Large-Scale In Vitro Expansion of Polyclonal Human Switched-Memory B Lymphocytes

Polyclonal preparations of therapeutic immunoglobulins, namely intravenous immunoglobulins (IVIg), are essential in the treatment of immunodeficiency and are increasingly used for the treatment of autoimmune and inflammatory diseases. Currently, patients’ accessibility to IVIg depends exclusively upon volunteer blood donations followed by the fractionation of pooled human plasma obtained from thousands of individuals. Presently, there are no in vitro cell culture procedures allowing the preparation of polyclonal human antibodies. All in vitro human therapeutic antibodies that are currently generated are based on monoclonal antibodies, which are mostly issued from genetic engineering or single cell antibody technologies. Here, we describe an in vitro cell culture system, using CD40-CD154 interactions, that leads to a 1×10⁶-fold expansion of switched memory B lymphocytes in approximately 50 days. These expanded cells secrete polyclonal IgG, which distribution into IgG₁, IgG₂, IgG₃ and IgG₄ is similar to that of normal human serum. Such in vitro generated IgG showed relatively low self-reactivity since they interacted moderately with only 24 human antigens among a total of 9484 targets. Furthermore, up to one liter of IgG secreting cells can be produced in about 40 days. This experimental model, providing large-scale expansion of human B lymphocytes, represents a critical step toward the in vitro production of polyclonal human IgG and a new method for the ex vivo expansion of B cells for therapeutic purposes.     

Antibody production: polyclonal-derived biotherapeutics

Antibody based therapies using monoclonal or polyclonal antibodies are emerging as an important therapeutic approach for the treatment of a number of diseases. With increasing emphasis on new technologies associated with monoclonal antibody expression and purification, the clinical need of polyclonal therapeutics for treatment of a variety of specific illnesses and infections is often overlooked. Despite being largely abandoned in the early twentieth century due to the development of antibiotics, polyclonal antibody therapeutics are today widely used in medicine for viral and toxin neutralization and for replacement therapy in patients with immunoglobulin deficiencies. Over the past 20 years, intravenous immunoglobulins have shown beneficial immunomodulatory and anti-inflammatory effects in many illnesses. Hyperimmune antibody preparations have been used over the past century for the treatment of a variety of infectious agents and medical emergencies, including digoxin toxicity, snake envenomation and spider bites. Here, we examine the contemporary techniques and applications, and assess the future therapeutic potential, for polyclonal-derived antibody therapeutics.     

The presence of non-isotype-specific antibodies in polyclonal anti-IgE reagents: demonstration of their binding to specifically selected Epstein-Barr virus-transformed cell lines

Polyclonal anti-human IgE reagents were earlier shown to contain variable amounts of nonisotype-specific antibodies depending on the strategy used for their preparation. The presence of these antibodies in two commercial anti-IgE reagents was demonstrated in this work by (a) their binding to human Ig-surface-positive lymphoblastoid cells specifically selected by one of the polyclonal anti-human IgE reagents and (b) their binding to the non-IgE immunoglobulins secreted by those lymphoblastoid cells. Peripheral blood B lymphocytes from two normal and two atopic patients were immortalized with Epstein-Barr virus (EBV) and then selected for cells that rosette with anti-IgE-coated erythrocytes. Selection was repeated four times and cells were then cloned. The cloned cells formed rosettes and their supernatants agglutinated erythrocytes coated with rabbit anti-IgE. The immunoglobulins of these clones were positive in an ELISA for IgE, using two different polyclonal anti-human IgE reagents. They were shown, however, to be 19 S IgMs. This discrepancy was due apparently to substantial contamination of anti-non-IgE-isotype-specific antibodies in the polyclonal anti-IgE reagents used both in the selection of cells and in the ELISA. The human monoclonal B-cell lines which were applied here as targets amplified the non-IgE-isotype specific antibody contamination present in the polyclonal anti-human IgE reagents. Because of the normally very low frequency of IgE-positive cells, the use of polyclonal anti-IgE reagents to detect these cells has to be carefully evaluated.     

