Publications by authors named "Benjamin J Hackel"

62 Publications

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

PLoS One 2021 18;16(3):e0247701. Epub 2021 Mar 18.

Aleta Biotherapeutics, Natick, MA, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0247701PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7971483PMC
March 2021

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

Biochemistry 2020 10 30;59(40):3856-3868. Epub 2020 Sep 30.

Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota 55455, United States.

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 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.
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http://dx.doi.org/10.1021/acs.biochem.0c00529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7658720PMC
October 2020

Mining and Statistical Modeling of Natural and Variant Class IIa Bacteriocins Elucidate Activity and Selectivity Profiles across Species.

Appl Environ Microbiol 2020 10 28;86(22). Epub 2020 Oct 28.

Department of Chemical Engineering and Materials Science, University of Minnesota-Twin Cities, Minneapolis, Minnesota, USA

Class IIa bacteriocin antimicrobial peptides (AMPs) are a compelling alternative to current antimicrobials because of potential specific activity toward antibiotic-resistant bacteria, including vancomycin-resistant enterococci. Engineering of these molecules would be enhanced by a better understanding of AMP sequence-activity relationships to improve efficacy and limit effects of off-target activity. Toward this goal, we experimentally evaluated 210 natural and variant class IIa bacteriocins for antimicrobial activity against six strains of enterococci. Inhibitory activity was ridge regressed to AMP sequence to predict performance, achieving an area under the curve of 0.70 and demonstrating the potential of statistical models for identifying and designing AMPs. Active AMPs were individually produced and evaluated against eight enterococcus strains and four strains to elucidate trends in susceptibility. It was determined that the mannose phosphotransferase system (manPTS) sequence is informative of susceptibility to class IIa bacteriocins, yet other factors, such as membrane composition, also contribute strongly to susceptibility. A broadly potent bacteriocin variant (lactocin DT1) from a genome was identified as the only variant with inhibitory activity toward all tested strains, while a novel enterocin variant (DT2) from an genome demonstrated specificity toward strains. Eight AMPs were evaluated for proteolytic stability to trypsin, chymotrypsin, and pepsin, and three C-terminal disulfide-containing variants, including divercin V41, were identified as compelling for future studies, given their high potency and proteolytic stability. Class IIa bacteriocin antimicrobial peptides (AMPs), an alternative to traditional small-molecule antibiotics, are capable of selective activity toward various Gram-positive bacteria, limiting negative side effects associated with broad-spectrum activity. This selective activity is achieved through targeting of the mannose phosphotransferase system (manPTS) of a subset of Gram-positive bacteria, although factors affecting this mechanism are not entirely understood. Peptides identified from genomic data, as well as variants of previously characterized AMPs, can offer insight into how peptide sequence affects activity and selectivity. The experimental methods presented here identify promising potent and selective bacteriocins for further evaluation, highlight the potential of simple computational modeling for prediction of AMP performance, and demonstrate that factors beyond manPTS sequence affect bacterial susceptibility to class IIa bacteriocins.
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http://dx.doi.org/10.1128/AEM.01646-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642072PMC
October 2020

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

ACS Comb Sci 2020 05 27;22(5):274-284. Epub 2020 Apr 27.

Department of Chemical Engineering and Materials Science, University of Minnesota-Twin Cities, 421 Washington Avenue Southeast, 356 Amundson Hall, Minneapolis, Minnesota 55455, United States.

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 ( = 2 nM) binder on cells with both high (10 per cell) target expression (9.6 ± 0.6% vs 3.2 ± 0.4%, < 0.0001) and mid (10) target expression (2.3 ± 0.5% vs 0.41 ± 0.09%, = 0.0008), as well as for a low affinity ( > 600 nM) binder on high target expression cells (2.0 ± 0.5% vs 0.017 ± 0.005%; = 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.
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http://dx.doi.org/10.1021/acscombsci.0c00036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263704PMC
May 2020

Spatial Distribution of PEO-PPO-PEO Block Copolymer and PEO Homopolymer in Lipid Bilayers.

Langmuir 2020 04 27;36(13):3393-3403. Epub 2020 Mar 27.

Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota 55455, United States.

