特异性抗-HC的McAb杂交瘤细胞株，经多次复苏传代仍能稳定分泌抗体，特异性强，效价高。与不同抗原蛋白的ELISA反应呈特异性阳性反应。分片断鉴定结果显示，所获5株McAb主要为抗HCV核心蛋白和非结构蛋白NS3。结论：此5株丙肝杂交瘤细胞株分泌的抗体对丙肝抗原具有特异亲和性，为丙型肝炎病毒抗原诊断试剂的研制提供了关键技术基础。     

Preparation and functional identification of a monoclonal antibody against the recombinant soluble human NKp30 receptor

NKp30 is an important activating receptor of human natural killer (NK) cells that participates in NK cell activation and cytotoxicity against tumor and infected cells. To study the function of NKp30, anti-human NKp30 monoclonal antibody was prepared. The human NKp30 ectodomain (rhNKp30) was expressed in Escherichia coli as inclusion bodies and refolded using the dilution method. The refolded rhNKp30 was purified by immobilized metal affinity chromatography. The activity of soluble rhNKp30 was confirmed by flow cytometry and NK cytotoxicity assays. Four hybridoma cell lines producing monoclonal antibodies against rhNKp30 were obtained. One of the monoclonal antibodies, designated as “3G5”, was highly specific and could be used in western blotting, immunoprecipitation, ELISA, and flow cytometry assays. The preparation of soluble rhNKp30 and a monoclonal antibody against NKp30 may provide useful tools for further functional studies of human NKp30.     

氯霉素单克隆抗体的制备与纯化

目的制备氯霉素(CAP)单克隆抗体。方法用人工合成抗原氯霉素-牛血清白蛋白(CAP-BSA)免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0按5∶1的比例融合,间接竞争ELISA法和有限稀释法进行单克隆杂交瘤细胞的筛选;制备腹水抗体;采用HiTrap rProtein A FF亲和色谱柱纯化抗体,用间接竞争ELISA法和间接ELISA测定抗体特异性。结果得到两株能稳定分泌单克隆抗体的杂交瘤细胞株,细胞培养上清中抗体效价达10-4以上,腹水抗体效价达10-7以上,纯化后的单克隆抗体纯度达98%,回收率达80%,抗体活性好并且与BSA、甲砜霉素、磺二甲基嘧啶等无交叉反应。结论成功获得两株能稳定分泌单克隆抗体的杂交瘤细胞,对动物性食品中CAP的检测具有较大的价值。     

嵌合抗体研究进展

单克隆抗体在疾病的诊断、治疗和预防中发挥着重要作用,但是在临床治疗中人抗鼠抗体反应的出现使鼠源性单克隆抗体的应用受到了很大限制.随着分子生物学、分子免疫学技术的飞速发展,抗体技术已发展到第三代抗体——基因工程抗体阶段,可利用基因工程技术对鼠源性抗体进行改造,保留或增强天然抗体的特异性和主要生物学活性,同时减少鼠源成分,以避免鼠源性单克隆抗体在临床应用方面的缺陷.此文就基因工程抗体中的重要组成部分嵌合抗体的研究进展做一综述.     

Patent Evaluation: High Affinity Humanized Monoclonal Antibodies

Summary

Novelty: A monoclonal antibody, or fragment thereof, wherein at least the hypervariable region is from a monoclonal antibody having an affinity of at least 10¹¹ l/M, and at least the constant region (or, if no constant region, the variable region) is derived from human immunoglobulin. The high-affinity monoclonal is from sheep or another mammal that gives higher affinity than a rodent. An antibody of the invention may be used in therapy, particularly for the treatment of cancer, inflammation, immunomodulation, for passive immunotherapy in the treatment of viruses, and to combat bacterial infections.

Biology: Particular advantages of high affinity antibodies for therapy are: (i) affinity is related to biological response; (ii) higher affinity antibodies will result in more rapid binding of the antibodies to the target cells or more rapid immunoneutralisation, ie. better localisation of antibody will be achieved by reducing non-specific binding to other sites; (iii) higher affinity means less antibodies can be used per dosage, leading to more economically viable therapies.

