Variable fragments of heavy chain antibodies (VHHs): a new magic bullet molecule of medicine?

 Serum of animals belonging to the Camelidae family (camels and llamas) contains fully active antibodies that are naturally devoid of light chains. Variable domains derived from heavy chain antibodies (hcAb) called VHHs or nanobodies™ can bind antigens as effectively as full-length antibodies and are easy to clone and express. Because of their potential, VHHs are being intensively studied as potential therapeutic, diagnostic and imaging tools. The paper reviews the molecular background of heavy chain antibodies and describes methods of obtaining recombinant fragments of heavy chain antibodies as well as their therapeutic, diagnostic and other applications.

Improvement in production and purification bioprocesses of bacterially expressed anti-alphaIIbbeta3 human single-chain FV antibodies.

Production of anti-alphaIIbbeta3 (anti-alphaIIbbeta3)-binding single-chain FV (scFv) fragments obtained from combinatorial libraries of IgG human antibodies is of broad interest for imaging and treatment of acute coronary syndromes. The objective of our work was to design an optimized production of one selected anti-alphaIIbbeta3-binding scFv fragment for subsequent in vivo animal studies. Fed-batch fermentation was initiated with 2TY media supplemented with 0.1 M glucose. This growing batch culture was used as a starting point for further fed-batch induction, in which a media without glucose containing 1 mM IPTG and 0.4 M saccharose was continuously added. Subsequent purification was performed on the whole cell extract in native conditions over an immobilized copper-ion affinity column. The improved conditions allowed the recovery of 5 mg of highly purified scFv fragments as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The bioactivity of the scFv fragments was further monitored by ELISA, cytometric and immunohistochemical methods.

Direct phage to intrabody screening (DPIS): demonstration by isolation of cytosolic intrabodies against the TES1 site of Epstein Barr virus latent membrane protein 1 (LMP1) that block NF-kappaB transactivation.

The expression of intracellular antibodies (intrabodies) in eukaryotic cells has provided a powerful tool to manipulate microbial and cellular signaling pathways in a highly precise manner. However, there have been several technical issues that have restricted their more widespread use. In particular, single-chain antibodies (sFv) have been reported to fold poorly in the reducing environment of the cytoplasm and as such there has been a reluctance to use sFv-phage libraries as a source of intrabodies unless a pre-selection step to identify these rare sFvs from natural libraries or libraries of engineering sFvs that could fold properly in the absence of disulfide bonds were used. Here, we investigated whether target specific sFvs that are isolated from a 15 billion member non-immune human sFv-phage display library could be directly screened in pools as intrabodies without prior knowledge of their individual identity or purity within pools of antigen-specific sFvs. As the target, we used a synthetic transformation effector site 1 (TES1) polypeptide comprising the membrane-most proximal 34 amino acid residues of the carboxy-terminal cytoplasmic tail of the oncogenic latent membrane protein 1 (LMP1) of Epstein Barr virus, which serves as a docking site for adapter proteins of the tumor necrosis factor (TNF) receptor (TNFR)-associated factor (TRAF) family. Anti-TES1 sFvs, initially identified by phage ELISA screens, were grouped into pools according to the absorbance reading of the antigen-specific phage ELISA assays and then transferred as pools into eukaryotic expression vectors and expressed as cytoplasmic intrabodies. Using the pooling strategy, there was no loss of individual anti-TES1 sFvs in the transfer from prokaryotic to eukaryotic expression vectors. In addition, the initial assignments into sFv pools based on phage ELISA readings allowed the segregation of individual anti-TES1 sFvs into discrete or minimally overlapping intrabody pools. Further assessment of the biological activity of the anti-TES1 intrabody pools demonstrated that they were all able to selectively block F-LMP1-induced NFkappaB activity that was mediated through the TES1-site and to bind LMP1 protein with high efficiency. This direct phage to intrabody screening (DPIS) strategy should allow investigators to bypass much of the in vitro sFv characterization that is often not predictive of in vivo intrabody function and provide a more efficient use of large native and synthetic sFv phage libraries already in existence to identify intrabodies that are active in vivo.

[Studies on the optimal expression condition, purification and its characterization of ScFv-2F3].

The expression vectors of the gene encoding ScFv-2F3 were transformed into E. coli BL21(DE3). Clones of higher expression were first selected, then were grown in the presence of IPTG at 37 degrees C to induce its expression. The culture conditions were carefully optimized. It was found that optimal conditions were as follows: the induction was started as OD590 reached to 1.0-1.8; the concentration of IPTG was 0.3-0.5 mmol/L and induction time is 7 h. The yield of ScFv-2F3 expressed in the selected clones is about 20% of the total proteins. The optimal culture conditions were successfully applied to fermenter of 50 L. The conditions of washing the inclusion bodies were also optimized. A two-step method was used to renature the inclusion body. The expression product of interest and its biological activities were characterized with Western blotting and ELISA. A novel selenium-containing single-chain abzyme with GPX activity was prepared.