Maintaining the integrity of cell membranes is indispensable for cellular viability. Poloxamer 188 (P188), a poly(ethylene oxide)--poly(propylene oxide)--poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymer with a number-average molecular weight of 8700 g/mol and containing 80% by mass PEO, protects cell membranes from various external injuries and has the potential to be used as a therapeutic agent in diverse applications. The membrane protection mechanism associated with P188 is intimately connected with how this block copolymer interacts with the lipid bilayer, the main component of a cell membrane. Here, we report the distribution of P188 in a model lipid bilayer comprising 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) using neutron reflectivity (NR) and atomic force microscopy (AFM). We also investigated the association of a PEO homopolymer (PEO8.4K; = 8400 g/mol) that does not protect living cell membranes. These experiments were conducted following incubation of a 4.5 mmol/L polymer solution in a buffer that mimics physiological conditions with supported POPC bilayer membranes followed by washing with the aqueous medium. In contrast to previous reports, which dealt with P188 and PEO in salt-free solutions, both P188 and PEO8.4K penetrate into the inner portion of the lipid bilayer as revealed by NR, with approximately 30% by volume occupancy across the membrane without loss of bilayer structural integrity. These results indicate that PEO is the chemical moiety that principally drives P188 binding to bilayer membranes. No defects or phase-separated domains were observed in either P188- or PEO8.4K-incubated lipid bilayers when examined by AFM, indicating that polymer chains mingle homogeneously with lipid molecules in the bilayer. Remarkably, the breakthrough force required for penetration of the AFM tip through the bilayer membrane is unaffected by the presence of the large amount of P188 and PEO8.4K.
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http://dx.doi.org/10.1021/acs.langmuir.9b03208DOI Listing
April 2020

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

J Phys Chem B 2020 03 16;124(12):2417-2424. Epub 2020 Mar 16.

Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota 55455, United States.

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)--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--glycero-3-phospho-(1'-rac-glycerol) sodium salt (POPG) liposomes than with 1-palmitoyl-2-oleoyl--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.
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http://dx.doi.org/10.1021/acs.jpcb.0c00553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7302449PMC
March 2020

Efficacy of Affibody-Based Ultrasound Molecular Imaging of Vascular B7-H3 for Breast Cancer Detection.

Clin Cancer Res 2020 05 10;26(9):2140-2150. Epub 2020 Jan 10.

Department of Radiology, Stanford University School of Medicine, Stanford, California.

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 was confirmed with soluble and cell-surface B7-H3 by flow cytometry. MB were functionalized with ABY or anti-B7-H3-antibody (Ab). Control and targeted MB were tested for binding to hB7-H3-expressing cells (MS1) under shear stress conditions. US imaging was performed with MB in an orthotopic mouse model of human MDA-MB-231 coimplanted with MS1 or control MS1 cells and a transgenic mouse model of breast cancer development.

Results: ABY specifically binds to MS1 and murine-B7-H3-expressing monocytes. MB (8.5 ± 1.4 MB/cell) and MB (9.8 ± 1.3 MB/cell) showed significantly higher ( < 0.0001) binding to the MS1 cells compared with control MB (0.5 ± 0.1 MB/cell) under shear stress conditions. , MB produced significantly higher ( < 0.04) imaging signal in orthotopic tumors coengrafted with MS1 (8.4 ± 3.3 a.u.) compared with tumors with MS1 cells (1.4 ± 1.0 a.u.). In the transgenic mouse tumors, MB (9.6 ± 2.0 a.u.) produced higher ( < 0.0002) imaging signal compared with MB (1.3 ± 0.3 a.u.), whereas MB signal in normal mammary glands and tumors with B7-H3 blocking significantly reduced ( < 0.02) imaging signal.

Conclusions: MB 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.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-1655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7196517PMC
May 2020

Computationally Aided Discovery of LysEFm5 Variants with Improved Catalytic Activity and Stability.

Appl Environ Microbiol 2020 02 3;86(4). Epub 2020 Feb 3.

