Grafting of protein L-binding activity onto recombinant antibody fragments

Recombinant antibody fragments consisting of variable domains can be easily produced in various host cells, but there is no universal system that can be used to purify and detect them in the free form or complexed with their antigen. Protein L (PpL) is a cell wall protein isolated from Peptostreptococcus magnus, which has been reported to interact with the V-KAPPA chain of some, but not all, antibodies. Here we grafted the V-KAPPA framework region 1 (FR1) sequence of a high-affinity PpL-binding antibody onto single-chain antibody fragments (scFvs), which have no reactivity with PpL. This substitution made it possible to purify and detect scFvs using PpL conjugates. It did not hinder scFv folding and expression in recombinant bacteria, and it did not interfere with their antigen-binding function. We also identified residue 12 as being potentially able to alter PpL binding. This study, therefore, suggests a way of engineering a PpL-binding site on any scFv without interfering with its function. This could provide a universally applicable method both for the rapid purification of functional recombinant antibody fragments and for their detection even when complexed with their antigen without requiring fusion to an epitope Flag.     

A role in cell-binding for the C-terminus of pneumolysin, the thiol-activated toxin of Streptococcus pneumoniae

Abstract Cell binding of pneumolysin to target cells is an important step in the lysis of cells by this toxin. We sought to locate the cell-binding region of pneumolysin. Deletion of the six C-terminal amino acids decreased cell-binding activity by 98%. Furthermore mutagenesis of an amino acid near the C-terminus decreased the cell-binding activity of full-length pneumolysin by 90%. The C-terminus of pneumolysin has an important role in cell-binding activity.     

Use of high-capacity surface with oriented recombinant antibody fragments in a 5-min immunoassay for thyroid-stimulating hormone

High-capacity surfaces can enhance analyte-binding kinetics and be beneficial for rapid immunoassays. Site-specifically immobilized, oriented recombinant single-chain Fv (scFv) and Fab antibody fragments were compared with a conventional, nonoriented monoclonal antibody (Mab) to capture antigen from serum to solid surface in a one-step, two-site thyroid-stimulating hormone (TSH) immunoassay with a 5-min incubation time. The assay used a ready-to-use dry reagent-based concept and time-resolved fluorescent measurement. TSH binding capacities were 3.0-fold (Fab) and at least 4.1-fold (scFv) higher when recombinant antibodies were used instead of Mab. Recombinant antibody fragments also produced faster kinetics (5 vs. 45-min saturation level) than Mab: 21-25% (Mab) versus 72-83% (scFv and Fab). Analytical sensitivities of the 5-min assay were 0.09 mIU/L TSH (Fab), 0.16 mIU/L TSH (scFv), and 0.26 mIU/L TSH (Mab). Between-run variabilities were 4.2-7.9% (Fab), 4.6-17.7% (scFv), and 5.5-7.2% (Mab). The assays correlated well with the AutoDELFIA hTSH (human TSH) Ultra assay (r = 0.99, n = 109). Fab was good in all aspects of immunoassay—capacity, kinetics, sensitivity, and analytical performance. As a homogeneous, stable, and small-sized binding molecule with optimized surface-coating properties as well as reduced risk for interference by heterophilic antibodies, Fab fragment is a promising and realistic immunoreagent for the future.     

A study of pathogenic factors of Streptococcus pneumoniae strains causing meningitis

Abstract Pneumococcal meningitis in St. Petersburg in the period 1985–1991 occurred in 1.7–2.3 children per 100 000 annually. The most common serotypes among pneumococcal strains isolated from patients with meningitis were 19, 1, 6, 15, and 2, whereas, among the capsulated strains isolated from carriers, type 3 predominated. Only one third of strains from cases of meningitis were highly virulent for mice (types 1, 2, 3). Hyaluronidase was produced by all the 39 studied strains, 22 (84.6±7.1%) out of 26 strains from patients with otitis media, and only by 15 (11.5±2.8%) out of 130 strains isolated from carriers. Non-capsulated strains lacked this enzyme. Results of intranasal inoculation of pneumococcal strains with different hyaluronidase activity and addition of exogenous hyaluronidase to strains which did not produce the enzyme confirm the hypothesis that this enzyme plays an important role in bacterial dissemination and breaching of the blood brain barrier by pneumococci. It was concluded that high hyaluronidase activity, presence of capsule, and pneumolysin or serotype (1, 2, and 19) despite hyaluronidase titer, are the most important factors contributing to the development of pneumococcal meningitis. The role of the mouse toxic factor is unclear.     

