Efficient Identification of Murine M2 Macrophage Peptide Targeting Ligands by Phage Display and Next-Generation Sequencing

Abstract

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 non-specific, 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.

IL-10 improves cardiac remodeling after myocardial infarction by stimulating M2 macrophage polarization and fibroblast activation

“…When myofibroblasts are highly proliferative, collagen production is actually dampened to balance collagen synthesis and degradation [1]. Higher hyaluronan is consistent with this finding, as higher hyaluronan in dermal tissue associates with increased fibroblast activation and less collagen scar formation [2]. Our results suggest that the myofibroblast activation stimulated by IL-10 infusion is a hyper-activated state represented by enriched hyaluronan and reduced collagen.…”

“…To demonstrate the effect of IL-10 on post-MI LV macrophages, whole transcriptome analysis by RNA-seq was performed as described previously [6, 29]. Briefly, samples passed quality parameters (minimum concentration and size range) and were used to develop RNA libraries ( n = 12 pooled index samples) using the TruSeq Stranded Total RNA with Ribo-Zero Kit, Set A (FC-122-2501, San Diego, CA) according to manufacturer’s instructions.…”

Nanomaterials for cancer immunotherapy

“…By phage display and high-throughput sequencing, Pun et al . identified a unique peptide sequence, M2pep, which preferentially binds to murine M2 cells, including TAMs, with low affinity for other leukocytes [203, 204]. This peptide sequence can be further applied for TAM-specific nanoparticle delivery.…”

Uncovering temporally sensitive targeting motifs for traumatic brain injury via phage display

“…Recent sequencing advancements in NGS capabilities are instrumental to the identification of candidate biological motifs in phage display libraries. NGS analysis provides an opportunity to uncover the entire population of phage display libraries at a sequencing space of 10 5 -10 7 in comparison to 20-100 for traditional Sanger sequencing methods [46]. This analysis also minimizes the probability of selecting false positive clones that may be overrepresented in the library due to propagation advantages, amplified as a consequence of repetitive screening [47,48].…”