Department of Chemical Engineering and Materials Science, University of Minnesota-Twin Cities, Minneapolis, Minnesota, USA

Bacteriophage-derived lysin proteins are potentially effective antimicrobials that would benefit from engineered improvements to their bioavailability and specific activity. Here, the catalytic domain of LysEFm5, a lysin with activity against vancomycin-resistant (VRE), was subjected to site-saturation mutagenesis at positions whose selection was guided by sequence and structural information from homologous proteins. A second-order Potts model with parameters inferred from large sets of homologous sequence information was used to predict the average change in the statistical fitness for mutant libraries with diversity at pairs of sites within the secondary catalytic shell. Guided by the statistical fitness, nine double mutant saturation libraries were created and plated on agar containing autoclaved VRE to quickly identify and segregate catalytically active (halo-forming) and inactive (non-halo-forming) variants. High-throughput DNA sequencing of 873 unique variants showed that the statistical fitness was predictive of the retention or loss of catalytic activity (area under the curve [AUC], 0.840 to 0.894), with the inclusion of more diverse sequences in the starting multiple-sequence alignment improving the classification accuracy when pairwise amino acid couplings (epistasis) were considered. Of eight random halo-forming variants selected for more sensitive testing, one showed a 1.8 (±0.4)-fold improvement in specific activity and an 11.5 ± 0.8°C increase in melting temperature compared to those of the wild type. Our results demonstrate that a computationally informed approach employing homologous protein information coupled with a mid-throughput screening assay allows for the expedited discovery of lysin variants with improved properties. Broad-spectrum antibiotics can indiscriminately kill most bacteria, including commensal species that are a part of the normal human flora. This can potentially lead to the proliferation of drug-resistant bacteria upon elimination of competing species and to unwanted autoimmune effects in patients. Bacteriophage-derived lysin proteins are an alternative to conventional antibiotics that have coevolved alongside specific bacterial hosts. Lysins are capable of targeting conserved substrates in the bacterial cell wall essential for its viability. To engineer these proteins to exhibit improved therapeutically relevant properties, homology-guided statistical approaches can be used to identify compelling sites for mutation and to quantify the functional constraints acting on these sites to direct mutagenic library creation. The platform described herein couples this informed approach with a visual plate assay that can be used to simultaneously screen hundreds of mutants for catalytic activity, allowing for the streamlined identification of improved lysin variants.
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http://dx.doi.org/10.1128/AEM.02051-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997734PMC
February 2020

Fine Epitope Mapping of the CD19 Extracellular Domain Promotes Design.

Biochemistry 2019 12 21;58(48):4869-4881. Epub 2019 Nov 21.

Department of Chemical Engineering and Materials Science , University of Minnesota-Twin Cities , 421 Washington Avenue SE , Minneapolis , Minnesota 55455 , United States.

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.
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http://dx.doi.org/10.1021/acs.biochem.9b00808DOI Listing
December 2019

Ligand Engineering via Yeast Surface Display and Adherent Cell Panning.

Methods Mol Biol 2020 ;2070:303-320

Department of Chemical Engineering and Materials Science, University of Minnesota-Twin Cities, Minneapolis, MN, USA.

High-throughput ligand discovery and evolution-via genotype-phenotype linkage strategies-empower molecularly targeted therapy, diagnostics, and fundamental science. Maintaining high-quality target antigen in these selections, particularly for membrane targets, is often a technical challenge. Panning yeast-displayed ligand libraries on intact mammalian cells expressing the molecular target has emerged as an effective strategy. Herein we describe the techniques used to select target-binding ligands via this approach including the use of target-negative cells to deplete non-specific binders and avidity reduction to preferentially select high-affinity ligands.
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http://dx.doi.org/10.1007/978-1-4939-9853-1_17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996137PMC
December 2020

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

Mol Pharm 2019 08 15;16(8):3544-3558. Epub 2019 Jul 15.

Aleta Biotherapeutics , 27 Strathmore Road , Natick , Massachusetts 01760 , United States.

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.
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http://dx.doi.org/10.1021/acs.molpharmaceut.9b00418DOI Listing
August 2019

Identification and elucidation of proline-rich antimicrobial peptides with enhanced potency and delivery.

Biotechnol Bioeng 2019 10 21;116(10):2439-2450. Epub 2019 Jul 21.

Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota.

