Predicting and Interpreting Protein Developability Via Transfer of Convolutional Sequence Representation.

Engineered proteins have emerged as novel diagnostics, therapeutics, and catalysts. Often, poor protein developability─quantified by expression, solubility, and stability─hinders utility. The ability to predict protein developability from amino acid sequence would reduce the experimental burden when selecting candidates. Recent advances in screening technologies enabled a high-throughput (HT) developability dataset for 105 of 1020 possible variants of protein ligand scaffold Gp2. In this work, we evaluate the ability of neural networks to learn a developability representation from a HT dataset and transfer this knowledge to predict recombinant expression beyond observed sequences. The model convolves learned amino acid properties to predict expression levels 44% closer to the experimental variance compared to a non-embedded control. Analysis of learned amino acid embeddings highlights the uniqueness of cysteine, the importance of hydrophobicity and charge, and the unimportance of aromaticity, when aiming to improve the developability of small proteins. We identify clusters of similar sequences with increased recombinant expression through nonlinear dimensionality reduction and we explore the inferred expression landscape via nested sampling. The analysis enables the first direct visualization of the fitness landscape and highlights the existence of evolutionary bottlenecks in sequence space giving rise to competing subpopulations of sequences with different developability. The work advances applied protein engineering efforts by predicting and interpreting protein scaffold expression from a limited dataset. Furthermore, our statistical mechanical treatment of the problem advances foundational efforts to characterize the structure of the protein fitness landscape and the amino acid characteristics that influence protein developability.

Consequences of poly(ethylene oxide) and poloxamer P188 on transcription in healthy and stressed myoblasts.

Poly(ethylene oxide) (PEO) and poloxamers, a class of poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers, have many personal and medical care applications, including the stabilization of stressed cellular membranes. Despite the widespread use, the cellular transcriptional response to these molecules is relatively unknown. C2C12 myoblasts, a model muscle cell, were subjected to short-term Poloxamer 188 (P188) and PEO181 (8,000 g/mol) treatment in culture. RNA was extracted and sequenced to quantify transcriptomic impact. The addition of moderate concentrations (14 µM) of either polymer to unstressed cells caused substantial differential gene expression, including at least twofold modulation of 357 and 588 genes, respectively. In addition, evaluation of the transcriptome response to osmotic stress without polymer treatment revealed dramatic change in RNA expression. Interestingly, the addition of polymer to stressed cells-at concentrations that provide physiological protection-did not yield a significant difference in expression of any gene relative to stress alone. Genome-scale expression analysis was corroborated by single-gene quantitative real-time PCR. Changes in protein expression were measured via western blot, which revealed partial alignment with the RNA results. Collectively, the significant changes to expression of multiple genes and resultant protein translation demonstrates an unexpectedly broad biochemical response to these polymers in healthy myoblasts in vitro. Meanwhile, the lack of substantial transcriptional response to polymer treatment in stressed cells highlights the physical nature of that protective mechanism.

Discovery of a Non-competitive TNFR1 Antagonist Affibody with Picomolar Monovalent Potency That Does Not Affect TNFR2 Function.

Tumor necrosis factor (TNF) is a key regulator of immune responses and plays a significant role in the initiation and maintenance of inflammation. Upregulation of TNF expression leads to several inflammatory diseases, such as Crohn's, ulcerative colitis, and rheumatoid arthritis. Despite the clinical success of anti-TNF treatments, the use of these therapies is limited because they can induce adverse side effects through inhibition of TNF biological activity, including blockade of TNF-induced immunosuppressive function of TNFR2. Using yeast display, we identified a synthetic affibody ligand (ABYTNFR1-1) with high binding affinity and specificity for TNFR1. Functional assays showed that the lead affibody potently inhibits TNF-induced NF-κB activation (IC50 of 0.23 nM) and, crucially, does not block the TNFR2 function. Additionally, ABYTNFR1-1 acts non-competitively─it does not block TNF binding or inhibit receptor-receptor interactions in pre-ligand-assembled dimers─thereby enhancing inhibitory robustness. The mechanism, monovalent potency, and affibody scaffold give this lead molecule uniquely strong potential as a therapeutic candidate for inflammatory diseases.

