Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

We developed an IgG1 domain-tethering approach to guide the correct assembly of 2 light and 2 heavy chains, derived from 2 different antibodies, to form bispecific monovalent antibodies in IgG1 format. We show here that assembling 2 different light and heavy chains by sequentially connecting them with protease-cleavable polypeptide linkers results in the generation of monovalent bispecific antibodies that have IgG1 sequence, structure and functional properties. This approach was used to generate a bispecific monovalent antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor that: 1) can be produced and purified using standard IgG1 techniques; 2) exhibits stability and structural features comparable to IgG1; 3) binds both targets simultaneously; and 4) has potent anti-tumor activity. Our strategy provides new engineering opportunities for bispecific antibody applications, and, most importantly, overcomes some of the limitations (e.g., half-antibody and homodimer formation, light chains mispairing, multi-step purification), inherent with some of the previously described IgG1-based bispecific monovalent antibodies.     

A high-throughput platform for population reformatting and mammalian expression of phage display libraries to enable functional screening as full-length IgG

Phage display antibody libraries are a rich resource for discovery of potential therapeutic antibodies. Single-chain variable fragment (scFv) libraries are the most common format due to the efficient display of scFv by phage particles and the ease by which soluble scFv antibodies can be expressed for high-throughput screening. Typically, a cascade of screening and triaging activities are performed, beginning with the assessment of large numbers of E. coli-expressed scFv, and progressing through additional assays with individual reformatting of the most promising scFv to full-length IgG. However, use of high-throughput screening of scFv for the discovery of full-length IgG is not ideal because of the differences between these molecules. Furthermore, the reformatting step represents a bottle neck in the process because each antibody has to be handled individually to preserve the unique VH and VL pairing. These problems could be resolved if populations of scFv could be reformatted to full-length IgG before screening without disrupting the variable region pairing. Here, we describe a novel strategy that allows the reformatting of diverse populations of scFv from phage selections to full-length IgG in a batch format. The reformatting process maintains the diversity and variable region pairing with high fidelity, and the resulted IgG pool enables high-throughput expression of IgG in mammalian cells and cell-based functional screening. The improved process led to the discovery of potent candidates that are comparable or better than those obtained by traditional methods. This strategy should also be readily applicable to Fab-based phage libraries. Our approach, Screening in Product Format (SiPF), represents a substantial improvement in the field of antibody discovery using phage display.     

GABAA Receptor Subtypes Expressed in Cerebellar Granule Cells: A Developmental Study

Abstract: The developmental properties of primary rat cerebellar granule cells have been characterised with respect to their expression of GABA_A receptor subtypes using both an immunological approach and radioligand binding assays. At day 1 in culture, the GABA_A receptor α1 subunit was detectable in immunoblots and increased in level up to day 9. The GABA_A receptor α6 subunit was not detectable at day 1; however, at days 3–5, a specific M_r 58,000 anti-α6 1–16 Cys immunoreactive species was present which further increased in level up to 9 days in culture. Similar qualitative results were obtained for the expression of the GABA_A receptor α6 subunit in age-matched rat cerebellar membranes. In parallel studies, it was found that although there was an overall increase in [³H]Ro 15–4513 binding sites with days in culture, the relative contributions of diazepam-sensitive and diazepam-in-sensitive [³H]Ro 15–4513 binding changed. A time-dependent enrichment of the diazepam-insensitive binding site up to a maximum of 74% of total [³H]Ro 15–4513 sites was found. This was concomitant with the appearance of the GABA_A receptor α6 subunit. These results are in agreement with the pharmacology described for α6βγ2 cloned receptors. They suggest a developmentally regulated expression of the GABA_A receptor α6 subunit gene at a time that is correlated in vivo with establishment of neuronal connections.     

酶联免疫吸附技术(ELISA)对大豆根瘤菌的鉴定

本文用直接ELISA法检测大豆根瘤菌USDA 110和RTt 50的纯培养菌体和根瘤。确定了该试验的最佳工作条件:酶标结合物HRP—Ab 110和HRP—Ab50的工作稀释度分别为1:3200和1:800,抗体Ab 110和Ab 50的工作稀释度分别为1:3200和1:800,抗原USDA 110和RTt 50的最适工作浓度均为6×10~7细胞/ml。该法能够特异地检测和区别慢生型和快生型大豆根瘤菌。在这两种类型的大豆根瘤菌中,同种内的少数菌株存在交叉反应,通过吸收可以消除,从而使ELISA的检测达到菌株     

Species specificity of barnacle settlement-inducing proteins

We previously isolated a larval settlement-inducing protein complex (SIPC) from adult extracts of the barnacle, Balanus amphitrite using a nitrocellulose membrane settlement assay. In the present study, we found that the extracts of other adult barnacles, Megabalanus rosa and Balanus eburneus, also induced the settlement of B. amphitrite cyprids although the inductive activity was slightly lower than that of conspecific extracts. Furthermore, we examined reactivity to anti-SIPC antibody in adult extracts from six species of Japanese barnacles other than B. amphitrite, brine shrimp and eight marine sessile organisms besides barnacles. The results showed that all barnacles examined contained SIPC-like proteins with slightly different molecular weight, while the other animals did not react to the antibody by immunoblot analysis. These findings suggest that species specificity in settlement-inducing proteins of barnacles is not so strict, but these proteins are characteristic to barnacle species.     