Proline-rich antimicrobial peptides (PrAMPs) kill bacteria via a nonlytic mechanism in which they permeate through the outer membrane, utilize protein-mediated transport across the inner membrane, and target the ribosome to inhibit protein synthesis. We previously reported that substitutions of oncocin ( ) with a pair of cationic residues improved the antimicrobial activity. In this study, we applied the design protocol to three other PrAMPs: apidaecin-1b, pyrrhocoricin, and bactenecin 7(1-16) and found that the substitutions (R4K and I8K/R) for apidaecin-1b improve the activity by twofold (p < .05) against nonpathogenic Escherichia coli. Moreover, the substitutions (L7K/R and R14K) for pyrrhocoricin improve the activity by 2-10-fold (p < .05) against some strains of E. coli and Salmonella Typhimurium. We also performed activity tests against inner membrane protein (SbmA or YgdD) knockout strains. The result is consistent with previous studies that SbmA is the major transporter for apidaecin-1b and pyrrhocoricin derivatives. However, bactenecin 7(1-16) functions independently of these transporters. In addition, several apidaecin-1b derivatives exhibit enhanced activity relative to wild-type only in the absence of SbmA, which is consistent with mutations that enhance transport across the inner membrane. A high performance liquid chromatography-based kinetic assay for cellular association and internalization demonstrates that the selected cationic mutations can improve cellular association in minimal media, but this enhanced association is not required for increased activity, which suggests the importance of inner membrane transport. These functional studies on cationic mutants of PrAMPs advance understanding of potency and mechanism and advance the ability to engineer improved antimicrobials as evidenced by the identification of the pyrrhocoricin mutant (L7R and R14K) with 10-fold elevated potency against pathogenic E. coli.
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http://dx.doi.org/10.1002/bit.27092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726534PMC
October 2019

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

Langmuir 2019 06 22;35(22):7231-7241. Epub 2019 May 22.

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.
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http://dx.doi.org/10.1021/acs.langmuir.9b00572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7050598PMC
June 2019

Systematic Mutagenesis of Oncocin Reveals Enhanced Activity and Insights into the Mechanisms of Antimicrobial Activity.

Mol Syst Des Eng 2018 Dec 8;3(6):930-941. Epub 2018 Oct 8.

Department of Chemical Engineering and Materials Science, University of Minnesota - Twin Cities, Minneapolis, MN 55455, USA.

Oncocin is a proline-rich antimicrobial peptide that inhibits protein synthesis by binding to the bacterial ribosome. In this work, the antimicrobial activity of oncocin was improved by systematic peptide mutagenesis and activity evaluation. We found that a pair of cationic substitutions (P4K and L7K/R) improves the activity by 2-4 fold (p<0.05) against multiple Gram-negative bacteria. An transcription / translation assay indicated that the increased activity was not because of stronger ribosome binding. Rather a cellular internalization assay revealed a higher internalization rate for the optimized analogs thereby suggesting a mechanism to increase potency. In addition, we found that the optimized peptides' benefit is dependent upon nutrient-depleted media conditions. The molecular design and characterization strategies have broad potential for development of antimicrobial peptides.
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http://dx.doi.org/10.1039/C8ME00051DDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6519479PMC
December 2018

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

Bioconjug Chem 2019 06 24;30(6):1677-1689. Epub 2019 May 24.

Department of Radiology , Stanford University , Stanford , California 94305 , United States.

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-ICG) showed significantly higher signal in mammary tumors compared to normal glands of mice. ABY-ICG is a compelling scaffold for molecular sPA imaging for breast cancer detection.
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http://dx.doi.org/10.1021/acs.bioconjchem.9b00239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6745046PMC
June 2019

Cardiac Muscle Membrane Stabilization in Myocardial Reperfusion Injury.

JACC Basic Transl Sci 2019 Apr 29;4(2):275-287. Epub 2019 Apr 29.

Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, Minnesota.

The phospholipid bilayer membrane that surrounds each cell in the body represents the first and last line of defense for preserving overall cell viability. In several forms of cardiac and skeletal muscle disease, deficits in the integrity of the muscle membrane play a central role in disease pathogenesis. In Duchenne muscular dystrophy, an inherited and uniformly fatal disease of progressive muscle deterioration, muscle membrane instability is the primary cause of disease, including significant heart disease, for which there is no cure or highly effective treatment. Further, in multiple clinical forms of myocardial ischemia-reperfusion injury, the cardiac sarcolemma is damaged and this plays a key role in disease etiology. In this review, cardiac muscle membrane stability is addressed, with a focus on synthetic block copolymers as a unique chemical-based approach to stabilize damaged muscle membranes. Recent advances using clinically relevant small and large animal models of heart disease are discussed. In addition, mechanistic insights into the copolymer-muscle membrane interface, featuring atomistic, molecular, and physiological structure-function approaches are highlighted. Collectively, muscle membrane instability contributes significantly to morbidity and mortality in prominent acquired and inherited heart diseases. In this context, chemical-based muscle membrane stabilizers provide a novel therapeutic approach for a myriad of heart diseases wherein the integrity of the cardiac muscle membrane is at risk.
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http://dx.doi.org/10.1016/j.jacbts.2019.01.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6488758PMC
April 2019

Validation and Stabilization of a Prophage Lysin of Clostridium perfringens by Using Yeast Surface Display and Coevolutionary Models.