Domain unfolding of monoclonal antibody fragments revealed by non-reducing SDS-PAGE

Monoclonal antibodies and derived fragments are used extensively both experimentally and therapeutically. Thorough characterization of such antibodies is necessary and includes assessment of their thermal and storage stabilities. Thus, assessment of the underlying conformational stabilities of the antibodies is also important. We recently documented that non-reducing SDS-PAGE can be used to assess both monoclonal and polyclonal IgG domain thermal unfolding in SDS. Utilizing this same h2E2 anti-cocaine mAb, in this study we generated and analyzed various mAb antibody fragments to delineate the structural domains of the antibody responsible for the observed discrete bands following various heating protocols and analysis by non-reducing SDS-PAGE. Previously, these domain unfolding transitions and gel bands were hypothesized to stem from known mAb structural domains based on the relative thermal stability of those CH2, CH3, and Fab domains in the absence of SDS, as measured by differential scanning calorimetry. In this study, we generated and analyzed F(ab’)₂, Fab, and Fc fragments, as well as a mAb consisting of only heavy chains, and examined the thermally induced domain unfolding in each of these fragments by non-reducing SDS-PAGE. The results were interpreted and integrated to generate an improved model of thermal unfolding for the mAb IgG in SDS. These results and the model presented should be generally applicable to many monoclonal and polyclonal antibodies and allow novel comparisons of conformational stabilities between chemically or genetically modified versions of a given antibody. Such modified antibodies and antibody drug conjugates are commonly utilized and important for experimental and therapeutic applications.     

Novel multispecific heterodimeric antibody format allowing modular assembly of variable domain fragments

Multispecific antibody formats provide a promising platform for the development of novel therapeutic concepts that could facilitate the generation of safer, more effective pharmaceuticals. However, the production and use of such antibody-based multispecifics is often made complicated by: 1) the instability of the antibody fragments of which they consist, 2) undesired inter-subunit associations, and 3) the need to include recombinant heterodimerization domains that confer distribution-impairing bulk or enhance immunogenicity. In this paper, we describe a broadly-applicable method for the stabilization of human or humanized antibody Fv fragments that entails replacing framework region IV of a V_κ1/V_H3-consensus Fv framework with the corresponding germ-line sequence of a λ-type V_L chain. We then used this stable Fv framework to generate a novel heterodimeric multispecific antibody format that assembles by cognate V_L/V_H associations between 2 split variable domains in the core of the complex. This format, termed multispecific antibody-based therapeutics by cognate heterodimerization (MATCH), can be applied to produce homogeneous and highly stable antibody-derived molecules that simultaneously bind 4 distinct antigens. The heterodimeric design of the MATCH format allows efficient in-format screening of binding domain combinations that result in maximal cooperative activity.     

Development of neutralising human recombinant antibodies to pertussis toxin

A phage antibody display library of single chain Fv (scFv) was derived from the peripheral blood of two patients recently recovered from pertussis infection. Ten scFv, differentiated by DNA fingerprinting, were isolated by panning the library against pertussis toxin. One scFv (type 1) accounted for 33% of clones after panning. Six of the panned scFv bound to pertussis toxin. The ability of the scFv to neutralise pertussis toxin was assessed using the Chinese hamster ovary cell assay. The predominant scFv (type 1) and two others (types IV and VIII) were able to neutralise the pertussis toxin.     

Method for generation of human hyperdiversified antibody fragment library

The selection of antibody fragments from libraries using in vitro screening technologies has proven to be a very good alternative to the classical hybridoma technology, and has overcome the laborious process of antibody humanization. However, the complexity of the library is critical in the probability of being able to directly isolate a high affinity antibody specific to a target. We report a method to make hyperdiversified antibody fragment libraries, based on human immunoglobulin variable genes mimicking the somatic hypermutation process. This mutagenesis technology, MutaGen, was used for the first time on the entire variable domain (frameworks and CDRs) of large repertoires of human variable antibody domains. Our MutaGen process uses low-fidelity human polymerases, known as mutases, suggested to be involved in the somatic hypermutation process of immunoglobulin genes. Depending on the mutases used, we generated complementary mutation patterns with randomly distributed mutations. The libraries were generated with an average of 1.8 mutations per 100 amino acids. The hyperdiversified antibody fragment libraries constructed with our process should enable the selection of antibody fragments specific to virtually any target.     

