The method of displaying recombinant proteins on the surface of Saccharomyces cerevisiae via genetic fusion to an abundant cell wall protein, a technology known as yeast surface display, or simply, ...yeast display, has become a valuable protein engineering tool for a broad spectrum of biotechnology and biomedical applications. This review focuses on the use of yeast display for engineering protein affinity, stability, and enzymatic activity. Strategies and examples for each protein engineering goal are discussed. Additional applications of yeast display are also briefly presented, including protein epitope mapping, identification of protein-protein interactions, and uses of displayed proteins in industry and medicine.
Display omitted
Growth factors are important morphogenetic proteins that instruct cell behavior and guide tissue repair and renewal. Although their therapeutic potential holds great promise in ...regenerative medicine applications, translation of growth factors into clinical treatments has been hindered by limitations including poor protein stability, low recombinant expression yield, and suboptimal efficacy. This review highlights current tools, technologies, and approaches to design integrated and effective growth factor-based therapies for regenerative medicine applications. The first section describes rational and combinatorial protein engineering approaches that have been utilized to improve growth factor stability, expression yield, biodistribution, and serum half-life, or alter their cell trafficking behavior or receptor binding affinity. The second section highlights elegant biomaterial-based systems, inspired by the natural extracellular matrix milieu, that have been developed for effective spatial and temporal delivery of growth factors to cell surface receptors. Although appearing distinct, these two approaches are highly complementary and involve principles of molecular design and engineering to be considered in parallel when developing optimal materials for clinical applications.
Growth factors are promising therapeutic proteins that have the ability to modulate morphogenetic behaviors, including cell survival, proliferation, migration and differentiation. However, the translation of growth factors into clinical therapies has been hindered by properties such as poor protein stability, low recombinant expression yield, and non-physiological delivery, which lead to suboptimal efficacy and adverse side effects. To address these needs, researchers are employing clever molecular and material engineering and design strategies to both improve the intrinsic properties of growth factors and effectively control their delivery into tissue. This review highlights examples of interdisciplinary tools and technologies used to augment the therapeutic potential of growth factors for clinical applications in regenerative medicine.
Protein-based therapeutics have been revolutionizing the oncology space since they first appeared in the clinic two decades ago. Unlike traditional small-molecule chemotherapeutics, protein biologics ...promote active targeting of cancer cells by binding to cell-surface receptors and other markers specifically associated with or overexpressed on tumors versus healthy tissue. While the first approved cancer biologics were monoclonal antibodies, the burgeoning field of protein engineering is spawning research on an expanded range of protein formats and modifications that allow tuning of properties such as target-binding affinity, serum half-life, stability, and immunogenicity. In this review we highlight some of these strategies and provide examples of modified and engineered proteins under development as preclinical and clinical-stage drug candidates for the treatment of cancer.
Selective protein degradation platforms have afforded new development opportunities for therapeutics and tools for biological inquiry. The first lysosome-targeting chimeras (LYTACs) targeted ...extracellular and membrane proteins for degradation by bridging a target protein to the cation-independent mannose-6-phosphate receptor (CI-M6PR). Here, we developed LYTACs that engage the asialoglycoprotein receptor (ASGPR), a liver-specific lysosome-targeting receptor, to degrade extracellular proteins in a cell-type-specific manner. We conjugated binders to a triantenerrary N-acetylgalactosamine (tri-GalNAc) motif that engages ASGPR to drive the downregulation of proteins. Degradation of epidermal growth factor receptor (EGFR) by GalNAc-LYTAC attenuated EGFR signaling compared to inhibition with an antibody. Furthermore, we demonstrated that a LYTAC consisting of a 3.4-kDa peptide binder linked to a tri-GalNAc ligand degrades integrins and reduces cancer cell proliferation. Degradation with a single tri-GalNAc ligand prompted site-specific conjugation on antibody scaffolds, which improved the pharmacokinetic profile of GalNAc-LYTACs in vivo. GalNAc-LYTACs thus represent an avenue for cell-type-restricted protein degradation.
Display omitted
•Knottins are small constrained peptides with ultra-high stability.•New binding epitopes can be introduced into natural knottins using protein engineering.•Knottins have been ...engineered to target a broad range of cancer-related targets.•Rapid tumor uptake and renal clearance make knottins ideal for molecular imaging.•Therapeutic pharmacokinetics and oral delivery are active research areas.
Inhibitor cystine-knots, also known as knottins, are a structural family of ultra-stable peptides with diverse functions. Knottins and related backbone-cyclized peptides called cyclotides contain three disulfide bonds connected in a particular arrangement that endows these peptides with high thermal, proteolytic, and chemical stability. Knottins have gained interest as candidates for non-invasive molecular imaging and for drug development as they can possess the pharmacological properties of small molecules and the target affinity and selectively of protein biologics. Naturally occurring knottins are clinically approved for treating chronic pain and GI disorders. Combinatorial methods are being used to engineer knottins that can bind to other clinically relevant targets in cancer, and inflammatory and cardiac disease. This review details recent examples of engineered knottin peptides; their use as molecular imaging agents, therapeutics, and drug delivery vehicles; modifications that can be introduced to improve peptide folding and bioactivity; and future perspectives and challenges in the field.