Engineered protein-small molecule conjugates empower selective enzyme inhibition.

Potent, specific ligands drive precision medicine and fundamental biology. Proteins, peptides, and small molecules constitute effective ligand classes. Yet greater molecular diversity would aid the pursuit of ligands to elicit precise biological activity against challenging targets. We demonstrate a platform to discover protein-small molecule (PriSM) hybrids to combine unique pharmacophore activities and shapes with constrained, efficiently engineerable proteins. In this platform, a fibronectin protein library is displayed on yeast with a single cysteine coupled to acetazolamide via a maleimide-poly(ethylene glycol) linker. Magnetic and flow cytometric sorts enrich specific binders to carbonic anhydrase isoforms. Isolated PriSMs exhibit potent, specific inhibition of carbonic anhydrase isoforms with efficacy superior to that of acetazolamide or protein alone, including an 80-fold specificity increase and 9-fold potency gain. PriSMs are engineered with multiple linker lengths, protein conjugation sites, and sequences against two different isoforms, which reveal platform flexibility and impacts of molecular designs. PriSMs advance the molecular diversity of efficiently engineerable ligands.

Extended yeast surface display linkers enhance the enrichment of ligands in direct mammalian cell selections.

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 >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.

Anti-CD19 CAR T cells potently redirected to kill solid tumor cells.

Successful CAR T cell therapy for the treatment of solid tumors requires exemplary CAR T cell expansion, persistence and fitness, and the ability to target tumor antigens safely. Here we address this constellation of critical attributes for successful cellular therapy by using integrated technologies that simplify development and derisk clinical translation. We have developed a CAR-CD19 T cell that secretes a CD19-anti-Her2 bridging protein. This cell therapy strategy exploits the ability of CD19-targeting CAR T cells to interact with CD19 on normal B cells to drive expansion, persistence and fitness. The secreted bridging protein potently binds to Her2-positive tumor cells, mediating CAR-CD19 T cell cytotoxicity in vitro and in vivo. Because of its short half-life, the secreted bridging protein will selectively accumulate at the site of highest antigen expression, ie. at the tumor. Bridging proteins that bind to multiple different tumor antigens have been created. Therefore, antigen-bridging CAR-CD19 T cells incorporate critical attributes for successful solid tumor cell therapy. This platform can be exploited to attack tumor antigens on any cancer.

Noncompetitive Allosteric Antagonism of Death Receptor 5 by a Synthetic Affibody Ligand.

Fatty acid-induced upregulation of death receptor 5 (DR5) and its cognate ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), promotes hepatocyte lipoapoptosis, which is a key mechanism in the progression of fatty liver disease. Accordingly, inhibition of DR5 signaling represents an attractive strategy for treating fatty liver disease. Ligand competition strategies are prevalent in tumor necrosis factor receptor antagonism, but recent studies have suggested that noncompetitive inhibition through perturbation of the receptor conformation may be a compelling alternative. To this end, we used yeast display and a designed combinatorial library to identify a synthetic 58-amino acid affibody ligand that specifically binds DR5. Biophysical and biochemical studies show that the affibody neither blocks TRAIL binding nor prevents the receptor-receptor interaction. Live-cell fluorescence lifetime measurements indicate that the affibody induces a conformational change in transmembrane dimers of DR5 and favors an inactive state of the receptor. The affibody inhibits apoptosis in TRAIL-treated Huh-7 cells, an in vitro model of fatty liver disease. Thus, this lead affibody serves as a potential drug candidate, with a unique mechanism of action, for fatty liver disease.

Magnetic Bead-Immobilized Mammalian Cells Are Effective Targets to Enrich Ligand-Displaying Yeast.

