Capturing the natural diversity of the human antibody response against vaccinia virus.

The eradication of smallpox (variola) and the subsequent cessation of routine vaccination have left modern society vulnerable to bioterrorism employing this devastating contagious disease. The existing, licensed vaccines based on live vaccinia virus (VACV) are contraindicated for a substantial number of people, and prophylactic vaccination of large populations is not reasonable when there is little risk of exposure. Consequently, there is an emerging need to develop efficient and safe therapeutics to be used shortly before or after exposure, either alone or in combination with vaccination. We have characterized the human antibody response to smallpox vaccine (VACV Lister) in immunized volunteers and isolated a large number of VACV-specific antibodies that recognize a variety of different VACV antigens. Using this broad antibody panel, we have generated a fully human, recombinant analogue to plasma-derived vaccinia immunoglobulin (VIG), which mirrors the diversity and specificity of the human antibody immune response and offers the advantage of unlimited supply and reproducible specificity and activity. The recombinant VIG was found to display a high specific binding activity toward VACV antigens, potent in vitro VACV neutralizing activity, and a highly protective efficacy against VACV challenge in the mouse tail lesion model when given either prophylactically or therapeutically. Altogether, the results suggest that this compound has the potential to be used as an effective postexposure prophylaxis or treatment of disease caused by orthopoxviruses.

Extensive restrictions in the VH sequence usage of the human antibody response against the Rhesus D antigen.

Anti-Rhesus D immunoglobulin purified from human sera is used as a prophylactic reagent in Rhesus D negative women at risk of alloimmunization during pregnancy. We are currently developing a Rhesus D antigen-specific recombinant polyclonal antibody drug lead for replacing the existing blood derived-products. By analyzing the RhD-specific antibody VH repertoires from eight alloimmunized women we found, in agreement with previous studies, a strong preference for the VH 3-33 "superspecies" gene segments which encompasses the IGHV3-30-3*01, IGHV3-30*18, and IGHV3-33*01 VH alleles. Even more extensive genetic restriction was observed among five donors, which produced antibodies of identical V-D-J usage and CDR3 loop length and joining regions of similar amino acid composition. In addition, we find a high degree of sequence relatedness to previously isolated anti-Rhesus D antibodies. Such close homology in VH domains indicates that significant structural restrictions are operating in the selection of antibodies recognizing RhD as seen for T cell receptors. Moreover, some VH domains were isolated in their germline configuration indicating that anti-RhD antibodies of relatively high affinity are present in the naïve antibody repertoire of Rhesus negative individuals which offers an explanation for the strong and clinically significant immunogenicity of the Rhesus D.

Reduced susceptibility of recombinant polyclonal antibodies to inhibitory anti-variable domain antibody responses.

The immunogenicity of therapeutic Abs is a concern as anti-drug Abs may impact negatively on the pharmacodynamics and safety profile of Ab drugs. The factors governing induction of anti-drug Abs are not fully understood. In this study, we describe a model based on mouse-human chimeric Abs for the study of Ab immunogenicity in vivo. Six chimeric Abs containing human V regions and mouse C regions were generated from six human anti-Rhesus D Abs and the Ag-binding characteristics of the parental human Abs were retained. Analysis of the immune response toward the individual chimeric Abs revealed the induction of anti-variable domain Abs including anti-idiotypic Abs against some of these, thereby demonstrating the applicability of the model for studying anti-drug Ab responses in vivo. Immunization of BALB/c, C57, and outbred NMRI mice with a polyclonal composition consisting of all six chimeric Abs demonstrated that the immunogenicity of the individual Abs was haplotype dependent. Chimeric Abs, which were nonimmunogenic when administered individually, did not become immunogenic as part of the polyclonal composition, implying the absence of epitope spreading. Ex vivo Ab-binding studies established a clear correlation between the level of immunogenicity of the Abs comprised in the composition and the impact on the pharmacology of the Abs. These analyses demonstrate that under these conditions this polyclonal Ab composition was generally less susceptible to blocking Abs than the respective mAbs.

Development of recombinant human polyclonal antibodies for the treatment of complex human diseases.

Antibodies are a central factor in the immunity against invading pathogens, such as bacteria and viruses, as well as against malignantly transformed cells. Natural antibody responses are polyclonal, comprising antibodies against several epitopes, thus increasing the probability of eliminating the invading pathogen or malignant cell. The pharmacological advantage of polyclonality is exploited in the plasma-derived immunoglobulin products used at present to treat a number of infectious diseases. However, the use of plasma-derived products is limited by their cost, inconvenience of use and potential for transferring diseases from the donor to the patient. Symphogen has developed technologies to capture the advantages of antibody polyclonality while eliminating the potential safety risk associated with the sourcing of human material. Hence, the Symplex technology has been developed to identify diverse repertoires of target-specific, fully human antibodies. For the controlled manufacture of recombinant polyclonal antibody drugs, Symphogen has developed the Sympress technology. Combined, these two technologies allow the identification and industrial manufacturing of recombinant human polyclonal antibodies for medical use in humans. The authors believe that this new class of therapeutic antibodies will be advantageous in the treatment of complex human diseases, such as cancer and infection, as it allows the combination of several treatment modalities in one drug.