Bivalent monoclonal IgY antibody formats by conversion of recombinant antibody fragments

Monoclonal IgY have the potential to become unique tools for diagnostic research and therapeutic purposes since avian antibodies provide several advantages due to their phylogenetic difference when compared to mammalian antibodies. The mechanism of avian immunoglobulin gene diversification renders chicken an excellent source for the generation of recombinant scFv as well as Fab antibody libraries of high diversity. One major limitation of these antibody fragments, however, is their monovalent format, impairing the functional affinity of the molecules and, thereby, their applicability in prevalent laboratory methods. In this study, we generated vectors for conversion of avian recombinant antibody fragments into different types of bivalent IgY antibody formats. To combine the properties of established mammalian monoclonal antibodies with those of IgY constant domains, we additionally generated bivalent murine/avian chimeric antibody constructs. When expressed in HEK-293 cells, all constructs yielded bivalent disulfide-linked antibodies, which exhibit a glycosylation pattern similar to that of native IgY as assessed by lectin blot analysis. After purification by one step procedures, the chimeric and the entire avian bivalent antibody formats were analyzed for antigen binding and interaction with secondary reagents. The data demonstrate that all antibody formats provide comparable antigen binding characteristics and the well established properties of avian constant domains.     

Improved affinity coupling for antibody microarrays: engineering of double-(His)6-tagged single framework recombinant antibody fragments

Antibody-based microarray is a novel technology with great promise in biomedicine that will provide unique means to perform global proteome analysis. In the process of designing the high-density antibody microarrays required, several critical key issues have been identified that remain to be resolved. In particular, there is a great need for specific and selective approaches enabling non-purified probes to be directly purified, orientated and coupled in a generic one-step procedure directly on the chip. In this study, we report on the successful design of affinity-tagged human recombinant single-chain fragment variable antibody fragments for improved affinity coupling in array applications. By replacing the standard single-histidine (His)(6)-tag with a consecutive double-(His)(6)-tag, the binding to Ni(2+)-nitrilotriacetic acid-coated substrates was significantly improved. Surface plasmon resonance analysis showed a significantly tighter binding with at least a threefold slower dissociation. The improved binding characteristics thus enabled non-purified probes even in the format of crude expression supernatants to be directly applied thereby eliminating the need for any time-consuming pre-purification step(s) prior to the immobilization. While the double-(His)(6)-tag probes were found to be expressed equally well as compared to the single-(His)(6)-tag probes, they displayed better long-term functional on-chip stability. Taken together, the results demonstrate the generic potential of double-(His)(6)-tag recombinant antibodies for the facile fabrication of high-density antibody microarrays.     

Ion exchange chromatography of antibody fragments

Effects of pH and conductivity on the ion exchange chromatographic purification of an antigen-binding antibody fragment (Fab) of pI 8.0 were investigated. Normal sulfopropyl (SP) group modified agarose particles (SP Sepharosetrade mark Fast Flow) and dextran modified particles (SP Sepharose XL) were studied. Chromatographic measurements including adsorption isotherms and dynamic breakthrough binding capacities, were complemented with laser scanning confocal microscopy. As expected static equilibrium and dynamic binding capacities were generally reduced by increasing mobile phase conductivity (1-25 mS/cm). However at pH 4 on SP Sepharose XL, Fab dynamic binding capacity increased from 130 to 160 (mg/mL media) as mobile phase conductivity changed from 1 to 5 mS/cm. Decreasing protein net charge by increasing pH from 4 to 5 at 1.3 mS/cm caused dynamic binding capacity to increase from 130 to 180 mg/mL. Confocal scanning laser microscopy studies indicate such increases were due to faster intra-particle mass transport and hence greater utilization of the media's available binding capacity. Such results are in agreement with recent studies related to ion exchange of whole antibody molecules under similar conditions.     

