Yeast surface display is a proven tool for the selection and evolution of ligands with novel binding activity. Selections from yeast surface display libraries against transmembrane targets are generally carried out using recombinant soluble extracellular domains. Unfortunately, these molecules may not be good models of their true, membrane-bound form for a variety of reasons. Such selection campaigns often yield ligands that bind recombinant target but not targetexpressing cells or tissues. Advances in cell-based selections with yeast surface display may aid the frequency of evolving ligands that do bind true, membrane-bound antigens. This study aims to evaluate ligand selection strategies using both soluble target-driven and cellular selection techniques to determine which methods yield translatable ligands most efficiently and generate novel binders against CD276 (B7-H3) and Thy1, two promising tumor vasculature targets. Out of four ligand selection campaigns carried out using only soluble extracellular domains, only an affibody library sorted against CD276 yielded translatable binders. In contrast, fibronectin domains against CD276 and affibodies against CD276 were discovered in campaigns that either combined soluble target and cellular selection methods or used cellular selection methods alone. A high frequency of non-target specific ligands discovered from the use of cellular selection methods alone motivated the development of a depletion scheme using disadhered, antigen-negative mammalian cells as a blocking agent. Affinity maturation of CD276-binding affibodies by errorprone PCR and helix walking resulted in strong, specific cellular CD276 affinity (K d = 0.9 ± 0.6 nM). Collectively, these results motivate the use of cellular selections in tandem with recombinant selections and introduces promising affibody molecules specific to CD276 for further applications.

Spectroscopic photoacoustic (sPA) molecular imaging has high potential for identification of exogenous contrast agents targeted to specific markers. Antibody-dye conjugates have recently been used extensively for preclinical sPA and other optical imaging modalities for highly specific molecular imaging of breast cancer. However, antibody-based agents suffer from long circulation times that limit image specificity. Here, the efficacy of a small protein scaffold, the affibody (ABY), conjugated to indocyanine green (ICG), a near-infrared fluorescence dye, as a targeted molecular imaging probe is demonstrated. In particular, B7-H3 (CD276), a cellular receptor expressed in breast cancer, was imaged via sPA and fluorescence molecular imaging to differentiate invasive tumors from normal glands in mice. Administration of ICG conjugated to an ABY specific to B7-H3 (ABY B7-H3 -ICG) showed significantly higher signal in mammary tumors compared to normal glands of mice. ABY B7-H3 -ICG is a compelling scaffold for molecular sPA imaging for breast cancer detection.

Purpose: Human B7-H3 (hB7-H3) is a promising molecular imaging target differentially expressed on the neovasculature of breast cancer and has been validated for preclinical ultrasound (US) imaging with anti-B7-H3-antibody-functionalized microbubbles (MB). However, smaller ligands such as affibodies (ABY) are more suitable for the design of clinical-grade targeted MB.Experimental Design: Binding of ABY B7-H3 was confirmed with soluble and cell-surface B7-H3 by flow cytometry. MB were functionalized with ABY B7-H3 or anti-B7-H3-antibody (Ab B7-H3 ). Control and targeted MB were tested for binding to hB7-H3-expressing cells (MS1 hB7-H3 ) under shear stress conditions. US imaging was performed with MB ABY-B7-H3 in an orthotopic mouse model of human MDA-MB-231 coimplanted with MS1 hB7-H3 or control MS1 WT cells and a transgenic mouse model of breast cancer development.Results: ABY B7-H3 specifically binds to MS1 hB7-H3 and murine-B7-H3-expressing monocytes. MB ABY-B7-H3 (8.5 AE 1.4 MB/cell) and MB Ab-B7-H3 (9.8 AE 1.3 MB/cell) showed significantly higher (P < 0.0001) binding to the MS1 hB7-H3 cells compared with control MB Non-targeted (0.5 AE 0.1 MB/cell) under shear stress conditions. In vivo, MB ABY-B7-H3 produced significantly higher (P < 0.04) imaging signal in orthotopic tumors coengrafted with MS1 hB7-H3 (8.4 AE 3.3 a.u.) compared with tumors with MS1 WT cells (1.4 AE 1.0 a.u.). In the transgenic mouse tumors, MB ABY-B7-H3 (9.6 AE 2.0 a.u.) produced higher (P < 0.0002) imaging signal compared with MB Non-targeted (1.3 AE 0.3 a.u.), whereas MB ABY-B7-H3 signal in normal mammary glands and tumors with B7-H3 blocking significantly reduced (P < 0.02) imaging signal.Conclusions: MB ABY-B7-H3 enhances B7-H3 molecular signal in breast tumors, improving cancer detection, while offering the advantages of a small size ligand and easier production for clinical imaging.

Yeast surface display empowers selection of protein binding ligands, typically using recombinant soluble antigens. However, ectodomain fragments of transmembrane targets may fail to recapitulate their true, membrane-bound form. Direct selections against adhered mammalian cells empower enrichment of genuine binders yet benefit from high target expression, robustly adherent mammalian cells, and nanomolar affinity ligands. This study evaluates a modified format with mammalian cells immobilized to magnetic beads; yeast-displayed fibronectin domain and affibody ligands of known affinities and cells with expression ranges of epidermal growth factor receptor (EGFR) and CD276 elucidate important parameters to ligand enrichment and yield in cell suspension panning with comparison to adherent panning. Cell suspension panning is hindered by significant background of nondisplaying yeast but exhibits yield advantages in model EGFR systems for a high affinity (K D = 2 nM) binder on cells with both high (10 6 per cell) target expression (9.6 ± 0.6% vs 3.2 ± 0.4%, p < 0.0001) and mid (10 5 ) target expression (2.3 ± 0.5% vs 0.41 ± 0.09%, p = 0.0008), as well as for a low affinity (K D > 600 nM) binder on high target expression cells (2.0 ± 0.5% vs 0.017 ± 0.005%; p = 0.001). Significant enrichment was observed for all EGFR systems except the low-affinity, high expression system. The CD276 system failed to provide significant enrichment, indicating that this technique may not be suitable for all targets. Collectively, this study highlights new approaches that yield successful enrichment of yeast-displayed ligands via panning on immobilized mammalian cells.

Selections of yeast-displayed ligands on mammalian cell monolayers benefit from high target expression and nanomolar affinity, which are not always available. Prior work extending the yeast–protein linker from 40 to 80 amino acids improved yield and enrichment but is hypothesized to be below the optimal length, prompting evaluation of an extended amino acid linker. A 641-residue linker provided enhanced enrichment with a 2-nM affinity fibronectin ligand and 105 epidermal growth factor receptors (EGFR) per cell (14 ± 2 vs. 8 ± 1, P = 0.008) and a &gt;600-nM affinity ligand, 106 EGFR per cell system (23 ± 7 vs. 0.8 ± 0.2, P = 0.004). Enhanced enrichment was also observed with a 310-nM affinity affibody ligand and 104 CD276 per cell, suggesting a generalizable benefit to other scaffolds and targets. Spatial modeling of the linker suggests that improved extracellular accessibility of ligand enables the observed enrichment under conditions not previously possible.