Aberrant bispecific antibody pharmacokinetics linked to liver sinusoidal endothelium clearance mechanism in cynomolgus monkeys

Bispecific antibodies (BsAbs) can affect multiple disease pathways, thus these types of constructs potentially provide promising approaches to improve efficacy in complex disease indications. The specific and non-specific clearance mechanisms/biology that affect monoclonal antibody (mAb) pharmacokinetics are likely involved in the disposition of BsAbs. Despite these similarities, there are a paucity of studies on the in vivo biology that influences the biodistribution and pharmacokinetics of BsAbs. The present case study evaluated the in vivo disposition of 2 IgG-fusion BsAb formats deemed IgG-ECD (extracellular domain) and IgG-scFv (single-chain Fv) in cynomolgus monkeys. These BsAb molecules displayed inferior in vivo pharmacokinetic properties, including a rapid clearance (> 0.5 mL/hr/kg) and short half-life relative to their mAb counterparts. The current work evaluated factors in vivo that result in the aberrant clearance of these BsAb constructs. Results showed the rapid clearance of the BsAbs that was not attributable to target binding, reduced neonatal Fc receptor (FcRn) interactions or poor molecular/biochemical properties. Evaluation of the cellular distribution of the constructs suggested that the major clearance mechanism was linked to binding/association with liver sinusoidal endothelial cells (LSECs) versus liver macrophages. The role of LSECs in facilitating the clearance of the IgG-ECD and IgG-scFv BsAb constructs described in these studies was consistent with the minimal influence of clodronate-mediated macrophage depletion on the pharmacokinetics of the constructs in cynomolgus monkeys The findings in this report are an important demonstration that the elucidation of clearance mechanisms for some IgG-ECD and IgG-scFv BsAb molecules can be unique and complicated, and may require increased attention due to the proliferation of these more complex mAb-like structures.     

Induction by chemically modified actin derivatives of antibody specificity: A relation between modified sites and antibody interactions with monomeric and filamentous actins

A comparison of specific antibodies induced by unfolded actins modified either by oxidation or by arylation of lysine residues was reported. We have focused our work on binding properties with filamentous actin and located its preferential antigenic sites for the anti-arylated-actin antibodies in the C-part of the molecule. An interference of anti-oxidized actin antibodies upon actin polymerisation has also been reported.     

Y10W beta(1-40) fluorescence reflects epitope exposure in conformers of Alzheimer's beta-peptide

The Alzheimer's beta-peptide in neutral aqueous solution is characterized variously as a random coil or a heterogeneous mixture of conformers. Under conditions of lowered pH characteristic of intracellular compartments such as endosomes or lysosomes, a different conformation is favored, which is reflected in the biophysical and biological properties of the peptide. The reactivity of the epitope of the monoclonal antibody 6F/3D, encompassing residues 9-14, is drastically reduced. The fluorescence of human sequence beta(1-40) with the tyrosine at position 10 substituted with tryptophan (Y10W beta(1-40)) is quenched nearly 50% when the peptide is shifted to pH 4.6. The exposure of the 6F/3D epitope parallels Y10W beta(1-40) fluorescence changes induced by a variety of perturbations. The linkage of the sensitivity of immunological detection with the potential for monitoring rapid changes by fluorescence offers convergence of biology and biophysics in the study of beta-amyloid peptide conformation.     

Ascaris suum: specific antibodies in isolated intestinal loop washings from immunized swine.

Six pigs had been immunized with multiple dose of embryonated eggs and an isolated intestinal loop was prepared in each animal. Specific antibodies to Ascaris suum were detected in the soluble protein fraction of washings from the intestinal loops using an indirect fluorescent antibody test. The specific antibodies belonged to the IgA, IgG and IgE classes of immunoglobulins. In contrast, specific antibodies were not detected in the soluble protein fraction from the accumulated fluid from the intestinal loop of one pig. Soluble proteins from the washings of intestinal loops consisted of serum albumin, a large molecular size glycoprotein, and variable amounts of several α-globulins, transferrin, and immunoglobulins. The individual soluble protein solutions were efficiently fractionated using DEAE-cellulose, Sephadex G-200, and Sepharose 6B Chromatographic columns.