An Empirical Approach towards the Efficient and Optimal Production of Influenza-Neutralizing Ovine Polyclonal Antibodies Demonstrates That the Novel Adjuvant CoVaccine HT? Is Functionally Superior to Freund's Adjuvant

Passive immunotherapies utilising polyclonal antibodies could have a valuable role in preventing and treating infectious diseases such as influenza, particularly in pandemic situations but also in immunocompromised populations such as the elderly, the chronically immunosuppressed, pregnant women, infants and those with chronic diseases. The aim of this study was to optimise current methods used to generate ovine polyclonal antibodies. Polyclonal antibodies to baculovirus-expressed recombinant influenza haemagglutinin from A/Puerto Rico/8/1934 H1N1 (PR8) were elicited in sheep using various immunisation regimens designed to investigate the priming immunisation route, adjuvant formulation, sheep age, and antigen dose, and to empirically ascertain which combination maximised antibody output. The novel adjuvant CoVaccine HT™ was compared to Freund’s adjuvant which is currently the adjuvant of choice for commercial production of ovine polyclonal Fab therapies. CoVaccine HT™ induced significantly higher titres of functional ovine anti-haemagglutinin IgG than Freund’s adjuvant but with fewer side effects, including reduced site reactions. Polyclonal hyperimmune sheep sera effectively neutralised influenza virus in vitro and, when given before or after influenza virus challenge, prevented the death of infected mice. Neither the age of the sheep nor the route of antigen administration appeared to influence antibody titre. Moreover, reducing the administrated dose of haemagglutinin antigen minimally affected antibody titre. Together, these results suggest a cost effective way of producing high and sustained yields of functional ovine polyclonal antibodies specifically for the prevention and treatment of globally significant diseases.     

Novel human antibody therapeutics: the age of the Umabs

Monoclonal antibodies represent a major and increasingly important category of biotechnology products for the treatment of human diseases. The state-of-the-art of antibody technology has evolved to the point where therapeutic monoclonal antibodies, that are practically indistinguishable from antibodies induced in humans, are routinely generated. We depict how our science-based approach can be used to further improve the efficacy of antibody therapeutics, illustrated by the development of three monoclonal antibodies for various cancer indications: zanolimumab (directed against CD4), ofatumumab (directed against CD20) and zalutumumab (directed against epidermal growth factor receptor).     

Production and characterisation of a neutralising chimeric antibody against botulinum neurotoxin A

Botulinum neurotoxins, produced by Clostridium botulinum bacteria, are the causative agent of botulism. This disease only affects a few hundred people each year, thus ranking it among the orphan diseases. However, botulinum toxin type A (BoNT/A) is the most potent toxin known to man. Due to their potency and ease of production, these toxins were classified by the Centers for Disease Control and Prevention (CDC) as Category A biothreat agents. For several biothreat agents, like BoNT/A, passive immunotherapy remains the only possible effective treatment allowing in vivo neutralization, despite possible major side effects. Recently, several mouse monoclonal antibodies directed against a recombinant fragment of BoNT/A were produced in our laboratory and most efficiently neutralised the neurotoxin. In the present work, the most powerful one, TA12, was selected for chimerisation. The variable regions of this antibody were thus cloned and fused with the constant counterparts of human IgG1 (kappa light and gamma 1 heavy chains). Chimeric antibody production was evaluated in mammalian myeloma cells (SP2/0-Ag14) and insect cells (Sf9). After purifying the recombinant antibody by affinity chromatography, the biochemical properties of chimeric and mouse antibody were compared. Both have the same very low affinity constant (close to 10 pM) and the chimeric antibody exhibited a similar capacity to its parent counterpart in neutralising the toxin in vivo. Its strong affinity and high neutralising potency make this chimeric antibody interesting for immunotherapy treatment in humans in cases of poisoning, particularly as there is a probable limitation of the immunological side effects observed with classical polyclonal antisera from heterologous species.     

Pathogen inactivation and removal procedures used in the production of intravenous immunoglobulins

Patients with immunodeficiencies or some types of autoimmune diseases rely on a safe therapy with intravenous immunoglobulins (IVIGs) manufactured from human plasma, the only available source for this therapeutic. Since plasma is predisposed to contamination by a variety of blood-borne pathogens, ascertaining and ensuring the pathogen safety of plasma-derived therapeutics is a priority among manufacturers. State-of-the-art manufacturing processes provide a high safety standard by incorporating virus elimination procedures into the manufacturing process. Based on their mechanism these procedures are grouped into three classes: partitioning, inactivation, and virusfiltration.     