Appl Environ Microbiol 2019 05 2;85(10). Epub 2019 May 2.

Department of Chemical Engineering and Materials Science, University of Minnesota-Twin Cities, Minneapolis, Minnesota, USA

Bacteriophage lysins are compelling antimicrobial proteins whose biotechnological utility and evolvability would be aided by elevated stability. Lysin catalytic domains, which evolved as modular entities distinct from cell wall binding domains, can be classified into one of several families with highly conserved structure and function, many of which contain thousands of annotated homologous sequences. Motivated by the quality of these evolutionary data, the performance of generative protein models incorporating coevolutionary information was analyzed to predict the stability of variants in a collection of 9,749 multimutants across 10 libraries diversified at different regions of a putative lysin from a prophage region of a genome. Protein stability was assessed via a yeast surface display assay with accompanying high-throughput sequencing. Statistical fitness of mutant sequences, derived from second-order Potts models inferred with different levels of sequence homolog information, was predictive of experimental stability with areas under the curve (AUCs) ranging from 0.78 to 0.85. To extract an experimentally derived model of stability, a logistic model with site-wise score contributions was regressed on the collection of multimutants. This achieved a cross-validated classification performance of 0.95. Using this experimentally derived model, 5 designs incorporating 5 or 6 mutations from multiple libraries were constructed. All designs retained enzymatic activity, with 4 of 5 increasing the melting temperature and with the highest-performing design achieving an improvement of +4°C. Bacteriophage lysins exhibit high specificity and activity toward host bacteria with which the phage coevolved. These properties of lysins make them attractive for use as antimicrobials. Although there has been significant effort to develop platforms for rapid lysin engineering, there have been numerous shortcomings when pursuing the ultrahigh throughput necessary for the discovery of rare combinations of mutations to improve performance. In addition to validation of a putative lysin and stabilization thereof, the experimental and computational methods presented here offer a new avenue for improving protein stability and are easily scalable to analysis of tens of millions of mutations in single experiments.
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http://dx.doi.org/10.1128/AEM.00054-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6498168PMC
May 2019

Biophysical Characterization Platform Informs Protein Scaffold Evolvability.

ACS Comb Sci 2019 04 18;21(4):323-335. Epub 2019 Feb 18.

Department of Chemical Engineering and Materials Science , University of Minnesota-Twin Cities , 421 Washington Avenue Southeast, 356 Amundson Hall , Minneapolis , Minnesota 55455 , United States.

Evolving specific molecular recognition function of proteins requires strategic navigation of a complex mutational landscape. Protein scaffolds aid evolution via a conserved platform on which a modular paratope can be evolved to alter binding specificity. Although numerous protein scaffolds have been discovered, the underlying properties that permit binding evolution remain unknown. We present an algorithm to predict a protein scaffold's ability to evolve novel binding function based upon computationally calculated biophysical parameters. The ability of 17 small proteins to evolve binding functionality across seven discovery campaigns was determined via magnetic activated cell sorting of 10 yeast-displayed protein variants. Twenty topological and biophysical properties were calculated for 787 small protein scaffolds and reduced into independent components. Regularization deduced which extracted features best predicted binding functionality, providing a 4/6 true positive rate, a 9/11 negative predictive value, and a 4/6 positive predictive value. Model analysis suggests a large, disconnected paratope will permit evolved binding function. Previous protein engineering endeavors have suggested that starting with a highly developable (high producibility, stability, solubility) protein will offer greater mutational tolerance. Our results support this connection between developability and evolvability by demonstrating a relationship between protein production in the soluble fraction of Escherichia coli and the ability to evolve binding function upon mutation. We further explain the necessity for initial developability by observing a decrease in proteolytic stability of protein mutants that possess binding functionality over nonfunctional mutants. Future iterations of protein scaffold discovery and evolution will benefit from a combination of computational prediction and knowledge of initial developability properties.
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http://dx.doi.org/10.1021/acscombsci.8b00182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6458986PMC
April 2019

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

ACS Comb Sci 2019 03 24;21(3):207-222. Epub 2019 Jan 24.

