High-throughput cytotoxicity and antigen-binding assay for screening small bispecific antibodies without purification.

The cytotoxicity of T cell-recruiting antibodies with their potential to damage late-stage tumor masses is critically dependent on their structural and functional properties. Recently, we reported a semi-high-throughput process for screening highly cytotoxic small bispecific antibodies (i.e., diabodies). In the present study, we improved the high-throughput performance of this screening process by removing the protein purification stage and adding a stage for determining the concentrations of the diabodies in culture supernatant. The diabodies were constructed by using an Escherichia coli expression system, and each diabody contained tandemly arranged peptide tags at the C-terminus, which allowed the concentration of diabodies in the culture supernatant to be quantified by using a tag-sandwich enzyme-linked immunosorbent assay. When estimated diabody concentrations were used to determine the cytotoxicity of unpurified antibodies, results comparable to those of purified antibodies were obtained. In a surface plasmon resonance spectroscopy-based target-binding assay, contaminants in the culture supernatant prevented us from conducting a quantitative binding analysis; however, this approach did allow relative binding affinity to be determined, and the relative binding affinities of the unpurified diabodies were comparable to those of the purified antibodies. Thus, we present here an improved high-throughput process for the simultaneous screening and determination of the binding parameters of highly cytotoxic bispecific antibodies.

A semi high-throughput method for screening small bispecific antibodies with high cytotoxicity.

Small bispecific antibodies that induce T-cell-mediated cytotoxicity have the potential to damage late-stage tumor masses to a clinically relevant degree, but their cytotoxicity is critically dependent on their structural and functional properties. Here, we constructed an optimized procedure for identifying highly cytotoxic antibodies from a variety of the T-cell-recruiting antibodies engineered from a series of antibodies against cancer antigens of epidermal growth factor receptor family and T-cell receptors. By developing and applying a set of rapid operations for expression vector construction and protein preparation, we screened the cytotoxicity of 104 small antibodies with diabody format and identified some with 103-times higher cytotoxicity than that of previously reported active diabody. The results demonstrate that cytotoxicity is enhanced by synergistic effects between the target, epitope, binding affinity, and the order of heavy-chain and light-chain variable domains. We demonstrate the importance of screening to determine the critical rules for highly cytotoxic antibodies.

Organic crystal-binding peptides: morphology control and one-pot formation of protein-displaying organic crystals.

Crystalline assemblies of fluorescent molecules have different functional properties than the constituent monomers, as well as unique optical characteristics that depend on the structure, size, and morphological homogeneity of the crystal particles. In this study, we selected peptides with affinity for the surface of perylene crystal particles by exposing a peptide-displaying phage library in aqueous solution to perylene crystals, eluting the surface-bound phages by means of acidic desorption or liquid-liquid extraction, and amplifying the obtained phages in Escherichia coli. One of the perylene-binding peptides, PeryBPb1: VQHNTKYSVVIR, selected by this biopanning procedure induced perylene molecules to form homogenous planar crystal nanoparticles by means of a poor solvent method, and fusion of the peptide to a fluorescent protein enabled one-pot formation of protein-immobilized crystalline nanoparticles. The nanoparticles were well-dispersed in aqueous solution, and Förster resonance energy transfer from the perylene crystals to the fluorescent protein was observed. Our results show that the crystal-binding peptide could be used for simultaneous control of perylene crystal morphology and dispersion and protein immobilization on the crystals.

Enzymatic fabrication of protein-decorated gold nanoparticles by the aid of artificial peptides with gold-binding affinity.

Here, we report a new approach for the biofabrication of protein-immobilized gold nanoparticles (Au NPs), using oxidoreductase with gold-binding peptide-tagged recombinant proteins. The reduction of Au ions to Au(0) was achieved using a natural electron-donating cofactor, nicotinamide adenine dinucleotide, which was regenerated by the glycerol dehydrogenase (GLD) enzyme. First, we selected the A3 peptide (AYSSGAPPMPPF) as a gold binding moiety. The A3 peptide was introduced to the C-terminus of fusion proteins of immunoglobulin G (IgG)-binding domains of protein G and protein A. In the presence of the recombinant protein, the GLD-catalyzed cofactor reduction resulted in the efficient in situ fabrication of Au NPs immobilized with the fusion protein. Moreover, the protein-immobilized Au NPs were shown to have IgG binding activity. Although the A3 peptide had the ability to stabilize Au NPs, the results suggested that its binding affinity for Au NPs was unexpectedly weaker than that of His-tag. A cysteine residue was thus introduced to a recombinant protein adjacent to the A3 peptide. Finally, an artificial peptide, comprising A3 sequence with the C-terminal single cysteine residue, enabled the stable display of a fusion protein while maintaining its IgG binding activity through the Au-S bond. This enzyme-assisted one-pot methodology for protein-Au NPs conjugation offers one potent route for the facile fabrication of biomolecule-decorated metal NPs.

Facile, rapid and efficient biofabrication of gold nanoparticles decorated with functional proteins.

We report a one-pot biological approach to fabricate gold nanoparticle (AuNP)-ZZ domain conjugates using peptide-functionalized proteins that can simultaneously direct both biomineralization and surface modification of AuNPs. In addition, immuno-AuNPs are readily prepared through the specific binding of antibodies to the ZZ domain on the AuNPs.