Comparison of methods for the generation of immunoreactive fragments of a monoclonal antibody (B72.3) reactive with human carcinomas.

Immunoglobulin fragments, whether of polyclonal or monoclonal antibodies, offer a number of advantages over the intact immunoglobulin. The generation of immunoreactive fragments from monoclonal antibodies (MAb) is not always a straightforward task. Both pepsin and papain can be used to digest MAb to a bivalent molecule with a Mr of 100,000. However, pepsin pepsin digestions does not always result in immunoreactive fragments and a stable consistent product by papain digestion is often difficult to obtain. MAb B72.3 is an example of both situations. MAb B72.3 reacts with a glycoprotein (TAG-72) with a molecular weight greater than 10(6). MAb B72.3 has been shown to exhibit a high degree of selective reactivity with colon, breast and ovarian carcinomas and has been used for radioimmunodiagnosis in model systems and in clinical trials. A third enzyme, bromelain, in the same family of sulfhydryl proteases as papain, has been used to generate a fragment of MAb B72.3, with a Mr of approximately 100,000. The bromelain-generated fragment of MAb B72.3 retained 100% immunoreactivity as measured in competitive solid-phase radioimmunoassays and could be generated with consistent results from one preparation to another. Both the bromelain- and papain-generated fragments were radiolabelled with 125I without significant loss of the MAb's reactivity to tumor extracts. Differences were observed between the bromelain- and papain-generated fragment when compared in vivo. Fragmentation of MAb B72.3 with bromelain has yielded a superior bivalent fragment for radioimmunolocalization.

[Experiences in the preparation of the secretory IgA and secretory component from human colostrum]. / Erfahrungen bei der Präparation von sekretorischem IgA und sekretorischer Komponente aus Humankolostrum.

With the isolation of secretory IgA and secretory component from human colostrum following the route communicated by Kobayashi some differences revealed with respect to the preparative results. Mainly problems arising from lactoferrin and IgM encouraged us to offer some varied laboratory instructions.

[Purification and characterization of the free secretory piece of human colostrum (author's transl)]. / Reinigung und Charakterisierung der freien sekretorischen Komponente aus Human-Kolostrum.

The free secretory piece is isolated from human colostrum by gel filtration and ion-exchange chromatography in high yield (200 mg/l colostrum). DEAE-Cellulose chromatography separates the free secretory piece in two fractions which are electrophoretically distinct, but otherwise have the same characteristics, like molecular weight, antigenic determinants, N-terminal sequence, peptide map and amino acid composition. It was therefore concluded that the protein part of the secretory piece is homogenous.

[Method of preparation and evaluation of antisera for the differential determination of secretory immunoglobulin A and free secretory component in biological fluids]. / Metodika polucheniia i otsenka antisyvorotok dlia differentsirovannogo opredeleniia sekretornogo immunoglobulina A i svobodnogo sekretornogo komponenta v biologicheskikh zhidkostiakh.

One of the pressing tasks in the study of local nonsusceptibility to infectious diseases and immunochemical analysis of the external secretion is recording of the level of various forms of the secretory IgA (SIgA) and of the secretory component (SC) in various biological fluids. Indication and measurment of the concentrations of the mentioned proteins encounter serious difficulties caused by heterogeneity of their molecular forms. It was shown that the antisera to the whole molecule of SIgA and SC are of no use. On the basis of a new method of purification of free SC and technology of preparation of monospecific antisera capable of separation of SIgA and free SC there were obtained diagnostic antisera for the quantitative recording and differentiation of various forms of IgA and SC in biological fluids. A reliable measurement of the SIgA and SC concentration in some external secretion was carried out with the aid of the mentioned preparations without any complicated chromatographic experiments.

Structural composition of canine secretory component and immunoglobulin A.

Dog serum and colostral immunoglobulin A (IgA) and free secretory component from colostrum were isolated using affinity chromatography. Both serum and colostral IgA showed similar susceptibility to reduction with dithiothreitol, but only colostral IgA released the additional subunit, bound secretory component. This released secretory component was identical with free secretory component with respect to electrophoretic migration, isoelectric focusing point, and molecular weight, but lacked some antigenic determinants. The amino acid composition and the N-terminal sequence of canine free secretory component was similar to that reported for the cow.

Secretory immunoglobulin A in oral and respiratory passages in man.

