Efficient generation of monoclonal antibodies from single rhesus macaque antibody secreting cells

Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n = 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs.     

Target-selective homologous recombination cloning for high-throughput generation of monoclonal antibodies from single plasma cells

Background  

Molecular cloning of functional immunoglobulin genes from single plasma cells is one of the most promising technologies for the rapid development of monoclonal antibody drugs. However, the proper insertion of PCR-amplified immunoglobulin genes into expression vectors remains an obstacle to the high-throughput production of recombinant monoclonal antibodies.     

A novel platform to produce human monoclonal antibodies: The next generation of therapeutic human monoclonal antibodies discovery

A new technology has been developed by immunologix that allows human antibodies to be quickly generated against virtually any antigen. Using a novel process, naïve human B cells are isolated from tonsil tissue and transformed with efficiency up to 85%, thus utilizing a large portion of the human VDJ/VJ repertoire. Through ex vivo stimulation, the B cells class switch and may undergo somatic hypermutation, thus producing a human “library” of different IgG antibodies that can then be screened against any antigen. Since diversity is generated ex vivo, sampling immunized or previously exposed individuals is not necessary. Cells producing the antibody of interest can be isolated through limiting dilution cloning and the human antibody from the cells can be tested for biological activity. No humanization is necessary because the antibodies are produced from human B cells. By eliminating immunization and humanization steps and screening a broadly diverse library, this platform should reduce both the cost and time involved in producing therapeutic monoclonal antibody candidates.Key words: human, antibody, monoclonal, novel platform, naïve, B cell, therapeutic     

A novel immunohistochemical technique for the detection of human oral carcinomas transplanted in nude mice using mouse monoclonal antibodies

 We have developed a new immunostaining technique specifically for the detection of human tumors transplanted into nude mice using mouse monoclonal antibodies (MoAbs) produced in our laboratory. The formation of a molecular complex consisting of three components (mouse MoAb or hybridoma supernatant, biotin-labeled anti-mouse immunoglobulins, and normal nude mouse serum) markedly reduced background staining and enhanced specific reaction with the transplanted tumors in nude mice. When MoAb production by electrofusion was screened with this new method, the incidence of hybridoma supernatant reactive with sections of transplanted tumor was 2.3 per 100 wells immunostained. These results suggest that production of MoAbs using transplanted tumors is immunohistochemically possible and that this method may provide a new means for developing useful tumor markers. Accepted: 30 September 1996     

分泌抗PRRSV抗体的猪源单克隆B细胞永生化方法的建立

猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome, PRRS)是由猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV)引起的高度接触性传染疾病,对社会经济造成重大损失。目前并未获得有效的中和抗体应用于科研及治疗中,所以建立一种筛选具有中和活性的单克隆抗体的方法,对PRRSV防治及抗原位点筛选具有重要意义。单克隆抗体在人类和动物的众多疾病治疗及诊断中得到广泛应用,而针对不同病原如何筛选出有效的中和抗体是目前急需解决的问题。在单克隆抗体筛选的众多方法中,B细胞永生化方法是一种有效的且大概率能获得单克隆中和抗体的方法。通过将bcl-6和bcl-xl两个基因中间通过f2a序列连接后构建到一个载体上,进行逆转录病毒包装。包装成熟的逆转录病毒感染免疫PRRSV的猪源淋巴细胞,并使用加入CD40L和IL21细胞因子的完全培养基培养,然后使用CD21作为筛选标记通过磁珠法进行B细胞筛选,最后对筛选得到的B细胞单克隆化并进行检测,验证是否有抗体分泌。结果...     

A strategy for the production of human monoclonal antibodies

The EBV-hybridoma system will be, at present, the best method for rescuing low-frequency tumor-reactive B-cell clones in cancer patients to produce human monoclonal antibodies reactive with tumor-related antigens. To improve this system, we established a nobel fusion partner, 3HL3-6J(C5), which produced hybridomas efficiently (greater than 2 x 10(-5)) after fusion with EBV-transformed B cell lines. TAPC-301 and C5TK1, anti-SRBC IgM and anti-HBs IgG producer, respectively, which provided by Prof. Y. Ono, were used as standard EBV-transformed B cell lines. Their hybridomas were good antibody producers (greater than 10 micrograms/10(6) cells/24 hrs) and their cloning was relatively easy. The fusion rate was improved further when electrofusion was carried out instead of the conventional PEG fusion. Using PBL from a hepatoma patient, human monoclonal autoantibodies reactive with some cytoskeleton structure were easily produced by means of this system.     

Production of monoclonal antibodies to staphylococcal alpha-toxin using the technique of in vitro immunization]

The aim of this study is production of monoclonal antibodies (MAB) to staphylococcal alpha-toxin (SAT). SAT was obtained from culture medium of S. aureus s. 0-15 with ion-exchange chromatography and chromatofocusing. SAT was conjugated with CH-sepharose 4B and used for in vitro immunization of spleen cells, extracted from intact BALB/c mouse.     

Immunological heterogeneity of rat apolipoprotein B epitope expression. A study using monoclonal antibodies to rat apolipoprotein B

Epitope expression of rat apolipoprotein B on lipoproteins was investigated with the help of six monoclonal antibodies produced from mice. Through a variety of techniques, which include cotitrations, ELISAs and quantitative immunoadsorption precipitation, we concluded that the six monoclonal antibodies recognize five different epitopes. LRB 110 and LRB 260 recognize epitopes that may be overlapping. LRB 240 and LRB 250 recognize epitopes that are preferentially expressed in triacylglycerol-rich particles. LRB 220 recognizes an epitope that is expressed by all apolipoprotein-B-containing lipoproteins. We have also determined that apolipoprotein B epitope expression in rat lipoproteins is very similar to its human counterpart. Both rat and human apolipoprotein B epitope expression on lipoproteins showed heterogeneities even in homologous lipoprotein preparations. We concluded that a variety of techniques are necessary to fully characterize monoclonal antibodies to apolipoproteins. The possible implications of epitope expression in pathophysiology are also discussed.     