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 (KD = 2 nM) binder on cells with both high (106 per cell) target expression (9.6 ± 0.6% vs 3.2 ± 0.4%, p < 0.0001) and mid (105) target expression (2.3 ± 0.5% vs 0.41 ± 0.09%, p = 0.0008), as well as for a low affinity (KD > 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.

Influence of the Headgroup on the Interaction of Poly(ethylene oxide)-Poly(propylene oxide) Block Copolymers with Lipid Bilayers.

The lipid headgroup plays an important role in the association of polymers with lipid bilayer membranes. Herein, we report how a glycerol headgroup versus a choline headgroup affects the interaction of poly(ethylene oxide)-b-poly(propylene oxide) (PEO-PPO) block copolymers with lipid bilayer vesicles. Unilamellar vesicles composed of phosphatidylcholine and phosphatidylglycerol at various molar ratios were used as model membranes. The interactions between the block copolymers and lipid bilayers were quantified by pulsed-field gradient nuclear magnetic resonance (PFG-NMR) based on the distinctly different mobilities of free and bound polymers. All the investigated polymer species showed significantly higher binding with 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) sodium salt (POPG) liposomes than with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes, indicating stronger association with the glycerol headgroup compared to the choline headgroup. This effect did not become significant until the composition of mixed POPC/POPG liposomes contained more than 20 mol % POPG. A plausible explanation for the enhanced polymer binding with POPG invokes the role of hydrogen bonding between the glycerol headgroup and the ether moieties of the polymers.

Efficacy of Affibody-Based Ultrasound Molecular Imaging of Vascular B7-H3 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 ABYB7-H3 was confirmed with soluble and cell-surface B7-H3 by flow cytometry. MB were functionalized with ABYB7-H3 or anti-B7-H3-antibody (AbB7-H3). Control and targeted MB were tested for binding to hB7-H3-expressing cells (MS1hB7-H3) under shear stress conditions. US imaging was performed with MBABY-B7-H3 in an orthotopic mouse model of human MDA-MB-231 coimplanted with MS1hB7-H3 or control MS1WT cells and a transgenic mouse model of breast cancer development. RESULTS: ABYB7-H3 specifically binds to MS1hB7-H3 and murine-B7-H3-expressing monocytes. MBABY-B7-H3 (8.5 ± 1.4 MB/cell) and MBAb-B7-H3 (9.8 ± 1.3 MB/cell) showed significantly higher (P < 0.0001) binding to the MS1hB7-H3 cells compared with control MBNon-targeted (0.5 ± 0.1 MB/cell) under shear stress conditions. In vivo, MBABY-B7-H3 produced significantly higher (P < 0.04) imaging signal in orthotopic tumors coengrafted with MS1hB7-H3 (8.4 ± 3.3 a.u.) compared with tumors with MS1WT cells (1.4 ± 1.0 a.u.). In the transgenic mouse tumors, MBABY-B7-H3 (9.6 ± 2.0 a.u.) produced higher (P < 0.0002) imaging signal compared with MBNon-targeted (1.3 ± 0.3 a.u.), whereas MBABY-B7-H3 signal in normal mammary glands and tumors with B7-H3 blocking significantly reduced (P < 0.02) imaging signal. CONCLUSIONS: MBABY-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.

Fine Epitope Mapping of the CD19 Extracellular Domain Promotes Design.

The B-cell surface protein CD19 is present throughout the cell life cycle and is uniformly expressed in leukemias, making it a target for chimeric antigen receptor engineered immune cell therapy. Identifying the sequence dependence of the binding of CD19 to antibodies empowers fundamental study and more tailored development of CD19-targeted therapeutics. To identify the antibody-binding epitopes on CD19, we screened a comprehensive single-site saturation mutation library of the human CD19 extracellular domain to identify mutations detrimental to binding FMC63-the dominant CD19 antibody used in chimeric antigen receptor development-as well as 4G7-2E3 and 3B10, which have been used in various types of CD19 research and development. All three antibodies had partially overlapping, yet distinct, epitopes near the published epitope of antibody B43. The FMC63 conformational epitope spans spatially adjacent, but genetically distant, loops in exons 3 and 4. The 3B10 epitope is a linear peptide sequence that binds CD19 with 440 pM affinity. Along with their primary goal of epitope mapping, the mutational tolerance data also empowered additional CD19 variant design and analysis. A designed CD19 variant with all N-linked glycosylation sites removed successfully bound antibody in the yeast display context, which provides a lead for aglycosylated applications. Screening for thermally stable variants identified mutations to guide further CD19 stabilization for fusion protein applications and revealed evolutionary affinity-stability trade-offs. These fundamental insights into CD19 sequence-function relationships enhance our understanding of antibody-mediated CD19-targeted therapeutics.