肺炎链球菌自溶素和溶血素基因的PCR法鉴定

肺炎链球菌是一种致病率和致死率很高的病原菌 ,若无丰富临床检测经验从临床标准中分离鉴定此菌较困难 ,本实验以寡核苷酸引物YH1 YH2、YH7 YH8分别扩增肺炎链球菌自溶素和溶血素基因的 35 4bp和 30 7bp的DNA片段 ,通过改变各种反应条件 ,建立了这两种病原因子基因的PCR检测方法。用此方法对 2 0株肺炎链球菌标准菌株及 7株对照菌株进行了鉴定 ;其扩增产物分别经限制性内切酶TthHB81和和AccI进行酶切以确认扩增产物是否正确 ;用酚 氯仿抽提纯化的全细胞DNA对PCR方法的检测灵敏度进行了测定 ;并利用此方法对 2 8份临床标本分离物进行了鉴定。结果　所有 2 0株肺炎链球菌均可分别用引物YH1 YH2、YH7 YH8扩增出 35 4bp和 30 7bp的DNA片段 ,而对照菌株均呈阴性 ;自溶素及溶血素基因的扩增产物分别经限制性内切酶TthHB81和AccI消化后产生的片段和预期的完全一致 ;两对产物均可从 10fg的全细胞DNA中扩增出目的DNA片段。所建立的两套PCR系统对 2 8份临床标本分离物进行鉴定 ,其中PCR阳性的 15份分离物经生化学特性检查被鉴定为肺炎链球菌。本试验所建立的两套PCR检测系统具有特异性强 ,灵敏度高及操作简单等优点 ,均可用于肺炎链球菌的鉴定。     

Affinity-matured recombinant antibody fragments analyzed by single-molecule force spectroscopy

For many applications, antibodies need to be engineered toward maximum affinity. Strategies are in demand to especially optimize this process toward slower dissociation rates, which correlate with the (un)binding forces. Using single-molecule force spectroscopy, we have characterized three variants of a recombinant antibody single-chain Fv fragment. These variants were taken from different steps of an affinity maturation process. Therefore, they are closely related and differ from each other by a few mutations only. The dissociation rates determined with the atomic force microscope differ by one order of magnitude and agree well with the values obtained from surface plasmon resonance measurements. However, the effective potential width of the binding complexes, which was derived from the dynamic force spectroscopy measurements, was found to be the same for the different mutants. The large potential width of 0.9 nm indicates that both the binding pocket and the peptide deform significantly during the unbinding process.     

“Cleavable” hapten-biotin conjugates: Preparation and use for the generation of anti-steroid single-domain antibody fragments

Antibody engineering technology has the potential to provide artificial antibodies with higher performance than conventional antibodies. Filamentous phage particles are often used to express a vast diversity of mutated antibody fragments from which clones displaying improved fragments can be isolated. We recently showed that hapten-biotin conjugates, combined via a linker involving a reductively cleavable disulfide bond, are useful for isolating phage clones displaying high-affinity anti-hapten antibody fragments. Here we prepare cleavable hapten-biotin conjugates and use them to isolate anti-hapten antibody fragments with relatively low affinities. Three diagnostically important steroids (estradiol-17β [E₂], cortisol, and 17α-hydroxyprogesterone) were each coupled with a biotin derivative containing a disulfide bond. These conjugates could be bound simultaneously by their relevant anti-steroid antibody and NeutrAvidin, and their linkers were easily cleaved by dithiothreitol (DTT) treatment. The E₂-biotin conjugate was used to generate anti-E₂ single-domain antibody fragments (sdAbs). Random point mutations were introduced by error-prone PCR into the gene fragment encoding the V_H domain of a mouse anti-E₂ antibody, and these products were expressed as phagemid particles that were reacted with the E₂-biotin conjugates that had already been immobilized on a solid-phase via NeutrAvidin. Thorough washing off of nonspecific phages and subsequent DTT treatment provided a phagemid clone that displayed a mutated sdAb with improved binding properties.     