Department of Chemical Engineering and Materials Science , University of Minnesota-Twin Cities , Minneapolis , Minnesota 55455 , United States.

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 = 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.
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http://dx.doi.org/10.1021/acscombsci.8b00156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411437PMC
March 2019

Improved mutant function prediction via PACT: Protein Analysis and Classifier Toolkit.

Bioinformatics 2019 08;35(16):2707-2712

Department of Chemical Engineering and Materials Science, University of Minnesota, Twin Cities, Minneapolis, MN, USA.

Motivation: Deep mutational scanning experiments have enabled the measurement of the sequence-function relationship for thousands of mutations in a single experiment. The Protein Analysis and Classifier Toolkit (PACT) is a Python software package that marries the fitness metric of a given mutation within these experiments to sequence and structural features enabling downstream analyses. PACT enables the easy development of user sharable protocols for custom deep mutational scanning experiments as all code is modular and reusable between protocols. Protocols for mutational libraries with single or multiple mutations are included. To exemplify its utility, PACT assessed two deep mutational scanning datasets that measured the tradeoff of enzyme activity and enzyme stability.

Results: PACT efficiently evaluated classifiers that predict protein mutant function tested on deep mutational scanning screens. We found that the classifiers with the lowest false positive and highest true positive rate assesses sequence homology, contact number and if mutation involves proline.

Availability And Implementation: PACT and the processed datasets are distributed freely under the terms of the GPL-3 license. The source code is available at GitHub (https://github.com/JKlesmith/PACT).

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/bty1042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6691332PMC
August 2019

Engineering an EGFR-binding Gp2 domain for increased hydrophilicity.

Biotechnol Bioeng 2019 03 30;116(3):526-535. Epub 2018 Dec 30.

Department of Chemical Engineering and Materials Science, University of Minnesota - Twin Cities, Minneapolis, Minnesota.

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  = 53-63 nM), recombinant yield (1.3 ± 0.8 mg/L), and thermal stability ( T  = 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  = 13-28 nM) and reasonable thermal stability ( T  = 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.
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http://dx.doi.org/10.1002/bit.26893DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6358468PMC
March 2019

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

J Am Chem Soc 2019 01 17;141(1):251-261. Epub 2018 Dec 17.

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 EpCAM (2.8-3.8 × 10 antigens/cell) and EpCAM (5.2 × 10 to 2.2 × 10 antigens/cell) tissues both in vitro and in vivo.
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http://dx.doi.org/10.1021/jacs.8b09198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6520051PMC
January 2019

Multispecies activity screening of microcin J25 mutants yields antimicrobials with increased specificity toward pathogenic Salmonella species relative to human commensal Escherichia coli.

Biotechnol Bioeng 2018 10 20;115(10):2394-2404. Epub 2018 Jul 20.

Department of Chemical Engineering and Materials Science, University of Minnesota-Twin Cities, Minneapolis, Minnesota.

Modern large-scale agricultural practices that incorporate high density farming with subtherapeutic antibiotic dosing are considered a major contributor to the rise of antibiotic-resistant bacterial infections of humans with species of Salmonella being a leading agriculture-based bacterial infection. Microcin J25, a potent and highly stable antimicrobial peptide active against Enterobacteriaceae, is a candidate antimicrobial against multiple Salmonella species. Emerging evidence supports the hypothesis that the composition of the microbiota of the gastrointestinal tract prevents a variety of diseases by preventing infectious agents from proliferating. Reducing clearance of off-target bacteria may decrease susceptibility to secondary infection. Of the Enterobacteriaceae susceptible to microcin J25, Escherichia coli are the most abundant within the human gut. To explore the modulation of specificity, a collection of 207 mutants encompassing 12 positions in both the ring and loop of microcin J25 was built and tested for activity against Salmonella and E. coli strains. As has been found previously, mutational tolerance of ring residues was lower than loop residues, with 22% and 51% of mutations, respectively, retaining activity toward at least one target within the target organism test panel. The multitarget screening elucidated increased mutational tolerance at position G2, G3, and G14 than previously identified in panels composed of single targets. Multiple mutations conferred differential response between the different targets. Examination of specificity differences between mutants found that 30% showed significant improvements to specificity toward any of the targets. Generation and testing of a combinatorial library designed from the point-mutant study revealed that microcin J25 reduces off-target activity toward commensal human-derived E. coli isolates by 81% relative to Salmonella enterica serovar Enteritidis. These in vitro specificity improvements are likely to improve in vivo treatment efficacy by reducing clearance of commensal bacteria in the gastrointestinal tract of hosts.
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http://dx.doi.org/10.1002/bit.26772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6185767PMC
October 2018