Retargeting CD19 Chimeric Antigen Receptor T Cells via Engineered CD19-Fusion Proteins.

CD19-targeted chimeric antigen receptor (CAR) T-cells (CAR19s) show remarkable efficacy in the treatment of relapsed/refractory acute lymphocytic leukemia and Non-Hodgkin's lymphoma. However, the use of CAR T-cell therapy against CD19-negative hematological cancers and solid tumors has been challenging. We propose CD19-fusion proteins (CD19-FPs) to leverage the benefits of CAR19s while retargeting this validated cellular therapy to alternative tumor antigens. We demonstrate the ability of a fusion of CD19 extracellular domain (ECD) and a human epidermal growth factor receptor 2 (HER2) single-chain antibody fragment to retarget CAR19s to kill HER2+ CD19- tumor cells. To enhance the modularity of this technology, we engineered a more robust CD19 ECD via deep mutational scanning with yeast display and flow cytometric selections for improved protease resistance and anti-CD19 antibody binding. These enhanced CD19 ECDs significantly increase, and in some cases recover, fusion protein expression while maintaining target antigen affinity. Importantly, CD19-FPs retarget CAR19s to kill tumor cells expressing multiple distinct antigens, including HER2, CD20, EGFR, BCMA, and Clec12A as N- or C-terminal fusions and linked to both antibody fragments and fibronectin ligands. This study provides fundamental insights into CD19 sequence-function relationships and defines a flexible and modular platform to retarget CAR19s to any tumor antigen.

Influence of Cholesterol and Bilayer Curvature on the Interaction of PPO-PEO Block Copolymers with Liposomes.

Interactions of nonionic poly(ethylene oxide)- b-poly(propylene oxide) (PEO-PPO) block copolymers, known as Pluronics or poloxamers, with cell membranes have been widely studied for a host of biomedical applications. Herein, we report how cholesterol within phosphatidylcholine (POPC) lipid bilayer liposomes and bilayer curvature affects the binding of several PPO-PEO-PPO triblocks with varying PPO content and a tPPO-PEO diblock, where t refers to a tert-butyl end group. Pulsed-field-gradient NMR was employed to quantify the extent of copolymer associated with liposomes prepared with cholesterol concentrations ranging from 0 to 30 mol % relative to the total content of POPC and cholesterol and vesicle extrusion radii of 25, 50, or 100 nm. The fraction of polymer bound to the liposomes was extracted from NMR data on the basis of the very different mobilities of the bound and free polymers in aqueous solution. Cholesterol concentration was manipulated by varying the molar percentage of this sterol in the POPC bilayer preparation. The membrane curvature was varied by adjusting the liposome size through a conventional pore extrusion technique. Although the PPO content significantly influences the overall amount of block copolymer adsorbed to the liposome, we found that polymer binding decreases with increasing cholesterol concentration in a universal fashion, with the fraction of bound polymer dropping 10-fold between 0 and 30 mol % cholesterol relative to the total content of POPC and cholesterol. Increasing the bilayer curvature (decreasing the radius of the liposome) in the absence of cholesterol increases polymer binding between 2- and 4-fold over the range of liposome sizes studied. These results demonstrate that cholesterol plays a dominant role, and bilayer curvature has a less significant impact as the curvature decreases, on polymer-membrane association.