Recombinant human polyclonal antibodies: A new class of therapeutic antibodies against viral infections.

The mammalian immune system eliminates pathogens by generating a specific antibody response. Polyclonality is a key feature of this immune response: the immune system produces antibodies which bind to different structures on a given pathogen thereby increasing the likelihood of its elimination. The vast majority of current recombinant antibody drugs rely on monospecific monoclonal antibodies. Inherently, such antibodies do not represent the benefits of polyclonality utilized by a natural immune system and this has impeded the identification of efficacious antibody drugs against infectious agents, including viruses. The development of novel technologies has allowed the identification and manufacturing of antigen-specific recombinant polyclonal human antibodies, so-called symphobodies. This review describes the rationale for designing drugs based on symphobodies against pathogenic viruses, including HIV, vaccinia and smallpox virus, and respiratory syncytial virus.

Allergen-specific polyclonal antibodies reduce allergic disease in a mouse model of allergic asthma.

BACKGROUND: Recombinant allergen-specific immunoglobulin G (IgG) antibody therapy can reduce allergic asthma symptoms by inhibiting the immunoglobulin E (IgE)-mediated allergic response. This study investigated the effect of intranasally administered allergen-specific monoclonal (mAb) and polyclonal (pAb) antibody on airway inflammation and hyperresponsiveness (AHR) in a mouse model of human asthma. METHODS: Ovalbumin (OVA)-specific IgG2b antibodies were generated by phage display using spleens from OVA-immunized mice, and screening against OVA and finally expressed in CHO cells. Sensitized mice were treated intranasally with either a recombinant anti-OVA mAb (gc32) or a polyclonal preparation comprising seven selected antibodies (including gc32). Control mice received diluent only, OVA only, a control polymeric IgG or dexamethasone. Following challenge with nebulized OVA, investigators assessed airway inflammation by histology and cellular composition of the bronchoalveolar fluid, and methacholine-induced airway hyperresponsiveness (AHR). Serum levels of total and OVA-specific IgE were measured by ELISA. RESULTS: Sensitized mice developed airway inflammation and AHR in response to OVA challenge. Intranasally administered OVA-specific murine polyclonal or monoclonal IgG2b antibodies both reduced OVA-induced lung inflammation. Polyclonal, but not anti-OVA mAb, also reduced AHR and eosinophil influx into the airway lumen. Both anti-OVA antibody preparations reduced levels of specific IgE with no effect on total IgE levels. CONCLUSIONS: Intranasal treatment with allergen-specific pAb reduces pulmonary inflammation and AHR in a mouse model of allergic asthma, but allergen-specific mAb reduces inflammation only. Allergen-specific recombinant pAb offers a potentially valuable therapeutic approach to the management of allergic asthma.

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.

Isolation of human antibody repertoires with preservation of the natural heavy and light chain pairing.

The humoral immune system in higher vertebrates is unique in its ability to generate highly diverse antibody responses against most pathogens as well as against certain malignancies. Several technologies have been developed to exploit this vast source of potentially therapeutic antibodies, including hybridoma technology, phage display and yeast display. Here, we present a novel, high-throughput technology (the Symplex Technology) for rapid direct cloning and identification of human antigen-specific high-affinity antibodies from single antibody-producing cells of immune individuals. The utility of the technology was demonstrated by isolation of diverse sets of unique high-affinity antibodies against tetanus toxoid and influenza virus from immunized volunteers. Hence, the Symplex Technology is a new method for the rapid isolation of high-affinity antibodies directly from humans.

Invariant V alpha 14+ NKT cells participate in the early response to enteric Listeria monocytogenes infection.

Invariant Valpha14(+) NKT cells are a specialized CD1-reactive T cell subset implicated in innate and adaptive immunity. We assessed whether Valpha14(+) NKT cells participated in the immune response against enteric Listeria monocytogenes infection in vivo. Using CD1d tetramers loaded with the synthetic lipid alpha-galactosylceramide (CD1d/alphaGC), we found that splenic and hepatic Valpha14(+) NKT cells in C57BL/6 mice were early producers of IFN-gamma (but not IL-4) after L. monocytogenes infection. Adoptive transfer of Valpha14(+) NKT cells derived from TCRalpha degrees Valpha14-Jalpha18 transgenic (TCRalpha degrees Valpha14Tg) mice into alymphoid Rag(null) gamma(c)(null) mice demonstrated that Valpha14(+) NKT cells were capable of providing early protection against enteric L. monocytogenes infection with systemic production of IFN-gamma and reduction of the bacterial burden in the liver and spleen. Rechallenge experiments demonstrated that previously immunized wild-type and Jalpha18null mice, but not TCRalpha(null) or TCRalpha(null) Valpha14Tg mice, were able to mount adaptive responses to L. monocytogenes. These data demonstrate that Valpha14(+) NKT cells are able to participate in the early response against enteric L. monocytogenes through amplification of IFN-gamma production, but are not essential for, nor capable of, mediating memory responses required to sterilize the host.

Recombinant polyclonal antibodies: therapeutic antibody technologies come full circle.

