enzymatic-modification-flow-cytometry-assessment-yeast-surface

Enzymatic Modification and Flow Cytometry Assessment of Yeast Surface Displayed Proteins

Yeast surface display is a genotype-phenotype linkage strategy that empowers high-throughput screening of protein function. Traditionally, yeast surface ...

University of South Florida

High-throughput protein selection methods are a cornerstone for protein engineering and pharmaceutical development. Traditional high-throughput selection strategies rely largely on recombinant antigen to generate target specificity. Though effective, this selection strategy can be limited by soluble target quality, particularly in the case of recombinant extracellular domains of transmembrane proteins. Recent advances in cell-based selection techniques provide new opportunities for improving the outcomes of ligand selection campaigns but can introduce technical challenges in maintaining antigen specificity due to the heterogeneity of biomacromolecule expression on the mammalian cell surface. Here, we describe a combination technique using recombinant antigen to "train" library target specificity followed by cell panning selections to ensure that isolated ligands bind cell-expressed target, as well as a facile microscopy technique for assessing target specificity on a clonal basis without the need to produce soluble ligand.

Ligand Selection by Combination of Recombinant and Cell Panning Selection Techniques.

The effector potency of chimeric antigen receptor (CAR) T cell therapeutic products is essential to their clinical antitumor responses, and potency monitoring is a critical quality control method for CAR T cell therapy platforms. While many in vitro assays enable high-throughput assessment of CAR T cell cytotoxicity, it has been challenging for these assays to reflect the in vivo therapeutic effect due to their nature as short-term methods that fail to recapitulate the high tumor burden environment. Here, we describe two in vitro co-culture methods to evaluate CAR T cell recursive killing potential at high tumor cell loads. In these assays, long-term cytotoxic function and proliferative capacity of CAR T cells are examined in vitro over 7days. Further, these assays are coupled with profiling CAR T cell expansion, cytokine production and phenotypes. These methods provide a facile approach to assess CAR T cell potency and to elucidate the functional variations across different CAR T cell products.

Potency monitoring of CAR T cells.

The ability to understand and characterize phosphorylation is important to the study of cell signaling and to synthetic biology approaches. Current methods for characterizing kinase-substrate interactions are limited by their inherently low throughput and the heterogeneity of samples analyzed. Recent advances in yeast surface display techniques provide new opportunities for studying individual kinase-substrate interactions in a stimulus-independent fashion. Here, we describe techniques for building substrate libraries into full-length domains of interest that, when co-localized intracellularly with individual kinases, result in the display of phosphorylated domains on the yeast surface, as well as fluorescence-activated cell sorting and magnetic bead selection techniques for enriching from these libraries based on phosphorylation state.

Tyrosine Phosphorylation Screening on the Yeast Surface by Magnetic Bead Selection and FACS.

Advancing chimeric antigen receptor (CAR)-engineered T cells for the treatment of solid tumors is a major focus in the field of cellular immunotherapy. Several hurdles have hindered similar CAR T cell clinical responses in solid tumors as seen in hematological malignancies. These challenges include on-target off-tumor toxicities, which have inspired efforts to optimize CARs for improved tumor antigen selectivity and overall safety. We recently developed a CAR T cell therapy targeting prostate stem cell antigen (PSCA) for prostate and pancreatic cancers, showing improved preclinical antitumor activity and T cell persistence by optimizing the intracellular co-stimulatory domain. Similar studies were undertaken to optimize HER2-directed CAR T cells with modifications to the intracellular co-stimulatory domain for selective targeting of breast cancer brain metastasis. In the present study, we evaluate various nonsignaling extracellular spacers in these CARs to further improve tumor antigen selectivity. Our findings suggest that length and structure of the extracellular spacer can dictate the ability of CARs to selectively target tumor cells with high antigen density, while sparing cells with low antigen density. This study contributes to CAR construct design considerations and expands our knowledge of tuning solid tumor CAR T cell therapies for improved safety and efficacy.

Article	Year
Ligand Selection by Combination of Recombinant and Cell Panning Selection Techniques. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 35482193	2022
Potency monitoring of CAR T cells. Methods in cell biology\| PubMed ID: 36653083	2023
Tyrosine Phosphorylation Screening on the Yeast Surface by Magnetic Bead Selection and FACS. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 37405653	2023
Nonsignaling extracellular spacer regulates tumor antigen selectivity of CAR T cells. Molecular therapy. Oncology\| PubMed ID: 38939825	2024

Lawrence A. Stern

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