Manufacture of recombinant polyclonal antibodies

Polyclonal antibody therapy in the form of hyper-immune serum has for more than a century been used for treatment of many infectious diseases. However, with the emergence of first antibiotics and later recombinant monoclonal antibody therapy, the use of hyper-immune serum has declined. The main reason for this is that methods for consistent manufacturing of safe hyper immune immunoglobulin products have been lacking. In contrast, manufacturing processes of recombinant monoclonal antibodies follow a well established schedule and it appears obvious to use similar methods to produce recombinant polyclonal products. However, the methods for monoclonal antibody manufacturing are, for several reasons, not directly applicable to generation and manufacture of polyclonal recombinant antibodies. A new production strategy based on recombinant mammalian producer cells has recently been developed to support consistent generation of recombinant polyclonal antibodies for therapeutic use. This review describes aspects of this novel technology with emphasis on the generation, production and characterization procedures employed, and provides comparison with alternative polyclonal and monoclonal antibody manufacturing strategies.     

Thiophilic paramagnetic particles as a batch separation medium for the purification of antibodies from various source materials

A preparation of thiophilic agarose-based paramagnetic particles (T-Gel) has been developed with physical characteristics (particle size and particle density) that facilitate its use as a batch separation medium suitable for the large-scale purification and isolation of immunoglobulins. The medium was used to extract immunoglobulins from a wide range of starting materials, including sera, ascites fluid, tissue culture medium, and whole blood. None of these starting materials required pretreatment such as clarification by centrifugation or filtration prior to antibody extraction. The antibody purity obtained using T-Gel compared well with that obtained using protein A agarose column chromatography. Yields were approximately 30 mg of immunoglobulins per milliliter of T-Gel, and little was required in the way of specialist equipment. The method is uncomplicated and involves a roll mix extraction overnight, followed by magnetic separation to facilitate supernatant removal and subsequent washing of the particles. Elution of bound antibodies was carried out at neutral pH to yield a concentration of immunoglobulins that was approximately 7 mg/ml. The method was found to be applicable to antibody purification from the blood serum of seven different mammalian species and for all immunoglobulin classes.     

Recombinant monoclonal antibodies for rabies post-exposure prophylaxis

Rabies virus is a prototypical neurotropic virus that causes one of the most dangerous zoonotic diseases in humans. Humanized or fully human monoclonal antibodies (mAb) that neutralize rabies virus would be the basis for powerful post-exposure prophylaxis of rabies in humans, having several significant benefits in comparison with human or equine rabies polyclonal immunoglobulins. The most advanced antibodies should broadly neutralize natural rabies virus isolates, bind with conserved antigenic determinants of the rabies virus glycoprotein, and show high neutralizing potency in assays in vivo. The antibodies should recognize nonoverlapping epitopes if they are used in combination. This review focuses on basic requirements for anti-rabies therapeutic antibodies. The urgency in the search for novel rabies post-exposure prophylaxis and methods of development of anti-rabies human mAb cocktail are discussed. The rabies virus structure and pathways of its penetration into the nervous system are also briefly described.     

单克隆抗体与多克隆抗体配对ELISA方法比较

以人绒毛膜促性腺激素(HCG)为抗原,制备出对HCG的多克隆抗体和特异性单克隆抗体,并进行抗体纯化和特性分析,利用辣根过氧化物酶(HRP)分别对其进行了标记.采用双抗夹心ELISA试验,探讨了多克隆抗体与单克隆抗体配对的若干事项.结果表明,利用单克隆抗体和酶标多克隆抗体配对,并用含动物血清的稀释液稀释酶标抗体,可实现对检测原的高特异性和高灵敏度检测.     

