Selection of High-Affinity Heterodimeric Antigen-Binding Fc Fragments from a Large Yeast Display Library.

Antigen-binding Fc (Fcab™) fragments, where a novel antigen binding site is introduced by the mutagenesis of the C-terminal loops of the CH3 domain, function as parts of bispecific IgG-like symmetrical antibodies when they replace their wild-type Fc. Their homodimeric structure typically leads to bivalent antigen binding. In particular, biological situations monovalent engagement, however, would be preferred, either for avoiding agonistic effects leading to safety issues, or the attractive option of combining a single chain (i.e., one half) of an Fcab fragment reactive with different antigens in one antibody. We present the strategies for construction and selection of yeast libraries displaying heterodimeric Fcab fragments and discuss the effects of altered thermostability of the basic Fc scaffold and novel library designs that lead to isolation of highly affine antigen binding clones.

Trispecific antibodies produced from mAb2 pairs by controlled Fab-arm exchange.

Bispecific antibodies and antibody fragments are therapeutics of growing importance. They are clinically applied for effector cell engagement, enhanced targeting selectivity, addressing of multiple cellular pathways and active transfer of certain activities into difficult-to-reach compartments. These functionalities could profit from a third antigen specificity. In this work we have employed symmetrical bispecific parental antibodies of mAb2 format, which feature a novel antigen binding site in the CH3 domains, and engineered them with a minimal number of point mutations to guide the formation of a controlled Fab-arm exchanged trispecific antibody at a high yield after reduction and re-oxidation. Two model antibodies, one reactive with EGFR, Her2 and VEGF, and one with Fab-arms binding to Ang2 and VEGF and an Fc fragment binding to VEGF, were prepared and examined for heterodimeric status, stability, antigen binding properties and biological activity. Resulting molecules were of good biophysical characteristics and retained antigen reactivity and biological activity of the parental mAb2 constructs.

A Tetravalent Biparatopic Antibody Causes Strong HER2 Internalization and Inhibits Cellular Proliferation.

The overexpression of tyrosine kinase HER2 in numerous cancers, connected with fierce signaling and uncontrolled proliferation, makes it a suitable target for immunotherapy. The acquisition of resistance to currently used compounds and the multiplicity of signaling pathways involved prompted research into the discovery of novel binders as well as treatment options with multiple targeting and multispecific agents. Here we constructed an anti-HER2 tetravalent and biparatopic symmetrical IgG-like molecule by combining the Fab of pertuzumab with a HER2-specific Fcab (Fc fragment with antigen binding), which recognizes an epitope overlapping with trastuzumab. In the strongly HER2-positive cell line SK-BR-3, the molecule induced a rapid and efficient reduction in surface HER2 levels. A potent anti-proliferative effect, specific for the HER2-positive cell line, was observed in vitro, following the induction of apoptosis, and this could not be achieved with treatment with the mixture of pertuzumab and the parental Fcab. The inhibitory cytotoxic effect of our antibody as a single agent makes it a promising contribution to the armory of anti-cancer molecules directed against HER2-addicted cells.

Bispecific antibodies with Fab-arms featuring exchanged antigen-binding constant domains.

Monoclonal antibodies can acquire the property of engagement of a second antigen via fusion methods or modification of their CDR loops, but also by modification of their constant domains, such as in the mAb2 format where a set of mutated amino acid residues in the CH3 domains enables a high-affinity specific interaction with the second antigen. We tested the possibility of introducing multiple binding sites for the second antigen by replacing the Fab CH1/CL domain pair with a pair of antigen-binding CH3 domains in a model scaffold with trastuzumab variable domains and VEGF-binding CH3 domains. Such bispecific molecules were produced in a "Fab-like" format and in a full-length antibody format. Novel constructs were of expected molecular composition using mass spectrometry. They were expressed at a high level in standard laboratory conditions, purified as monomers with Protein A and gel filtration and were of high thermostability. Their high-affinity binding to both target antigens was retained. Finally, the Her2/VEGF binding domain-exchanged bispecific antibody was able to mediate a potentiated surface Her2-internalization effect on the Her2-overexpressing cell line SK-BR-3 due to improved level of cross-linking with the endogenously secreted cytokine. To conclude, bispecific antibodies with Fabs featuring exchanged antigen-binding CH3 domains offer an alternative solution in positioning and valency of antigen binding sites.

Cell-Free Synthesis of Dopamine and Serotonin in Two Steps with Purified Enzymes.

The synthesis of serotonin and dopamine with purified enzymes is described. Both pathways start from an amino acid substrate and synthesize the monoamine neurotransmitter in two enzymatic steps. The enzymes human tryptophan hydroxylase isoform 2, Rattus norvegicus tyrosine hydroxylase, Chlamydia pneumoniae Cpn1046, and aromatic amino acid decarboxylase from Drosophila melanogaster are recombinantly expressed, purified, and shown to be functional in vitro. The hydroxylases efficiently convert L-DOPA (L-dihydroxy-phenylalanine) and 5-HTP (5-hydroxytryptophan) from L-tyrosine and L-tryptophan, respectively. A single aromatic amino acid decarboxylase is capable of converting both hydroxylated intermediates into the final neurotransmitter. The platform described here may facilitate future efforts to generate medically useful artificial cells and nanofactories.