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

ACS Comb Sci 2018 07 5;20(7):423-435. Epub 2018 Jun 5.

University of Minnesota-Twin Cities , Department of Chemical Engineering and Materials Science , 421 Washington Avenue Southeast , Minneapolis , Minnesota 55455 , United States.

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 ( K = 6-9 nM).
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http://dx.doi.org/10.1021/acscombsci.8b00010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6051759PMC
July 2018

Surface Plasmon Resonance Study of the Binding of PEO-PPO-PEO Triblock Copolymer and PEO Homopolymer to Supported Lipid Bilayers.

Langmuir 2018 06 1;34(23):6703-6712. Epub 2018 Jun 1.

Poloxamer 188 (P188), a poly(ethylene oxide)- b-poly(propylene oxide)- b-poly(ethylene oxide) triblock copolymer, protects cell membranes against various external stresses, whereas poly(ethylene oxide) (PEO; 8600 g/mol) homopolymer lacks protection efficacy. As part of a comprehensive effort to elucidate the protection mechanism, we used surface plasmon resonance (SPR) to obtain direct evidence of binding of the polymers onto supported lipid bilayers. Binding kinetics and coverage of P188 and PEO were examined and compared. Most notably, PEO exhibited membrane association comparable to that of P188, evidenced by comparable association rate constants and coverage. This result highlights the need for additional mechanistic understanding beyond simple membrane association to explain the differential efficacy of P188 in therapeutic applications.
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http://dx.doi.org/10.1021/acs.langmuir.8b00873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6055929PMC
June 2018

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

Bioconjug Chem 2018 05 5;29(5):1646-1658. Epub 2018 Apr 5.

University of Minnesota - Twin Cities , Department of Chemical Engineering and Materials Science , 421 Washington Avenue SE , Minneapolis , Minnesota 55455 , United States.

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 ( T = 87 ± 1 °C), improving binding specificity, and maintaining affinity ( K = 8.8 ± 0.6 nM). This molecule was conjugated with 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid for Cu chelation and evaluated for physiological distribution in mice with xenografted A431 (EGFR) and MDA-MB-435 (EGFR) tumors. Excised tissue gamma counting and positron emission tomography/computed tomography imaging revealed good EGFR tumor signal (4.7 ± 0.5%ID/g) at 2 h post-injection and molecular specificity evidenced by low uptake in EGFR 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.
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http://dx.doi.org/10.1021/acs.bioconjchem.8b00144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6051758PMC
May 2018

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

Bioconjug Chem 2018 04 23;29(4):1291-1301. Epub 2018 Mar 23.

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.
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http://dx.doi.org/10.1021/acs.bioconjchem.8b00058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5937253PMC
April 2018

Evaluation of affibody charge modification identified by synthetic consensus design in molecular PET imaging of epidermal growth factor receptor.

Mol Syst Des Eng 2018 Feb 9;3(1):171-182. Epub 2017 Nov 9.

Department of Chemical Engineering and Materials Science, University of Minnesota - Twin Cities, Minneapolis, MN 55455.