Chemistry: A product of the invention may be prepared by a process comprizing immunization of a suitable animal, e.g. sheep, rabbit, cow, using an antigen and obtaining B cells secreting an antibody to the antigen. Secondly, high affinity monoclonal antibodies to the genes coding for them are obtained. Thirdly, these antibodies are humanized.     

Shorter development of immunoassay for drugs: application of the novel RIMMS technique enables rapid production of monoclonal antibodies to ranitidine

High affinity, specific murine monoclonal antibodies have been produced for ranitidine using the novel RIMMS (repetitive immunizations, multiple sites) technique. We demonstrate that this technique can be employed to produce high affinity monoclonal antibodies to drug haptens in approximately 1 month; whereas, conventional techniques typically require 3-9 months. Polyclonal antiserum development typically requires at least 6 months. Consequently, RIMMS has a clear impact allowing reagent antibodies to be available earlier in the drug development process. Isotyping studies demonstrated that the developed antibodies are either IgG1 or IgG2b immunoglobulins which confirms that the technique produces class-switched, affinity matured reagent antibodies. The most promising monoclonal antibody for quantitative applications afforded similar sensitivity, by competitive ELISA, to the established sheep polyclonal anti-ranitidine sera. The calibration range, estimated as the limits between the asymptotic regions of calibration graphs, is 0.5-41.2 ng ranitidine per well. Specificity studies indicated that the monoclonal antibody afforded superior selectivity, yielding only 4.1% cross-reactivity with the ranitidine sulphoxide metabolite; the corresponding value for the antiserum was 8.6%. Both reagents had similar cross-reactivities with the N-oxide metabolite.     

柯萨奇病毒A组10型抗原双抗体夹心ELISA定量方法的建立及应用

目的 建立柯萨奇病毒A组10型（coxsackievirus A10,CV-A10）抗原双抗体夹心ELISA定量检测方法,用于CV-A10生产过程样品和四价手足口病疫苗的质量控制。方法  以纯化的CV-A10抗原分别免疫绵羊和小鼠获得羊免疫血清和杂交瘤单克隆抗体(单抗)细胞株,以杂交瘤细胞株免疫小鼠制备腹水,羊血清和小鼠腹水分别经亲和层析后获得纯化羊抗CV-A10多克隆抗体（多抗）和小鼠抗CV-A10单抗。采用微量细胞病变法测定纯化多抗和单抗中和抗体效价。分别以纯化羊多抗作为包被抗体、小鼠单抗作为检测抗体,进行配对筛选研究,建立CV-A10抗原定量双抗体夹心ELISA;对方法的线性、专属性、准确度、精密度、范围进行验证,并对CV-A10抗原生产过程中的样品和成品进行检测。结果 纯化抗CV-A10单抗和多抗均具有中和抗体活性。以多抗作为包被抗体、7株单抗作为检测抗体进行配对,其中5株单抗配对成功,选择CY166-14R作为检测抗体用于CV-A10抗原定量检测ELISA的建立;对检测方法进行优化,确定以羊多抗作为包被抗体的使用稀释比例为1：5 000〜1：10 000、小鼠单抗检测抗体稀释比例1 ：2 000〜1：4 000。建立的CV-A10抗原定量检测方法范围为0.42〜10. 00 U/ml,线性决定系数≥0. 99;该方法仅能特异性检测CV-A10抗原,与其他抗原或物质（肠道病毒71型、CV-A6、CV-A16、M199、DMEM、Vero细胞蛋白、牛血清）无交叉反应;对高、中、低浓度样品进行测定,测定值/理论值在95%〜110%之间,相对标准偏差在15%以内。结论 建立了 CV-A10抗原定量双抗体夹心ELISA,该方法在一定范围内的线性、专属性、准确度、精密度均较好,可用于CV-A10抗原生产过程样品和成品的质量控制,为CV-A10抗原的体外效力评价提供方法学基础。     

Detection of sulfur mustard adducts in human callus by phage antibodies

As part of a research program to develop novel methods for diagnosis of sulfur mustard exposure in the human skin the suitability of phage display was explored. Phage display is a relative new method that enables researchers to quickly evaluate a huge range of potentially useful antibodies, thereby bypassing the more costly and time-consuming hybridoma technique. The Tomlinson I and J phage libraries were used to select phage antibodies exhibiting affinity for sulfur mustard adducts on keratins, isolated from human callus. Two kinds of phage antibodies were obtained: antibodies recognizing keratin and antibodies recognizing keratin which was exposed to sulfur mustard. These phage antibodies retained activity after repeated culturing and culturing in larger volumes. For the first time antibody phage display was successfully applied for immunodiagnostics of a chemical warfare agent.     