Rabbit immune repertoires as sources for therapeutic monoclonal antibodies: the impact of kappa allotype-correlated variation in cysteine content on antibody libraries selected by phage display

The rabbit immune repertoire has long been a rich source of diagnostic polyclonal antibodies. Now it also holds great promise as a source of therapeutic monoclonal antibodies. On the basis of phage display technology, we recently reported the first humanization of a rabbit monoclonal antibody. The allotypic diversity of rabbit immunoglobulins prompted us to compare different rabbit immune repertoires for the generation and humanization of monoclonal antibodies that bind with strong affinity to antigens involved in tumor angiogenesis. In particular, we evaluated the diversity of unselected and selected chimeric rabbit/human Fab libraries that were derived from different kappa light chain allotypes. Most rabbit light chains have an extra disulfide bridge that links the variable and constant domains in addition to the two intrachain disulfide bridges shared with mouse and human kappa light chains. Here we evaluate the impact of this increased disulfide bridge complexity on the generation and selection of chimeric rabbit/human Fab libraries. We demonstrate that rabbits with mutant bas and wild-type parental b9 allotypes are excellent sources for therapeutic monoclonal antibodies. Featured among the selected clones with b9 allotype is a rabbit/human Fab that binds with a dissociation constant of 1nM to both human and mouse Tie-2, which will facilitate its evaluation in mouse models of human cancer. Examination of 228 new rabbit antibody sequences allowed for a comprehensive comparison of the LCDR3 and HCDR3 length diversity in rabbits. This study revealed that rabbits exhibit an HCDR3 length distribution more closely related to human antibodies than mouse antibodies.     

Generation and application of chicken egg-yolk antibodies

Despite the fact that the use of chicken as immunization host brings many advantages to the production of polyclonal antibodies, the generation of egg yolk immunoglobulins (IgY) is rarely chosen. In this review, we report on the fast and efficient method for generation and affinity purification of IgY, in this case raised against the alpha-subunit of hypoxia-inducible factor-1 (HIF-1). The IgY antibody was successfully applied in a variety of methods and a number of different species for HIF-1alpha detection. In electrophoretic mobility shift assays, the IgY antibody recognized the native HIF-1 complex. The IgY antibody also detected HIF-1alpha protein on Western blots with extracts derived from human, monkey, pig, dog and mouse cell lines grown under hypoxic conditions. Immunofluorescence and immunoprecipitation experiments using the IgY antibody allowed detection and subcellular localization of HIF-1alpha in the nuclei of hypoxic cells. Chicken antibody production brings great benefit concerning the welfare of the immunized animals, due to non-invasive antibody harvesting with the added convenience of simple egg collection. An additional advantage is the fast and simple IgY isolation from egg yolk. IgY technology is a great improvement and should be considered as a good alternative to conventional polyclonal antibody production in mammals.     

Evaluation of new affinity chromatography resins for polyclonal,oligoclonal and monoclonal antibody pharmaceuticals

The performance of MabSelect SuRe and IgSelect affinity chromatography resins designed for process-scale purification of antibodies was investigated. Various antibodies (4 human monoclonal, 1 human polyclonal and 1 bovine polyclonal antibody and 1 Fc-fusion protein) were used to evaluate the elution pH and dynamic binding capacity of the resins. The elution pH for each human antibody was similar on MabSelect SuRe and IgSelect (pH 3.5–3.8). No significant differences in dynamic binding capacity were observed among human antibodies on MabSelect SuRe (∼20–40 mg/mL resin) and IgSelect (∼10–30 mg/mL resin). The binding capacity order for the human antibodies was the same on MabSelect SuRe and IgSelect. Using a linear pH gradient, both resins were able to partially separate monomeric and aggregated forms of the antibodies. The results indicate that these new affinity resins are powerful tools for the purification of human polyclonal antibodies from transgenic animals and oligoclonal antibodies from CHO cell cultures.     

Direct detection of herceptin/trastuzumab binding on breast tissue sections.

