Cellular-Based Selections Aid Yeast-Display Discovery of Genuine Cell-Binding Ligands: Targeting Oncology Vascular Biomarker CD276

Abstract

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 a recombinant target but not target-expressing 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 error-prone 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 introduce promising affibody molecules specific to CD276 for further applications.

Keywords: CD276; cell panning; ligand engineering; yeast display.

Figure 1.. Ligand discovery methods.

An affibody library and a fibronectin domain library were sorted for ligands that bound CD276 or Thy1 specifically. Libraries were sorted by five different schemes: 1) magnetic bead selection with recombinant extracellular domains followed by FACS with recombinant extracellular domains, 2) magnetic bead selection followed by FACS with detergent solubilized cell lysate, 3) magnetic bead selection followed by cell panning selection, 4) cell panning selection with magnetic bead depletion, and 5) cell panning selection.

Figure 2.. Enriched ligands evaluated for soluble extracellular domain and detergent-solubilized cell lysate binding.

Yeast-displayed ligand populations were enriched for binders against CD276 and Thy1 using soluble extracellular domains immobilized on magnetic beads followed by FACS with either soluble extracellular domains or detergent-solubilized cell lysates. Binding specificity for each enriched population was assessed by comparison to negative controls. For FACS with soluble extracellular domains, yeast were labeled with 100 nM soluble CD276 or Thy1-Fc extracellular domains (blue) or irrelevant negative control proteins (300 nM streptavidin for CD276 or 100 nM human IgG for Thy1-Fc; orange) (A-D). For selection of ligands with detergent-solubilized cell lysates against CD276, yeast were labeled with MS1-CD276 lysate (blue) or MS1-Thy1 lysate (orange). For selection of ligands with detergent-solubilized cell lysates against Thy1, yeast were labeled with MS1-Thy1 lysate (blue) or MS1-CD276 lysate (orange) (E-H).

Figure 3.. Enriched ligands evaluated for cellular binding.

Yeast-displayed ligand populations were enriched through sequential rounds of selection for binding against CD276 and Thy1 using cell panning methods. Rec. + Panning indicates recombinant target-coated magnetic bead selections followed by cellular panning. Depleted Panning and Panning indicate cellular panning with or without, respectively, depletion of non-specific binders via streptavidin-coated magnetic beads. Binding specificity for each enriched population was assessed by cell panning. Yeast were panned for binders on monolayers of target-positive MS1 cells (blue) or target-negative MS1 cells as a negative control (orange). In the depleted panning case, recovery of yeast from the first (white) and second (gray) magnetic beads was also quantified. Data represent single-run analyses during the course of each discovery campaign.

Figure 4.. Clonal assessment of specificity for cellular target by yeast-displayed cell panning.

(A) Forty-eight individual clones from each sorted population were panned for binding to target-expressing and target-negative MS1 cells and characterized by phase microscopy. Binding specificity was characterized as described in the text. Relative binding strength was classified by yeast density observed in a random microscopy field. Sequence diversity of specific binders was determined by Sanger sequencing random hits. Each box contains results for four scaffold/target pairs as detailed in the legend at the right. (B) Representative images of yeast displaying “+++”, “++”, “+”, and “-” clones are shown.

Figure 5.. Optimization of incubation time for yeast-displayed ligand enrichment.

Yeast displaying affibody clones LS (A) or HS (B) were panned for binding to adherent MS1-CD276 with varying incubation times. Recoveries are presented as the mean ± error deviation of 7–12 trials.

Figure 6.. Sequential depletion of non-specific binders with mammalian cell monolayers.

Mixtures of high-yield (A) or low-yield (B) CD276-specific, non-specific, and non-binding yeast were subjected to selection with 0, 2, 4, or 6 depletion steps followed by a single enrichment step. All individual enrichment ratios are shown for 3–10 trials as well as the average.

Figure 7.. Depletion of non-specific binders with mammalian cell pre-blocking.

Mixtures of high-yield (A) or low-yield (B) CD276-specific, non-specific, and non-binding yeast were incubated with either CD276-negative disadhered MS1-Thy1 cells (mammalian cell pre-block) or buffer only (no depletion) followed by incubation with MS1-CD276 cell monolayers. Enrichment ratios for CD276-specific (black), non-specific (gray), or non-binding (white) clones are shown as the mean ± standard deviation of 6–12 trials. (C). Specific (black) or non-target-specific (grey) clones were subjected to selection with or without mammalian cell pre-blocking for a total of 13 clone. Each point represents the yield of a single well of selection using pre-blocking normalized by a corresponding well selected without pre-blocking. Each clone was panned either in duplicate or triplicate.

Figure 8.. Evolved populations of yeast-displayed ligands show increasing binding to CD276 lysate while maintaining target specificity.

(A) Yeast populations collected after preliminary sorting on recombinant target beads (first column) or final sorting on target-positive lysate (second column) were labelled with 150 nM target cell lysate and analyzed for binding by flow cytometry. Yeast collected after sorting on target-positive lysate (third column) were labelled with 150 nM target-negative cell lysate and analyzed for specificity by flow cytometry. Substantial binding improvement can be observed between preliminary sorting and the final population, with low cross-reactivity to target-negative lysate. (B) A mixture of the merged library and triple-sorted single-helix library was labelled with 0.5 nM target lysate (left) or 50 nM target-negative lysate (right) and analyzed for binding by flow cytometry. The population shows significantly binding to target-positive lysate with minimal cross-reactivity to target-negative lysate.

Figure 9.. Characterization of parental and evolved CD276-binding Affibodies.

(A) affibody variants AC2, AC9, AC12, and AC16 were characterized for their binding affinity (K_D). Blue lettering indicates diversified residues in the helix-walking libraries. (B) Purified affibody variants AC2 (upper-left), AC9 (upper-right), AC12 (lower-left), and AC16 (lower-right) were used to label MS1-CD276 cells at the indicated concentrations. Binding was quantified by flow cytometry. The best-fit estimate of K_D and 68% confidence interval are indicated by solid and dashed lines, respectively. (C) Purified affibody AC12 was analyzed by circular dichroism spectroscopy in triplicate between 200 and 260 nm wavelengths before (solid) and after (dashed) thermal denaturation and cooling. (D) Purified affibody AC12 was scanned at a wavelength of 220 nm during heating from 20 to 98 °C (1 °C/min). The midpoint of thermal denaturation (T_m) was calculated by linear least-squares regression using a two-state protein unfolding model.

Figure 10.. Deep sequencing of the merged library after enrichment reveals substantially improved mutants compared to parental.

(A) Top unique sequences of the merged library listed by number of reads. Parental AC2 is supplied as a reference. Blue lettering indicates diversified residues in the helix-walking libraries and dashes indicate parental amino acids at the indicated position. Clone names are supplied for sequences that were pursued for characterization. (B) Sitewise amino acid enrichments for the merged library. Amino acid frequencies were calculated by grouping, counting, and quad-root dampening identical sequences. Values shown are change in amino acid frequency in sorted populations compared to theoretical amino acid diversity of the naïve library. Amino acids not allowed by library design are shown in grey, except in cases where they are substantially enriched or depleted.