Affibody-Indocyanine Green Based Contrast Agent for Photoacoustic and Fluorescence Molecular Imaging of B7-H3 Expression in Breast Cancer.

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 (ABYB7-H3-ICG) showed significantly higher signal in mammary tumors compared to normal glands of mice. ABYB7-H3-ICG is a compelling scaffold for molecular sPA imaging for breast cancer detection.

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

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 ( Kd = 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.

Engineering an EGFR-binding Gp2 domain for increased hydrophilicity.

The Gp2 domain is a 45 amino-acid scaffold that has been evolved for specific, high-affinity binding towards multiple targets and was proven useful in molecular imaging and biological antagonism. It was hypothesized that Gp2 may benefit from increased hydrophilicity for improved physiological distribution as well as for physicochemical robustness. We identified seven exposed hydrophobic sites for hydrophilic mutations and experimentally evaluated single mutants, which yielded six mutations that do not substantially hinder expression, binding affinity or specificity (to epidermal growth factor receptor), and thermal stability. Eight combinations of these mutations improved hydrophilicity relative to the parental Gp2 clone as assessed by reverse-phase high-performance liquid chromatography (p < 0.05). Secondary structures and refolding abilities of the selected single mutants and all multimutants were unchanged relative to the parental ligand. A variant with five hydrophobic-to-hydrophilic mutations was identified with enhanced solubility as well as reasonable binding affinity ( K d = 53-63 nM), recombinant yield (1.3 ± 0.8 mg/L), and thermal stability ( T m = 53 ± 3°C). An alternative variant with a cluster of three leucine-to-hydrophilic mutations was identified with increased solubility, nominally increased binding affinity ( K d = 13-28 nM) and reasonable thermal stability ( T m = 54.0 ± 0.6°C) but reduced yield (0.4 ± 0.3 mg/L). In addition, a ≥7°C increase in the midpoint of thermal denaturation was observed in one of the single mutants (T21N). These mutants highlight the physicochemical tradeoffs associated with hydrophobic-to-hydrophilic mutation within a small protein, improve the solubility and hydrophilicity of an existent molecular imaging probe, and provide a more hydrophilic starting point for discovery of new Gp2 ligands towards additional targets.

Multivalent Ligand Binding to Cell Membrane Antigens: Defining the Interplay of Affinity, Valency, and Expression Density.

Nature uses multivalency to govern many biological processes. The development of macromolecular and cellular therapies has largely been dependent on engineering similar polyvalent interactions to enable effective targeting. Such therapeutics typically utilize high-affinity binding domains that have the propensity to recognize both antigen-overexpressing tumors and normal-expressing tissues, leading to "on-target, off-tumor" toxicities. One strategy to improve these agents' selectivity is to reduce the binding affinity, such that biologically relevant interactions between the therapeutic and target cell will only exist under conditions of high avidity. Preclinical studies have validated this principle of avidity optimization in the context of chimeric antigen receptor (CAR) T cells; however, a rigorous analysis of this approach in the context of soluble multivalent targeting scaffolds has yet to be undertaken. Using a modular protein nanoring capable of displaying ≤8 fibronectin domains with engineered specificity for a model antigen, epithelial cell adhesion molecule (EpCAM), this study demonstrates that binding affinity and ligand valency can be optimized to afford discrimination between EpCAMHigh (2.8-3.8 × 106 antigens/cell) and EpCAMLow (5.2 × 104 to 2.2 × 105 antigens/cell) tissues both in vitro and in vivo.

Constrained Combinatorial Libraries of Gp2 Proteins Enhance Discovery of PD-L1 Binders.