The protooncogene product HER-2/neu is the target of the humanized monoclonal antibody trastuzumab (Herceptin). Several tests are used clinically to identify patients with HER-2/neu overexpression based on evaluation by pathologists of gene amplification by fluorescence in situ hybridization or protein expression using immunohistochemistry (IHC). A simple technique has been developed for staining formalin-fixed, paraffin-embedded breast cancer tissue using unmodified Herceptin/trastuzumab as the primary antibody. Results were compared with staining with the commercial kit, HercepTest, as well as with polyclonal anti-HER-2/neu antibodies and with biotinylated trastuzumab. These procedures were tested using four breast cancer microarrays. There were 854 cores that were stained with all four antibodies, representing 325 cases. A standard 4-point scoring system (0-3) was used. A total of 156 cases (48%) were scored as 0 by all the methods used and 31 (9.5%) were positive (3+) by all methods. Of interest, three cases scored negative using polyclonal anti-HER-2/neu antibodies but were positive using unmodified trastuzumab. To clarify this discrepancy, whole sections of tumors were examined with both antibodies using double labeling. There were some tumors that demonstrated a mosaic pattern of staining with neighboring cells or groups of cells stained exclusively with one antibody or the other. These results demonstrate that unmodified humanized or human therapeutic antibodies could be used for preclinical testing or in a clinical laboratory setting for IHC-based selection of patients for treatment, and results of such selection could be different from those obtained using polyclonal antibody-based IHC procedure.     

The differential inhibition of hemopoietic growth factor activity by cytotoxins and interferon-gamma

The effects of recombinant human tumor necrosis factor (TNF), lymphotoxin (LT), and interferon-gamma (IFN-gamma) on the growth of human hemopoietic progenitor cells in clonal culture have been examined. Colony growth was induced by using granulocyte colony-stimulating factor (G-CSF), or granulocyte-macrophage colony-stimulating factor (GM-CSF). A suppressive effect of TNF, LT, and IFN-gamma on the development of granulocyte, macrophage, and mixed granulocyte/macrophage colonies was shown. Suppression of colonies formed after stimulation with G-CSF was greater than that observed after stimulation with GM-CSF. In the presence of a monoclonal antibody to TNF, or polyclonal antibodies to either LT or IFN-gamma, the inhibitory effect of the molecule to which the antibody was directed was abrogated. These findings suggest that progenitor cells responsive to G-CSF or GM-CSF have different sensitivities to the effects of TNF, LT, and IFN-gamma. Defining the interactions of growth factors and inhibitors should increase understanding of mechanisms underlying diseases associated with suppression of normal hemopoiesis, and in predicting the effects in vivo of these bioregulatory molecules in clinical medicine.     

Neutralization of vascular endothelial growth factor prevents collagen-induced arthritis and ameliorates established disease in mice

Vascular endothelial growth factor (VEGF) is implicated in the pathogenesis of inflammatory joint diseases, including rheumatoid arthritis (RA). To determine the importance of this cytokine in vivo, the effect of administering VEGF-neutralizing antibodies to mice with collagen-induced arthritis (CIA), which has many immunological and pathological similarities to human RA, has been investigated. Either saline, normal rabbit immunoglobulin or anti-human VEGF121 rabbit polyclonal antibody was administered to mice subcutaneously either before the onset of arthritis or after the establishment of clinical disease. Anti-VEGF antibody administered prior to disease onset significantly delayed the development of arthritis and decreased clinical score and paw thickness as well as histological severity. On the other hand, the frequency of occurrence of disease compared to either the control group administered saline or normal rabbit immunoglobulin was not altered. Anti-VEGF antibody also significantly ameliorated clinical and histological parameters even when administered after disease onset. These results indicate a possible therapeutical potential for anti-VEGF treatment in human arthritis.     

Targeted localized use of therapeutic antibodies: a review of non-systemic,topical and oral applications

