Camel single-domain antibodies as modular building units in bispecific and bivalent antibody constructs

Single-domain antibodies against various antigens are isolated from the unique heavy-chain antibodies of immunized camels and llamas. These minimal sized binders are very robust and bind the antigen with high affinity in a monomeric state. We evaluated the feasibility to produce soluble, functional bispecific and bivalent antibodies in Escherichia coli with camel single-domain antibody fragments as building blocks. Two single-domain antibody fragments were tethered by the structural upper hinge of a natural antibody to generate bispecific molecules. This linker was chosen for its protease resistance in serum and its natural flexibility to reorient the upstream and downstream located domains. The expression levels, ease of purification, and the solubility of the recombinant proteins were comparable with those of the constituent monomers. The individual moieties fully retain the binding capacity and the binding characteristics within the recombinant bispecific constructs. The easy generation steps and the biophysical properties of these bispecific and bivalent constructs based on camel single-domain antibody fragments makes them particularly attractive for use in therapeutic or diagnostic programs.     

Actinoplanes teichomyceticus ATCC 31121 as a cell factory for producing teicoplanin

Background

Single-domain antibody fragments possess structural features, such as a small dimension, an elevated stability, and the singularity of recognizing epitopes non-accessible for conventional antibodies that make them interesting for several research and biotechnological applications.

Results

The discovery of the single-domain antibody's potentials has stimulated their use in an increasing variety of fields. The rapid accumulation of articles describing new applications and further developments of established approaches has made it, therefore, necessary to update the previous reviews with a new and more complete summary of the topic.

Conclusions

Beside the necessary task of updating, this work analyses in detail some applicative aspects of the single-domain antibodies that have been overseen in the past, such as their efficacy in affinity chromatography, as co-crystallization chaperones, protein aggregation controllers, enzyme activity tuners, and the specificities of the unconventional single-domain fragments.     

Indication of antigens and antibodies using the reaction of compound immune adsorption and reactivation of the phage.

A method of determining antibodies by their adsorption on large-pore or surface immunosorbents with subsequent treatment of the carrier with anti-immunoglobulin serum and antiphage serum isologous to the antibodies and then with the bacteriophage has been presented. The adsorbed virions are split off by means of papain-induced hydrolysis of the antibody complex. The antigens are determined by the reaction of phage fixing inhibition. The method permits to determine small amounts of antibodies to proteins, haptenes and cells with objective calculation of results.     

Determination of antibody affinity by using antigenic and antibody immunosorbents

To determine the affinity of the active centers of antibodies, cellulose immunosorbents for antibodies and antigens have been used. The fixation of serum proteins on the sorbent, the interaction of fixed antibodies with a monovalent antigen and the graphic analysis of the results thus obtained allows one to assess not only the concentration of the effective active centers on the sorbent, but also all known characteristics of antibody affinity: the average association constant K0, the common association constant Kt, the geometric association constant Kg, the average association constants which determine the affinity of different antibody groups. The use of antigenic immunosorbent permits one to determine the value of the average internal association constant K0. The determination of antibody affinity in hyperimmune antiplague sera by means of immunosorbents and red blood cells coated with capsular antigen has resulted in obtaining similar values of affinity indices.     

Plasma and bile antibodies of the teleost Trematomus bernacchii specific for the nematode Pseudoterranova decipiens

We investigated the occurrence of antibodies against protein antigens of the nematode parasite Pseudoterranova decipiens in the plasma and bile of the Antarctic teleost Trematomus bernacchii. Three different P. decipiens protein solutions were prepared: excreted/secreted proteins from live larvae (ESP); surface-associated proteins obtained by mild extraction of larval bodies (SAP); and cuticular soluble proteins recovered by extraction in strong reducing conditions (CSP). Using different immunoassays, these 3 preparations were tested for their ability to bind fish antibody. As determined by ELISA, the specific antibody binding activity was higher in SAP than in CSP. As determined by dot-blot immunoassay, the specific antigen binding activity versus SAP was higher in bile than in plasma antibodies. A different number of antigenic components of SAP and ESP were identified by immunoblotting performed with plasma or bile antibodies. These results led to the conclusion that T. bernacchii parasitism by nematodes involves plasma and bile anti-parasite antibodies. Furthermore bile antibodies were found to be more reactive and more heterogeneous than plasma.     

Wild - type Pseudomonas aeruginosa phage AP 34 transducing gentamicin-tobramycin resistance and autoplaque formation.

A method of determining antibodies by their adsorption on large-pore or surface immunosorbents with subsequent treatment of the carrier with anti-immunoglobulin serum and antiphage serum isologous to the antibodies and then with the bacteriophage, has been presented. The adsorbed virions are split off by means of papain-induced hydrolysis of the antibody complex. The antigens are determined by the reaction of phage fixing inhibition. The method permits to determine small amounts of antibodies to proteins, haptenes and cells with objective calculation of results.     