Single-chain antibody fragments: Purification methodologies

At present, single-chain variable fragments (scFv) of antibodies are considered one of the most important tools in human therapies. Wide applications of antibodies are being exploited in different medical, pharmaceutical and research areas. These molecules maintain the same binding functionality that full length antibodies but possess several advantageous features as quickness to penetrate the tissues, easy manipulation, fast elimination of their immunocomplex and the possibility of being produced in simple expression systems like bacteria and yeast. The increasing demand in antibody based methodologies is driving advances in the production and purification of genetically engineered antibodies and antibody fragments. While advances in expression systems allow the production of high titers of antibodies, there exist some limits imposed by the downstream methodologies which are not efficient enough to ensure their industrialization.The main aim of this review is to highlight the principal characteristics of single-chain variable fragments of antibodies addressing advances and perspectives on scFv purification.     

Influence of molecular size on tissue distribution of antibody fragments

Biodistribution coefficients (BC) allow estimation of the tissue concentrations of proteins based on the plasma pharmacokinetics. We have previously established the BC values for monoclonal antibodies. Here, this concept is extended by development of a relationship between protein size and BC values. The relationship was built by deriving the BC values for various antibody fragments of known molecular weight from published biodistribution studies. We found that there exists a simple exponential relationship between molecular weight and BC values that allows the prediction of tissue distribution of proteins based on molecular weight alone. The relationship was validated by a priori predicting BC values of 4 antibody fragments that were not used in building the relationship. The relationship was also used to derive BC50 values for all the tissues, which is the molecular weight increase that would result in 50% reduction in tissue uptake of a protein. The BC50 values for most tissues were found to be ~35 kDa. An ability to estimate tissue distribution of antibody fragments based on the BC vs. molecular size relationship established here may allow better understanding of the biologics concentrations in tissues responsible for efficacy or toxicity. This relationship can also be applied for rational development of new biotherapeutic modalities with optimal biodistribution properties to target (or avoid) specific tissues.     

Generation and characterisation of functional recombinant antibody fragments against RNA replicase NIb from plum pox virus

A monoclonal antibody (mAb 2A) able to react against the RNA replicase NIb from plum pox virus (PPV) was obtained and used for generating a specific scFv fragment. The VH and VL coding sequences were cloned and expressed as a fusion scFv protein to alkaline phosphatase. This fusion protein was able to recognise viral NIb in both Western and tissue-print ELISA blots. The affinity and specificity of scFv2A for NIb was similar to that of the parental mAb and the region YLEAFY from PPV-NIb was identified by PEPSCAN assay as the putative epitope. Isolated VH domains from scFv2A were also expressed as fusion to alkaline phosphatase. However, their ability to react against NIb was greatly altered. scFv2A fragments were transiently expressed in the cytosol of Nicotiana benthamiana and although they accumulated to low levels, inhibition-ELISA results indicated that they retained antigen-binding activity.     

PiuA and PiaA, iron uptake lipoproteins of Streptococcus pneumoniae, elicit serotype independent antibody responses following human pneumococcal septicaemia

Streptococcus pneumoniae causes considerable morbidity and mortality worldwide. The need for a cheap and effective pneumococcal vaccine has necessitated the evaluation of common virulence-associated proteins as potential vaccine antigens. PiuA and PiaA are the lipoprotein components of two pneumococcal iron ABC transporters. Here, we show that patients with culture confirmed pneumococcal septicaemia have elevated levels of antibody to PiuA and PiaA in convalescent-phase, compared with acute-phase serum. Additionally, sera from septicaemic patients infected with 13 pneumococcal strains covering eight different serotypes, cross-reacted with recombinant PiuA-His(6) and PiaA-His(6) from a single pneumococcal strain, indicating that this immune response is serotype independent. Anti-PiuA and anti-PiaA antibodies were also found in healthy seven-month-old infants, indicating that they are immunogenic at a very early age.     

Application of recombinant antibody fragments for troponin I measurements

The cardiac isoform of troponin I is a reliable biomarker of damaged cardiomyocytes that accompanies such severe cardiovascular diseases as myocardial infarction. Monoclonal antibody 19C7 recognizes troponin I in the blood-stream with high affinity and specificity. Recombinant antibodies can be used to improve detection systems based on monoclonal antibodies produced with hybridoma technology. In the present study, we compare the properties of monoclonal anti-body 19C7 and its recombinant fragments. It is shown that the recombinant antibody fragments demonstrate similar affinity values as monoclonal antibodies and can be applied for troponin I detection.