Our understanding of translation underpins our capacity to engineer living systems. The canonical start codon (AUG) and a few near-cognates (GUG, UUG) are considered as the 'start codons' for ...translation initiation in Escherichia coli. Translation is typically not thought to initiate from the 61 remaining codons. Here, we quantified translation initiation of green fluorescent protein and nanoluciferase in E. coli from all 64 triplet codons and across a range of DNA copy number. We detected initiation of protein synthesis above measurement background for 47 codons. Translation from non-canonical start codons ranged from 0.007 to 3% relative to translation from AUG. Translation from 17 non-AUG codons exceeded the highest reported rates of non-cognate codon recognition. Translation initiation from non-canonical start codons may contribute to the synthesis of peptides in both natural and synthetic biological systems.
Antibody–drug conjugates (ADCs) offer increased efficacy and reduced toxicity compared to systemic chemotherapy. Less attention has been paid to peptide–drug delivery, which has the potential for ...increased tumor penetration and facile synthesis. We report a knottin peptide–drug conjugate (KDC) and demonstrate that it can selectively deliver gemcitabine to malignant cells expressing tumor‐associated integrins. This KDC binds to tumor cells with low‐nanomolar affinity, is internalized by an integrin‐mediated process, releases its payload intracellularly, and is a highly potent inhibitor of brain, breast, ovarian, and pancreatic cancer cell lines. Notably, these features enable this KDC to bypass a gemcitabine‐resistance mechanism found in pancreatic cancer cells. This work expands the therapeutic relevance of knottin peptides to include targeted drug delivery, and further motivates efforts to expand the drug‐conjugate toolkit to include non‐antibody protein scaffolds.
Tied up in knottins: Tumor‐targeted knottin peptide‐drug conjugates (KDC) are significantly smaller than antibody–drug conjugates, allowing for facile synthesis and conjugation. A KDC bearing the nucleoside gemcitabine is internalized by an integrin‐mediated mechanism, releases its payload intracellularly, and is shown to be a highly potent inhibitor of several malignant cell lines.
Display omitted
•Ligands and receptors have been identified as important oncology targets.•Biodistribution, regulation, and function help dictate whether to target a ligand or receptor.•MAbs have ...been most often used to modulate the activity of ligands and receptors.•Ligands or receptors themselves are being developed as promising cancer therapeutics.•Protein engineering is often used to create optimized cancer therapeutics.
The biological importance and druggable properties of receptors and their cognate ligands have designated them as especially useful clinical targets. This significance continues to expand as new molecular insights underlying disease pathophysiology are uncovered. While both ligands and receptors have been exploited as drug targets, their differing biochemical properties require nuanced considerations for drug development, including where in the body they are located and how they are regulated on a cellular and molecular level. In this review we will discuss ligands and receptors as therapeutics targets, including their biodistribution and biological function. We provide examples of monoclonal antibodies (mAbs) used to modulate the activity of these targets, and discuss approaches for using engineered versions of ligands and receptors themselves for therapeutic intervention in cancer.
V-domain immunoglobulin (Ig) suppressor of T cell activation (VISTA) is an immune checkpoint protein that inhibits the T cell response against cancer. Similar to PD-1 and CTLA-4, a blockade of VISTA ...promotes tumor clearance by the immune system. Here, we report a 1.85 Å crystal structure of the elusive human VISTA extracellular domain, whose lack of homology necessitated a combinatorial MR-Rosetta approach for structure determination. We highlight features that make the VISTA immunoglobulin variable (IgV)-like fold unique among B7 family members, including two additional disulfide bonds and an extended loop region with an attached helix that we show forms a contiguous binding epitope for a clinically relevant anti-VISTA antibody. We propose an overlap of this antibody-binding region with the binding epitope for V-set and Ig domain containing 3 (VSIG3), a purported functional binding partner of VISTA. The structure and functional epitope presented here will help guide future drug development efforts against this important checkpoint target.
Display omitted
•The crystal structure of the human VISTA extracellular domain was determined•VISTA contains two unique disulfides and an extended C-C′ loop•The epitope of an inhibitor antibody was mapped to a three-residue surface•The antibody epitope was found to overlap with the VISTA-VSIG3 binding interface
Using a combinatorial MR-Rosetta approach, Mehta et al. solve the crystal structure of human V-domain immunoglobulin (Ig) suppressor of T cell activation (VISTA), an important checkpoint protein in cancer immunotherapy. The authors use yeast display to map the epitope of a clinical anti-VISTA antibody and demonstrate its overlap to the VISTA/V-set and Ig domain containing 3 (VSIG3) binding interface.
PDF