Engineered antibody fragments and the rise of single domains

With 18 monoclonal antibody (mAb) products currently on the market and more than 100 in clinical trials, it is clear that engineered antibodies have come of age as biopharmaceuticals. In fact, by 2008, engineered antibodies are predicted to account for >30% of all revenues in the biotechnology market. Smaller recombinant antibody fragments (for example, classic monovalent antibody fragments (Fab, scFv)) and engineered variants (diabodies, triabodies, minibodies and single-domain antibodies) are now emerging as credible alternatives. These fragments retain the targeting specificity of whole mAbs but can be produced more economically and possess other unique and superior properties for a range of diagnostic and therapeutic applications. Antibody fragments have been forged into multivalent and multi-specific reagents, linked to therapeutic payloads (such as radionuclides, toxins, enzymes, liposomes and viruses) and engineered for enhanced therapeutic efficacy. Recently, single antibody domains have been engineered and selected as targeting reagents against hitherto immunosilent cavities in enzymes, receptors and infectious agents. Single-domain antibodies are anticipated to significantly expand the repertoire of antibody-based reagents against the vast range of novel biomarkers being discovered through proteomics. As this review aims to show, there is tremendous potential for all antibody fragments either as robust diagnostic reagents (for example in biosensors), or as nonimmunogenic in vivo biopharmaceuticals with superior biodistribution and blood clearance properties.     

Preparation of Solid-Phase Immunosorbents by Coupling Human Serum Proteins to Cyanogen Bromide-Activated Agarose

The method of preparing solid-phase immunosorbents by covalently attaching proteins from whole human serum to cyanogen bromide-activated agarose has been investigated to determine optimum concentrations of cyanogen bromide and protein, and the optimum pH for the maximum attachment of proteins from serum. Two systems in which the above immunosorbents have proved useful are described: the removal of antibodies to normal serum proteins from anti-hepatitis B serum and the removal of light chain antibodies from anti-human immunoglobulin M serum.     

Single-Domain Antibodies Used to Pretreat the Human Urinary Proteome in Cancer Biomarker Testing

Molecular Biology - A number of single-domain antibodies (nanobodies) obtained previously to major marker blood proteins were tested as tools to preprocess urine samples from patients with bladder...     

Comparative profiling of serum glycoproteome by sequential purification of glycoproteins and 2-nitrobenzensulfenyl (NBS) stable isotope labeling: a new approach for the novel biomarker discovery for cancer

The recent progress in various proteomic technologies allows us to screen serum biomarker including carbohydrate antigens. However, only a limited number of proteins could be detected by current conventional methods such as shotgun proteomics, primarily because of the enormous concentration distribution of serum proteins and peptides. To circumvent this difficulty and isolate potential cancer-specific biomarkers for diagnosis and treatment, we established a new screening system consisting of the sequential steps of (1) immunodepletion of 6 high-abundance proteins, (2) targeted enrichment of glycoproteins by lectin column chromatography, and (3) the quantitative proteome analysis using 12C6- or 13C6-NBS (2-nitrobenzenesulfenyl) stable isotope labeling followed by MALDI-QIT-TOF mass spectrometric analysis. Through this systematic analysis for five serum samples derived from patients with lung adenocarcinoma, we identified as candidate biomarkers 34 serum glycoproteins that revealed significant difference in alpha1,6-fucosylation level between lung cancer and healthy control, clearly demonstrating that the carbohydrate-focused proteomics could allow for the detection of serum components with cancer-specific features. In addition, we developed a more simplified and practical technique, mass spectrometry-based glycan structure analysis and lectin blotting, in order to validate glycan structure of candidate biomarkers that could be applicable in clinical use. Our new glycoproteomic strategy will provide highly sensitive and quantitative profiling of specific glycan structures on multiple proteins, which should be useful for serum biomarker discovery.     

Evaluation of three principally different intact protein prefractionation methods for plasma biomarker discovery

The aim of this study was to evaluate three principally different top-down protein prefractionation methods for plasma: high-abundance protein depletion, size fractionation and peptide ligand affinity beads, focusing in particular on compatibility with downstream analysis, reproducibility and analytical depth. Our data clearly demonstrates the benefit of high-abundance protein depletion. However, MS/MS analysis of the proteins eluted from the high-abundance protein depletion column show that more proteins than aimed for are removed and, in addition, that the depletion efficacy varies between the different high-abundance proteins. Although a smaller number of proteins were identified per fraction using the peptide ligand affinity beads, this technique showed to be both robust and versatile. Size fractionation, as performed in this study, focusing on the low molecular weight proteome using a combination of gel filtration chromatography and molecular weight cutoff filters, showed limitations in the molecular weight cutoff precision leading detection of high molecular weight proteins and, in the case of the cutoff filters, high variability. GeLC-MS/MS analysis of the fractionation methods in combination with pathway analysis demonstrates that increased fractionation primarily leads to high proteome coverage of pathways related to biological functions of plasma, such as acute phase reaction, complement cascade and coagulation. Further, the prefractionation methods in this study induces limited effect on the proportion of tissue proteins detected, thereby highlighting the importance of extensive or targeted downstream fractionation.     