Therapeutic antibodies provide important tools in the “medicine chest” of today’s clinician for the treatment of a range of disorders. Typically monoclonal or polyclonal antibodies are administered in large doses, either directly or indirectly into the circulation, via a systemic route which is well suited for disseminated ailments. Diseases confined within a specific localized tissue, however, may be treated more effectively and at reduced cost by a delivery system which targets directly the affected area. To explore the advantages of the local administration of antibodies, we reviewed current alternative, non-systemic delivery approaches which are in clinical use, being trialed or developed. These less conventional approaches comprise: (a) local injections, (b) topical and (c) peroral administration routes. Local delivery includes intra-ocular injections into the vitreal humor (i.e. Ranibizumab for age-related macular degeneration), subconjunctival injections (e.g. Bevacizumab for corneal neovascularization), intra-articular joint injections (i.e. anti-TNF alpha antibody for persistent inflammatory monoarthritis) and intratumoral or peritumoral injections (e.g. Ipilimumab for cancer). A range of other strategies, such as the local use of antibacterial antibodies, are also presented. Local injections of antibodies utilize doses which range from 1/10th to 1/100th of the required systemic dose therefore reducing both side-effects and treatment costs. In addition, any therapeutic antibody escaping from the local site of disease into the systemic circulation is immediately diluted within the large blood volume, further lowering the potential for unwanted effects. Needle-free topical application routes become an option when the condition is restricted locally to an external surface. The topical route may potentially be utilized in the form of eye drops for infections or corneal neovascularization or be applied to diseased skin for psoriasis, dermatitis, pyoderma gangrenosum, antibiotic resistant bacterial infections or ulcerated wounds. Diseases confined to the gastrointestinal tract can be targeted directly by applying antibody via the injection-free peroral route. The gastrointestinal tract is unusual in that its natural immuno-tolerant nature ensures the long-term safety of repeatedly ingesting heterologous antiserum or antibody materials. Without the stringent regulatory, purity and clean room requirements of manufacturing parenteral (injectable) antibodies, production costs are minimal, with the potential for more direct low-cost targeting of gastrointestinal diseases, especially with those caused by problematic antibiotic resistant or toxigenic bacteria (e.g. Clostridium difficile, Helicobacter pylori), viruses (e.g. rotavirus, norovirus) or inflammatory bowel disease (e.g. ulcerative colitis, Crohn’s disease). Use of the oral route has previously been hindered by excessive antibody digestion within the gastrointestinal tract; however, this limitation may be overcome by intelligently applying one or more strategies (i.e. decoy proteins, masking therapeutic antibody cleavage sites, pH modulation, enzyme inhibition or encapsulation). These aspects are additionally discussed in this review and novel insights also provided. With the development of new applications via local injections, topical and peroral routes, it is envisaged that an extended range of ailments will increasingly fall within the clinical scope of therapeutic antibodies further expanding this market.     

靶向人白介素-6蛋白的治疗性单克隆抗体研究进展*

白介素-6(interleukin-6,IL-6)作为一种多效的细胞因子,参与机体内众多生理与病理过程。研究表明,IL-6首先与自身受体(IL-6R、gp130)形成异源六聚体复合物,进而激活下游信号转导通路,最终发挥生物学功能。 IL-6信号通路异常活化及功能失调与多种疾病密切相关,如自身免疫疾病、慢性炎症、恶性肿瘤等。另外IL-6的异常表达在新型冠状病毒肺炎(COVID-19)细胞因子风暴综合征(CSS)中也扮演重要角色。一般而言,阻断IL-6信号通路上的各关键节点,均可用于IL-6相关疾病的治疗。有别于阻断IL-6R或gp130等公共受体分子,阻断IL-6蛋白的治疗性单克隆抗体特异性更高,在临床研究中,部分品种显示出其独有的治疗特点及有益的疗效。现阶段只有1个靶向IL-6蛋白的单克隆抗体药物获美国FDA批准上市,以及超过8个治疗性单克隆抗体在临床研究阶段。重点对国内外靶向人IL-6蛋白的治疗性单克隆抗体及其临床应用进行综述。     

Production of target-specific recombinant human polyclonal antibodies in mammalian cells

We describe the expression and consistent production of a first target-specific recombinant human polyclonal antibody. An anti-Rhesus D recombinant polyclonal antibody, Sym001, comprised of 25 unique human IgG1 antibodies, was produced by the novel Sympress expression technology. This strategy is based on site-specific integration of antibody genes in CHO cells, using the FRT/Flp-In recombinase system. This allows integration of the expression construct at the same genomic site in the host cells, thereby reducing genomic position effects. Different bioreactor batches of Sym001 displayed highly consistent manufacturing yield, antibody composition, binding potency, and functional activity. The results demonstrate that diverse recombinant human polyclonal antibody compositions can be reproducibly generated under conditions directly applicable to industrial manufacturing settings and present a first recombinant polyclonal antibody which could be used for treatment of hemolytic disease of the newborn and/or idiopathic thrombocytopenic purpura.