Screening cDNA expression libraries with monoclonal and polyclonal antibodies using an amplified biotin-avidin-peroxidase technique

A chromogenic method using biotinylated secondary antibodies and peroxidase-coupled avidin for screening cDNA expression libraries is described. This method offers increased sensitivity over peroxidase-coupled secondary antibodies and rapid processing of samples, and avoids preparation and handling of radioactive materials. All materials for the chromogenic assay are available commercially and the method offers a fast and easy way to screen lambda and plasmid expression libraries with mono- and polyclonal antibodies.     

A strategy for production of monoclonal antibodies to Echinococcus granulosus antigen 5 and antigen B.

Serum antibody responses to sheep hydatid cyst fluid (SHCF) and a purified Antigen 5 (Ag5) were examined in ELISA, immunoelectrophoresis (IEP) and immunoprecipitation (IP) to facilitate production of monoclonal antibodies (MAb) to E. granulosus Ag5 and Antigen B (AgB). Although sera from mice immunized with SHCF contained antibodies of various classes, the fusions using these donor mice resulted in mainly anti-AgB MAb, possibly due to the preferential selection of MAb to AgB by the SHCF-based ELISA screening system. Donor mice immunized with Ag5 also produced several classes of antibodies, and the resultant fusions enabled selection of IgG MAb to Ag5.     

Growth inhibition of tumor cells in vitro by using monoclonal antibodies against gonadotropin-releasing hormone receptor

As the continuation of a previous study, synthetic peptides corresponding to the extracellular domains of human gonadotropin-releasing hormone (GnRH) receptor were used to generate additional monoclonal antibodies which were further characterized biochemically and immunologically. Among those identified to recognize GnRH receptor, monoclonal antibodies designated as GHR-103, GHR-106 and GHR-114 were found to exhibit high affinity (Kd ≤ 1 × 10⁻⁸ M) and specificity to GnRH receptor as judged by the whole cell binding immunoassay and Western blot assay. Both anti-GnRH receptor monoclonal antibodies and GnRH were shown to compete for the same binding site of GnRH receptor on the surface of cultured cancer cells. Growth inhibitions of cancer cells cultured in vitro were demonstrated by cellular apoptosis experiments (TUNEL and MTT assays) under different conditions of treatment with GHR-106 monoclonal antibody or GnRH analogs. It was generally observed that both GnRH I and GHR-106 effectively induce the apoptosis of cultured cancer cells as determined by TUNEL and MTT assays. Consistently, suppressions of gene expressions at mRNA levels were demonstrated with several ribosomal proteins (P0, P1, P2 and L37), when cancer cells were incubated with GnRH or GHR-106. The widespread expressions of GnRH receptor in almost all of the studied human cancer cell lines were also demonstrated by RT-PCR and Western blot assay, as well as indirect immunofluorescence assay with either of these monoclonal antibodies as the primary antibody. In view of the longer half life of antibodies as compared to that of GnRH or its analogs, anti-GnRH receptor monoclonal antibodies in humanized forms could function as GnRH analogs and serve as an ideal candidate of anti-cancer drugs for therapeutic treatments of various cancers in humans as well as for fertility regulations.     

A new technique for mapping epitope specificities of monoclonal antibodies using quasi-elastic light scattering spectroscopy

Quasi-elastic light scattering (QLS) was used to determine relative epitope specificities of a group of anti-bovine serum albumin (BSA) monoclonal antibodies (MAb). QLS is a non-invasive technique which can determine the mean size and size distributions of macromolecular scatterers by analysis of the fluctuations in the intensity of laser light scattering. When two MAbs are mixed together with antigen, QLS detects the complex formation which results from the Ag-Ab reaction, and can easily distinguish between the large complexes formed by interaction of non-competitive MAbs and the smaller complexes formed by competitive MAbs. In this report, the competitive or non-competitive behavior of six anti-BSA MAbs were assessed by radioimmunoassay (RIA) and QLS analysis. The results obtained by QLS analysis confirmed the RIA findings indicating that the six MAbs examined can be categorized into three distinct, non-interacting groups. QLS analysis represents a simple, and extremely rapid technique for epitope mapping studies.     

Human monoclonal antibodies by immortalization of memory B cells

The administration of hyper immune sera to prevent or treat life-threatening infections is a remarkable milestone in medicine and biotechnology that has been achieved more than a century ago. Yet, the therapeutic use of monoclonal antibodies in this field has developed slowly over the last decades. Here we compare and contrast current methods to generate human monoclonal antibodies and highlight the advantages of exploiting the human antibody repertoire using a novel method that allows efficient immortalization and cloning of human memory B cells. This method, which has been successfully applied to isolate broadly neutralizing antibodies against SARS and H5N1 influenza viruses, is expected to accelerate the development of therapeutics in the field of infectious diseases not only by providing neutralizing antibodies for passive serotherapy, but also by generating relevant information for vaccine design.     

A novel micromanipulation technique for measuring the bursting strength of single mammalian cells

Summary Information about the bursting strength of animal cells is essential if the mechanisms of cell damage in bioreactors are to be understood, and if cell mechanical properties are ever to be related to cell structure and physiology. We have developed a novel cell compression technique that makes it possible to directly measure the bursting strength of single mammalian cells, and to infer information about cell mechanical properties. Offprint requests to: C. R. Thomas