基于细胞的抗单增李斯特菌单域重链抗体的筛选及鉴定

【目的】获得针对单增李斯特菌的特异性单域重链抗体,并对筛选过程中特异性克隆的富集规律进行分析,为筛选具有种属特异性的噬菌体展示抗体提供参考。【方法】采用固相筛选技术,以热灭活的单增李斯特菌菌体为抗原,通过四轮常规筛选和一轮消减筛选,从驼源天然噬菌体展示文库中筛选针对单增李斯特菌的单域重链抗体。采用Phage-ELISA法,对后四轮筛选洗脱物中随机挑选的噬菌体进行鉴定,阳性克隆进行基因测序及结合特异性分析。通过多序列比对分析将获得的基因序列进行分组和统计。【结果】成功筛选到2株单增李斯特菌特异性的单域重链抗体。【结论】在优化的筛选条件下,基于全细胞的筛选方法能够获得特异性识别单增李斯特菌的单域重链抗体,消减筛选对于去除非特异性克隆是有效的和必要的。     

An alternative strategy for high throughput generation and characterization of monoclonal antibodies against human plasma proteins using fractionated native proteins as immunogens

Construction of a monoclonal antibody (mAb) bank containing a vast variety of antibodies against human tissue proteins is important for proteomic research. A novel strategy of subtractive immunization using fractionated native proteins was developed for high throughput generation of mAb against human plasma proteins. By this novel approach, the bottleneck of antigen preparation can be overcome by combining repeated immunization of animals with subtracted fractions of plasma or tissue proteins and identification of target antigen by immunoprecipitation/mass spectrum strategies. Plasma freshly collected from healthy adults was pooled and three fractions were prepared by size exclusion chromatography. Mice were immunized with the fractionated plasma proteins, and 205 strains of hybridomas secreting mAb were obtained after two-round subtractive immunizations and cell fusions. In the first round, 110 strains of hybridomas were established, in which 77 strains secreting mAb were identified against 10 human plasma high-abundant proteins. In the second round, plasma fraction I was absorbed with mAb against IgM, IgG, ceruloplasmin and haptoglobin. The absorbed fraction I was used as immunogen for the second round immunization and cell fusion. Ninety-five strains of hybridomas secreting mAb were obtained. Although the target antigens of mAb from 82 strains of hybridomas were identified as IgM, IgA, alpha2-macroglobulin and fibrinogen, about 85% antibodies obtained from this round were identified as new antibodies when compared with mAb obtained in the first round immunization with plasma fraction I. The results suggest that subtractive immunization with fractionated plasma proteins followed by identification of antigens with immunoprecipitation/mass spectrum may be an effective approach for rapid preparation of mAb against high-and medium-abundant plasma or tissue proteins.     

Nature of the inhibitory serum factor in mice injected with isologous antibodies conjugated with cellulose

The formation of antigen-specific serum inhibitory factor was induced by injection of covalently bound to cellulose syngeneic antibodies to sheep red blood cells into (CBA X C57BL/6)F1 mice. This factor was absorbed by cellulose immunosorbents immobilized with antibodies against sheep red blood cells and with rabbit antibodies against mouse gamma-globulin and was not absorbed by immunosorbents immobilized with immunoglobulins of intact mice or immunoglobulin containing antibodies against rat red blood cells. These data, and evidence obtained by the authors previously, indicate that inhibitory factor of the serum is likely to be due to idiotypic antibodies.     

A synthetic library for rapid isolation of humanized single-domain antibodies

Relative to conventional full-length immunoglobulin G (IgG) antibodies and antibody fragments, single-domain antibodies, derived from the antigen-binding domain of the immunoglobulin of camelid species or cartilaginous fish, hold great potential for many biotechnological applications due to their small size and excellent physicochemical properties. To bypass animal immunization and facilitate the isolation of antigen-specific single-domain antibodies with ease, we have constructed a synthetic single-domain antibody library comprising three diversified synthetic complementarity determining regions (CDRs) grafted into a humanized camelid heavy- chain antibody VH (VHH) framework. Using three types of model antigens, interleukin-1β (IL-1β), amyloid-β, and vascular endothelial growth factor, the constructed single-domain antibody library, which has a vast diversity of approximately 1.8 × 10¹⁰, was evaluated, and single-domain antibody sequences against them were identified.     