Monoclonal antibody technology

The development, production, limitations, and uses of monoclonal antibody (MoAb) technology are presented. The first MoAbs were developed in 1975 using a process whereby the antibody-producing spleen cells of mice that had been immunized against sheep red blood cells were fused with the cells of a mouse myeloma cell line, producing hybridomas. These hybridoma cells are used to produce MoAbs, which are antibodies that will bind to only one specific target site on an antigen. Large quantities of MoAbs are grown, either in cell cultures or in the peritoneum of mice, and harvested. Although large quantities of MoAbs can be produced, these techniques are limited because of the potential for contamination by mouse viruses and the inability of the hybridomas to yield sufficient quantities of MoAbs. MoAbs are currently used in diagnostic techniques, including pregnancy tests and drug assays, as well as in tests for detecting viral and bacterial infections and cancer. MoAbs, coupled with dyes or radioactive isotopes, can be used in imaging techniques. Other possible applications of MoAbs include tissue typing, purification, therapy of cancer and autoimmune diseases, and treatment of drug toxicities. As the use of MoAbs in health care increases, pharmacists will need to have a good understanding of the functions and applications of these agents.     

治疗性单克隆抗体类药物分析技术及其应用

治疗性单克隆抗体(单抗)类药物是高度均一的生物大分子,抗原表位、理化性质和生物活性等方面的均一性程度高,能够通过特异性地与靶标分子结合发挥治疗作用。单抗类药物治疗过程中不良反应发生率低、患者耐受性高。单抗类药物是生物药物领域的热点和焦点,已成为现代生物制药行业中占比最大、增长最快的细分领域。根据生产技术和工艺的不同,单抗类药物发展已经经历了4代。我国最近批准上市的信迪利单抗注射液、特瑞普利单抗注射液、注射用卡瑞利珠单抗和替雷利珠单抗注射液等均属于第3代人源化单克隆抗体药物。单抗类药物分子结构复杂,容易发生二聚体、多聚体、末端氨基酸突变等不均一性变化,严重影响临床用药的安全性和有效性。对单抗类药物的原液和成品进行有效的质量控制是确保该类产品安全性和有效性的重要措施。目前,单抗类产品质量控制与分析的主要方法包括高效液相色谱(HPLC)法、酶联免疫吸附测定(ELISA)法、毛细管区带电泳(CZE)法、毛细管等电聚焦电泳(cIEF)法、成像毛细管等电聚焦电泳(iCIEF)法和十二烷基硫酸钠-毛细管电泳(CE-SDS)法等。本文就单抗类药物质量控制分析方法及应用进行综述,为相关研究及产品质量控制提供参考。     

多重特异性单克隆抗体研究进展

疾病的形成通常是涉及多个关键介质的复杂过程,因此针对单一成分的抗体药物有时并不能获得较好的临床疗效。多抗原靶向性治疗策略有望提高对一些复杂疾病的治疗效果,它们包括抗体的联合使用、多克隆抗体或混合抗体以及双特异性抗体等。近年来,双重或多重特异性单克隆抗体的出现为多抗原靶向性治疗提供了新策略和方法,此文对多重特异性单克隆抗体的研究进展做一综述。     

治疗性单克隆抗体类制品质量控制标准的思考

治疗性单克隆抗体制品在肿瘤、自身免疫、器官移植和感染性疾病的治疗中均取得了显著疗效,其品种和市场份额逐年显著提高。随着新的治疗性单克隆抗体制品的研发和上市,不同产品的质量控制研究,特别是新技术在质量控制中的应用被提到新的高度。由于治疗性单克隆抗体制品结构和生产的复杂性,使得质量控制的复杂程度也相应提高。本文结合治疗性单克隆抗体质量控制的工作经验和国际上的最新进展,对治疗性单克隆抗体质量控制项目设定、标准和方法进展进行论述。