Current antibody therapeutics can be grouped into two generations, each distinguished by a unique feature of the immune system: diversity and specificity. Antibodies from human blood (immunoglobulin) represent the first generation, and are characterized by the natural diversity of human antibody responses. The second generation consists of recombinant monoclonal antibodies (mAbs), which are characterized by high specificity toward a single, often well-described antigen. The natural immune response comprises a plurality of specificities, many of which do not compete for binding, whereas molecules in a mAb all compete for binding to the same epitope. Thus, the epitope is more likely to become a limiting factor for mAb binding to complex targets compared with a polyclonal antibody. Also, epitope-escape by mutation or natural variation is less likely to be a problem for polyclonal antibodies. Technologies attempting to develop truly human recombinant antigen-specific polyclonal antibodies, such as the Sympress technology, are closing a natural circle between the first generations of antibody technologies.

The long-acting glucagon-like peptide-1 analogue, liraglutide, inhibits beta-cell apoptosis in vitro.

We here show that GLP-1 and the long-acting GLP-1 analogue, liraglutide, interfere with diabetes-associated apoptotic processes in the beta-cell. Studies using primary neonatal rat islets showed that native GLP-1 and liraglutide inhibited both cytokine- and free fatty acid-induced apoptosis in a dose-dependent manner. The anti-apoptotic effect of liraglutide was mediated by the GLP-1 receptor as the specific GLP-1 receptor antagonist, exendin(9-39), blocked the effects. The adenylate cyclase activator, forskolin, had an anti-apoptotic effect similar to those of GLP-1 and liraglutide indicating that the effect was cAMP-mediated. Blocking the PI3 kinase pathway using wortmannin but not the MAP kinase pathways by PD98059 inhibited the effects of liraglutide. In conclusion, GLP-1 receptor activation has anti-apoptotic effect on both cytokine, and free fatty acid-induced apoptosis in primary islet-cells, thus suggesting that the long-acting GLP-1 analogue, liraglutide, may be useful for retaining beta-cell mass in both type 1 and type 2 diabetic patients.

Pathogen-specific recombinant human polyclonal antibodies: biodefence applications.

The potential use of biological agents such as viruses, bacteria or bacterial toxins as weapons of mass destruction has fuelled significant national and international research and development in novel prophylactic or therapeutic countermeasures. Such measures need to be fast-acting and broadly specific, a hallmark of target-specific polyclonal antibodies (pAbs). As reviewed here, pathogen-specific antibodies in the form of human or animal serum have long been recognised as effective therapies in a number of infectious diseases. This review focuses in particular on the potential biowarfare agents prioritised by the National Institute of Allergy and Infectious Diseases and the Centers for Disease Control and Prevention (CDC), referred to as the category A organisms. Furthermore, it is propose that the last decade of development in recombinant antibody technologies offers the possibility for developing highly specific human monoclonal or polyclonal pathogen-specific antibodies. In particular, pathogen-specific polyclonal human antibodies offer certain advantages over existing hyperimmune serum products, monoclonal antibodies, small molecule drugs and vaccines. Here, the rationale for designing pAb-based therapeutics against the CDC category A microbial agents causing anthrax, botulism, plague, smallpox, tularaemia and viral haemorrhagic fevers, as well as the overall design of such therapeutics, are discussed.

Genetic fusion of human insulin B-chain to the B-subunit of cholera toxin enhances in vitro antigen presentation and induction of bystander suppression in vivo.

The pentameric B-subunit of cholera toxin (CTB) can be used as an efficient mucosal carrier of either immunogenic or tolerogenic T-cell epitopes. In this study a series of fusions was constructed between the genes encoding CTB and the B-chain of human insulin (InsB). The resulting fusion proteins were expressed in Escherichia coli and isolated as cytoplasmic inclusion bodies that were then dissolved and assembled in vitro. GM1 enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses showed that the protein construct in which InsB was fused to the C-terminus of a CTB monomer (CI) assembled into structures that both bound to the receptor GM1 ganglioside and reacted with monoclonal antibodies to CTB and insulin. Fusion of InsB to the N-terminus of CTB resulted in protein that could not assemble into pentameric CTB. In vitro assays showed that the CI fusion protein was 300-fold more potent than native insulin at inducing interleukin-2 (IL-2) production by an insulin-specific T-cell hybridoma. When administered orally, the CI fusion protein induced efficient immunological suppression of ovalbumin-specific T-cell responses in mice co-immunized parenterally with insulin and ovalbumin. These results demonstrate the stability, GM1 receptor-binding activity and antigenic authenticity of the CI fusion protein as well as its ability to elicit insulin-specific T-cell responses in vitro. In addition, we demonstrate that the CI fusion protein induces efficient immunosuppression after oral administration, raising the possibility of using such constructs in the treatment of type-1 diabetes.

A new model for analyzing immunomodulation of T cell responses induced by oral administration of islet cell antigens.

Based on the coimmunization of BALB/c mice with insulin and ovalbumin, we here describe a feasible efficacy model for analyzing the tolerogenic effect of the oral administration of sequestered antigens-for example, islet-specific proteins such as insulin and GAD65.