Hexapeptide combinatorial ligand libraries: the march for the detection of the low-abundance proteome continues

After 10 years of extensive proteomic research, it has become increasingly apparent that new technologies are sorely needed for detecting the low-abundance proteome-those proteins (up to 50% in any proteome) whose concentration in tissues or cells falls below the detection limits of currently available methodologies. Here we survey one such method: a combinatorial ligand library (called ProteoMiner), comprising dozens of millions of hexapeptides capable of interacting with most, if not all, proteins in any given proteome. They act by drastically reducing the signal of high-abundance species while increasing the level of the low-abundance components to bring their signal within the detection limit of present-day tools. Such a library has been tested against a number of human biological fluids, such as sera, urine, cerebrospinal fluid as well as against cell lysates (e.g., platelets, red blood cells) with interesting results.     

Depletion of the high-abundance plasma proteins

Summary. Body fluids, like plasma and urine, are comparatively easy to obtain and are useful for the detection of novel diagnostic markers by applying new technologies, like proteomics. However, in plasma, several high-abundance proteins are dominant and repress the signals of the lower-abundance proteins, which then become undetectable either by two-dimensional gels or chromatography. Therefore, depletion of the abundant proteins is a prerequisite for the detection of the low-abundance components. We applied affinity chromatography on blue matrix and Protein G and removed the most abundant human plasma proteins, albumin and the immunoglobulin chains. The plasma proteins, prior to albumin and immunoglobulin depletion, as well the eluates from the two chromatography steps were analyzed by two-dimensional electrophoresis and the proteins were identified by MALDI-TOF-MS. The analysis resulted in the identification of 83 different gene products in the untreated plasma. Removal of the high-abundance proteins resulted in the visualization of new protein signals. In the eluate of the two affinity steps, mostly albumin and immunoglobulin spots were detected but also spots representing several other abundant plasma proteins. The methodology is easy to perform and is useful as a first step in the detection of diagnostic markers in body fluids by applying proteomics technologies.Current address: Foundation for Biomedical Research of the Academy of Athens, Greece     

High throughput monoclonal antibody generation by immunizing multiple antigens

Recognizing proteins via the production of highly specific monoclonal antibodies (mAbs) is crucial to identifying proteins for proteomic research. However, traditional mAb generation is time-consuming with low efficiency. In this study, we assessed the high throughput method of producing mAbs by immunizing mice with multiple antigens in order to obtain hybridomas against these multiple antigens in one cell fusion. We selected eight proteins that play important roles in human physiological or pathological processes. These proteins were mixed and simultaneously administered to one mouse. We observed the immunizing period for 10 d, and determined the effect of liquid medium and semi-solid medium in hybridoma generation. As a result, all eight immunogens induced antibodies in the immunized mouse in one cell fusion, we obtained hybridomas specific to all eight proteins by enzyme-linked immuno sorbent assay (ELISA) screening, hybridomas against five out of eight showed specific positive in Western-blotting assays. This indicates that we generated mAbs specific to eight proteins in one cell fusion, greatly increasing the efficiency of mAb generation. Furthermore, we observed that hybridomas selected from the liquid medium and semi-solid medium showed different reactivity to antigens. Our study established high-throughput and time-saving methods for production of mAbs. These results provide alternative approaches for increasing the efficacy of mAb generation.     

Large scale depletion of the high-abundance proteins and analysis of middle- and low-abundance proteins in human liver proteome by multidimensional liquid chromatography

An unbiased method for large-scale depletion of high-abundance proteins and identification of middle- or low-abundance proteins by multidimensional LC (MDLC) was demonstrated in this paper. At the protein level, the MDLC system, coupling the first dimensional strong cation exchange (SCX) chromatography with the second dimensional RP-HPLC, instead of immunoaffinity technology, was used to deplete high-abundance proteins. Sixty-two fractions from SCX were separated further by RPLC. UV absorption spectra were observed to differentiate high-abundance proteins from middle- or low-abundance proteins. After the depletion of high-abundance proteins, middle- or low-abundance proteins were enriched, digested, and separated by online 2D-micro-SCX/cRPLC. The eluted peptides were deposited on the MALDI target and detected by MALDI-TOF/TOF MS. This depletion strategy was applied to the proteome of the normal human liver (NHL) provided by the China Human Liver Proteome Project (CHLPP). In total, 58 high-abundance proteins were depleted in one experiment. The strategy increases greatly the number of identified proteins and around 1213 proteins were identified, which was about 2.7 times as that of the nondepletion method.