Engineered protein ligands are used for molecular therapy, diagnostics, and industrial biotechnology. The Gp2 domain is a 45-amino acid scaffold that has been evolved for specific, high-affinity binding to multiple targets by diversification of two solvent-exposed loops. Inspired by sitewise enrichment of select amino acids, including cysteine pairs, in earlier Gp2 discovery campaigns, we hypothesized that the breadth and efficiency of de novo Gp2 discovery will be aided by sitewise amino acid constraint within combinatorial library design. We systematically constructed eight libraries and comparatively evaluated their efficacy for binder discovery via yeast display against a panel of targets. Conservation of a cysteine pair at the termini of the first diversified paratope loop increased binder discovery 16-fold ( p < 0.001). Yet two other libraries with conserved cysteine pairs, within the second loop or an interloop pair, did not aid discovery thereby indicating site-specific impact. Via a yeast display protease resistance assay, Gp2 variants from the loop one cysteine pair library were 3.3 ± 2.1-fold ( p = 0.005) more stable than nonconstrained variants. Sitewise constraint of noncysteine residues-guided by previously evolved binders, natural Gp2 homology, computed stability, and structural analysis-did not aid discovery. A panel of binders to programmed death ligand 1 (PD-L1), a key target in cancer immunotherapy, were discovered from the loop 1 cysteine constraint library. Affinity maturation via loop walking resulted in strong, specific cellular PD-L1 affinity ( Kd = 6-9 nM).

Engineering Reversible Cell-Cell Interactions with Lipid Anchored Prosthetic Receptors.

Membrane-engineered cells displaying antigen-targeting ligands are useful as both scientific tools and clinical therapeutics. While genetically encoded artificial receptors have proven efficacious, their scope remains limited, as this approach is not amenable to all cell types and the modification is often permanent. Our group has developed a nongenetic method to rapidly, stably, and reversibly modify any cell membrane with a chemically self-assembled nanoring (CSAN) that can function as a prosthetic receptor. Bifunctional CSANs displaying epithelial cell adhesion molecule (EpCAM)-targeted fibronectin domains were installed on the cell membrane through hydrophobic insertion and remained stably bound for ≥72 h in vitro. These CSAN-labeled cells were capable of recognizing EpCAM-expressing target cells, forming intercellular interactions that were subsequently reversed by disassembling the nanoring with the FDA-approved antibiotic, trimethoprim. This study demonstrates the use of this system to engineer cell surfaces with prosthetic receptors capable of directing specific and reversible cell-cell interactions.

Engineered Charge Redistribution of Gp2 Proteins through Guided Diversity for Improved PET Imaging of Epidermal Growth Factor Receptor.

The Gp2 domain is a protein scaffold for synthetic ligand engineering. However, the native protein function results in a heterogeneous distribution of charge on the conserved surface, which may hinder further development and utility. We aim to modulate charge, without diminishing function, which is challenging in small proteins where each mutation is a significant fraction of protein structure. We constructed rationally guided combinatorial libraries with charge-neutralizing or charge-flipping mutations and sorted them, via yeast display and flow cytometry, for stability and target binding. Deep sequencing of functional variants revealed effective mutations both in clone-dependent contexts and broadly across binders to epidermal growth factor receptor (EGFR), insulin receptor, and immunoglobulin G. Functional mutants averaged 4.3 charge neutralizing mutations per domain while maintaining net negative charge. We evolved an EGFR-targeted Gp2 mutant that reduced charge density by 33%, maintained net charge, and improved charge distribution homogeneity while elevating thermal stability ( Tm = 87 ± 1 °C), improving binding specificity, and maintaining affinity ( Kd = 8.8 ± 0.6 nM). This molecule was conjugated with 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid for 64Cu chelation and evaluated for physiological distribution in mice with xenografted A431 (EGFRhigh) and MDA-MB-435 (EGFRlow) tumors. Excised tissue gamma counting and positron emission tomography/computed tomography imaging revealed good EGFRhigh tumor signal (4.7 ± 0.5%ID/g) at 2 h post-injection and molecular specificity evidenced by low uptake in EGFRlow tumors (0.6 ± 0.1%ID/g, significantly lower than for non-charge-modified Gp2, p = 0.01). These results provide charge mutations for an improved Gp2 framework, validate an effective approach to charge engineering, and advance performance of physiological EGFR targeting for molecular imaging.