Tumor overexpression of epidermal growth factor receptor (EGFR) correlates to therapeutic response in select patient populations. Thus, molecular positron emission tomography (PET) imaging of EGFR could stratify responders versus non-responders. We previously demonstrated effectiveness of a "synthetic consensus" design principle to identify six neutralizing mutations within a 58-amino acid EGFR-targeted affibody domain. Herein, we extend the approach to identify additional neutralized variants that vary net charge from -2 to either -4 or +4 while retaining high affinity (1.6 ± 1.2 nM and 2.5 ± 0.7 nM), specific binding to EGFR, secondary structure, and stability (T = 68 °C and 59 °C). We radiolabeled the resultant collection of five charge variants with Cu and evaluated PET imaging performance in murine models with subcutaneously xenografted EGFR and EGFR tumors. All variants exhibited good EGFR tumor imaging as early as 1 h, with EA35S (+3/-5) achieving 7.7 ± 1.4 %ID/g tumor at 4 h with 1.5 ± 0.3%ID/g EGFR tumor, 34 ± 5 tumor:muscle and 12 ± 3 tumor:blood ratios. The positively charged EA62S mutant (+6/-2) exhibited 2.2-3.3-fold higher liver signal than the other variants (p<0.01). The EA68 variant with higher charge density was more stable to human and mouse serum than neutralized variants. In a comparison of radiometal chelators, 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA) exhibited superior physiological specificity to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). In total, these studies comparatively evaluated a set of EGFR-targeted affibodies varying in net charge and charge density, which revealed functional variations that are useful in engineering an ideal probe for translational studies.
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http://dx.doi.org/10.1039/C7ME00095BDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715147PMC
February 2018

Quantifying Binding of Ethylene Oxide-Propylene Oxide Block Copolymers with Lipid Bilayers.

Langmuir 2017 11 25;33(44):12624-12634. Epub 2017 Oct 25.

Department of Chemical Engineering and Materials Science, ‡Department of Integrative Biology and Physiology, and §Department of Chemistry, University of Minnesota , Minneapolis, Minnesota 55455, United States.

Block copolymers composed of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) have been widely used in cell membrane stabilization and permeabilization. To explore the mechanism of interaction between PPO-PEO block copolymers and lipid membranes, we have investigated how polymer structure influences the polymer-lipid bilayer association by varying the overall molecular weight, the hydrophobic and hydrophilic block lengths, and the end-group structure systematically, using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) unilamellar liposomes as model membranes. Pulsed-field-gradient NMR (PFG-NMR) was employed to probe polymer diffusion in the absence and presence of liposomes. The echo decay curves of free polymers in the absence of liposomes are single exponentials, indicative of simple translational diffusion, while in the presence of liposomes, the decays are biexponential, with the slower decay corresponding to polymers bound to liposomes. The binding percentage of polymer to the liposome was quantified by fitting the echo decay curves to a biexponential model. The NMR experiments show that increasing the total molecular weight and hydrophobicity of the polymer can significantly enhance the polymer-lipid bilayer association, as the binding percentage and liposome surface coverage both increase. We hypothesize that the hydrophobic PPO block inserts into the lipid bilayer due to the fact that little molecular exchange between bound and free polymers occurs on the time scale of the diffusion experiments. Additionally, as polymer concentration increases, the liposome surface coverage increases and approaches a limit. These results demonstrate that PFG-NMR is a simple yet powerful method to quantify interactions between polymers and lipid bilayers.
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http://dx.doi.org/10.1021/acs.langmuir.7b02279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6055234PMC
November 2017

Chemical End Group Modified Diblock Copolymers Elucidate Anchor and Chain Mechanism of Membrane Stabilization.

Mol Pharm 2017 07 12;14(7):2333-2339. Epub 2017 Jun 12.

Integrative Biology and Physiology, University of Minnesota Medical School , Minneapolis, Minnesota 55455, United States.

Block copolymers can be synthesized in an array of architectures and compositions to yield diverse chemical properties. The triblock copolymer Poloxamer 188 (P188), the family archetype, consisting of a hydrophobic poly(propylene oxide) core flanked by hydrophilic poly(ethylene oxide) chains, can stabilize cellular membranes during stress. However, little is known regarding the molecular basis of membrane interaction by copolymers in living organisms. By leveraging diblock architectural design, discrete end-group chemistry modifications can be tested. Here we show evidence of an anchor and chain mechanism of interaction wherein titrating poly(propylene oxide) block end group hydrophobicity directly dictates membrane interaction and stabilization. These findings, obtained in cells and animals in vivo, together with molecular dynamics simulations, provide new insights into copolymer-membrane interactions and establish the diblock copolymer molecular architecture as a valuable platform to inform copolymer-biological membrane interactions. These results have implications for membrane stabilizers in muscular dystrophy and for other biological applications involving damaged cell membranes.
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http://dx.doi.org/10.1021/acs.molpharmaceut.7b00197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5648059PMC
July 2017