Secretory IgA (SIgA) is the predominant immunoglobulin in certain external secretions and may have an important role in immunological mucosal resistance. SIgA differs in chemical and immunological properties from serum IgA. The present study was undertaken to investigate the antigenic relationship between SIgA, free secretory component (FSC) and serum IgA and the localization of SIgA as well as other immunological classes in tissues of oral and respiratory passages by use of immunofluorescence technique. SIgA and FSC were highly purified from human colostrum and rabbit anti-SIgA and anti-SC antisera were prepared. On the basis of antigenic relationships between SIgA, FSC and serum IgA, it was emphasized that individual specific antisera for SC and IgA and/or SIgA should be used in immunochemical or immunohistological investigations for SIgA. The present study failed to detect SC determinants in palatine and lingual tonsils. However, it was evident that cells present in the pharyngeal tonsillar epithelium contain SC determinants. SC molecules may be synthesized in certain secretory cells of mucous membrane and glandular epithelium and the combining of SC with IgA could occur in the cytoplasm of epithelial cells, the intercellular spaces and/or in the lumens of glandular acini and ductules.

A study of the association of human secretory component with IgA and IgM proteins.

Human secretory component (SC) was isolated from colostral whey, and the binding of 125I-SC to purified IgA and IgM monoclonal proteins was studied using two methods to separate free from immunoglobulin-bound 125I-SC: a) gel filtration on Sephadex G-200, and b) precipitation of 125I-SC-Ig complexes with anti-Ig antibody. Both IgA dimeric proteins and IgM pentamers bound 125I-SC with approximately one SC-binding site per mole of polymer and similar affinity. Assuming a reversible equilibrium, an apparent association constant congruent to 10-8 M-1 was calculated to govern the binding of 125I-SC to immunoglobulin polymers. The assignment of a single association constant may be an oversimplication, particularly for the case of IgA polymers, since evidence was obtained that disulfide bonds were formed in the 125I-SC-IgA complex. Despite the complexity of the reaction, binding of 125I-SC to both IgA and IgM polymers could be analyzed by standard methods of saturation analysis, and both were shown to have a similar affinity for 125I-SC. No differences were noted in the affinity of 125I-SC binding to the IgA1 and IgA2 subclasses. Binding of monomeric IgA and IgM proteins could not be measured and was at least 100-fold lower than that found for IgA and IgM polymers. Complexes of 125I-SC with IgA dimers were presumed to involve covalent bond formation, since these complexes did not dissociate in guanidine-HCl. One IgA2 trimer did not form a covalent bond since it was completely dissociated in guanidine. In contrast, 125I-SC-IgM complexes were dissociated in denaturing solvent, indicating that such complexes were held together primarily by non-covalent bonds. Experiments with (Fc)5 mu isolated by high temperature tryptic digestion of IgM showed that binding of 125I-SC was to the Fc region of IgM proteins. It was suggested that the binding of SC with similar affinity to both IgA and IgM polymers may be important in the biologic function of both these immunoglobulin classes.

Human J-chain: isolation and molecular weight studies.

PIP: J-chain, a polypeptide chain unique to polymeric immunoglobulins (Igs) which has been postulated to be responsible for joining monomeric subunits into the polymeric forms, was isolated from human IgA, secretory IgA, and IgM by 3 different procedures, disc electrophoresis, immunoadsorbent radioimmunoassay, and dialysis against distilled water. Precipitation in water was the simplest and yielded satisfactory results. Molecular weights of the various J-chain isolates were studied by using 2-species plot analysis of sedimentation equilibrium data. 2 populations of molecules were found: 1 had a molecular weight of 13,300-17,700 and the other of 6400-11,500. Variations in these data from those of other investigators are discussed in terms of isolation procedure differences.^ieng

Localization of idiotypic antigenic determinants in the Fv region of murine myeloma protein MOPC-315.

Rabbit antisera were prepared against the idiotypic determinants of the mouse IgA myeloma protein-315, its purified heavy and light chains, and the Fv fragment comprising the variable region of both heavy and light chains. Agar diffusion demonstrated a line of identity between protein-315 and its Fv fragment against either homologous antiserum. Protein-315 and Fv fragment were labeled with (125)I and reacted with their anti-idiotypic antisera. Inhibition studies confirmed that the Fv fragment contained all the idiotypic specificities present in the intact protein. Fv was as effective as protein-315 on a weight basis in inhibiting the reaction between anti-idiotypic antiserum and protein-315. Protein-315 has high affinity for the 2,4-dinitrophenyl group, and such ligands can inhibit the reaction between protein-315 and its anti-idiotypic antibodies. Hapten inhibition was also obtained with the Fv fragment and its anti-idiotypic antiserum, implying that Fv contains an intact combining site. In this system with protein-315 the antigenic determinants expressed as idiotypic specificities are entirely within the variable region and are not influenced in their expression by the constant region. We therefore suggest that, in general, idiotypic determinants are antigenic determinants of the Fv protions of immunoglobulins.