6 research outputs found
Efficient Identification of Murine M2 Macrophage Peptide Targeting Ligands by Phage Display and Next-Generation Sequencing
Efficient Identification of Murine M2 Macrophage Peptide Targeting Ligands by Phage Display and Next-Generation Sequencing
Peptide ligands are used to increase
the specificity of drug carriers to their target cells and to facilitate
intracellular delivery. One method to identify such peptide ligands,
phage display, enables high-throughput screening of peptide libraries
for ligands binding to therapeutic targets of interest. However, conventional
methods for identifying target binders in a library by Sanger sequencing
are low-throughput, labor-intensive, and provide a limited perspective
(<0.01%) of the complete sequence space. Moreover, the small sample
space can be dominated by nonspecific, preferentially amplifying “parasitic
sequences” and plastic-binding sequences, which may lead to
the identification of false positives or exclude the identification
of target-binding sequences. To overcome these challenges, we employed
next-generation Illumina sequencing to couple high-throughput screening
and high-throughput sequencing, enabling more comprehensive access
to the phage display library sequence space. In this work, we define
the hallmarks of binding sequences in next-generation sequencing data,
and develop a method that identifies several target-binding phage
clones for murine, alternatively activated M2 macrophages with a high
(100%) success rate: sequences and binding motifs were reproducibly
present across biological replicates; binding motifs were identified
across multiple unique sequences; and an unselected, amplified library
accurately filtered out parasitic sequences. In addition, we validate
the Multiple Em for Motif Elicitation tool as an efficient and principled
means of discovering binding sequences
Synthesis and evaluation of multivalent M2pep peptides for targeting alternatively activated M2 macrophages
MMP-10 Regulates Collagenolytic Activity of Alternatively Activated Resident Macrophages
A modular IgG-scFv bispecific antibody topology
Here we present a bispecific antibody (bsAb) format in which a disulfide-stabilized scFv is fused to the C-terminus of the light chain of an IgG to create an IgG-scFv bifunctional antibody. When expressed in mammalian cells and purified by one-step protein A chromatography, the bsAb retains parental affinities of each binding domain, exhibits IgG-like stability and demonstrates in vivo IgG-like tumor targeting and blood clearance. The extension of the C-terminus of the light chain of an IgG with an scFv or even a smaller peptide does appear to disrupt disulfide bond formation between the light and heavy chains; however, this does not appear to affect binding, stability or in vivo properties of the IgG. Thus, we demonstrate here that the light chain of an IgG can be extended with an scFv without affecting IgG function and stability. This format serves as a standardized platform for the construction of functional bsAbs