ACS Bio & Med Chem Au

Targeting GC-rich gene loci is a major challenge owing to their high duplex stability, repetitive sequence composition, and propensity to adopt alternative DNA structures. Triplex-forming oligonucleotides (TFOs) provide a programmable strategy towards the recognition of GC-rich DNA, but their application is restricted by the limited recognition capabilities of natural nucleobases in a cellular setting. Here, we overcome this barrier using parallel-binding TFOs containing the synthetic nucleobase 6-amino-5-nitropyridin-2-one (Z), which enables pH-independent recognition of G-C base pairs. Using two structurally distinct regulatory elements within the MYC promoter, we show that Z-modified TFOs form stable, sequence-selective triplexes that repress promoter activity by 50-80% in both episomal reporter assays and at endogenous gene loci. Notably, the greatest repression was observed at a GC-rich quadruplex-forming element that functions as a structural hub for transcription factor recruitment. To our knowledge, this represents the first demonstration that a simple nucleobase modification alone is sufficient to enable parallel-binding TFOs to repress expression of an endogenous gene, establishing a general strategy for targeting GC-rich regulatory elements through programmable DNA recognition. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/741700v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1d23b1corg.highwire.dtl.DTLVardef@126de76org.highwire.dtl.DTLVardef@d75631org.highwire.dtl.DTLVardef@15bd46d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Achieving predictable, tunable, and temporal control over mRNA function would grant direct regulation of gene expression, facilitating the development of new therapeutics and biotechnologies. Although several approaches for stimuli-responsive control of nucleic acids have been explored, most are limited to short oligonucleotides, lack a timed-release mechanism, or both. We envisioned a complementary method using glyoxal as a caging reagent. Glyoxal readily reacts with amidine groups found on the faces of nucleobases to give stable bis-hemiaminal adducts, directly disrupting hydrogen bonding. Fortuitously, this reaction is readily reversible, enabling spontaneous time-release decaging that varies with temperature. However, when applied previously to full-length mRNAs, the sequence length and excessive adduct formation resulted in no reactivation under relevant physiological conditions. To address this challenge, we developed chemical lithography in which portions of longer RNAs are "masked" through hybridization to complementary DNAs, permitting selective caging on only non-masked regions and preventing excessive adduct formation. We present an optimized glyoxalation protocol applied to EGFP as a model mRNA sequence and evaluate masking effectiveness through qualitative and quantitative studies. Using EGFP fluorescence, we monitored and assessed the ability of selective glyoxalation to control gene expression over time in vitro. We demonstrate the direct dependence of both the initial inhibited expression and the respective activity recovery based on the amount and location of glyoxalation. We also highlight distinct caging patterns exhibiting total inhibition upon initial treatment and complete reactivation following decaging. We anticipate that this approach will improve the mechanistic study of mRNA and gene expression and also facilitate new investigations and methods within chemical biology and biomedicine.

Antisense oligonucleotides (ASO) are nucleotide polymers that hybridize to sense strands and have been successful in treating a variety of diseases. A wide range of strategies have been investigated to optimize and develop ASO for clinical studies. A key objective for this study was to provide an overview of the range of detailed data that get be obtained and provide an updated method review on how to design surface plasmon resonance (SPR) kinetic experiments for DNA oligonucleotide hybridization studies that can also be applied to other ASO including peptide nucleic acids (PNA). We describe many lessons learned from published literature and provide a state-of-the-art strategy and methods for generating not only kinetic but also thermodynamic characterizations of oligonucleotide hybridization. In this study we have performed an SPR kinetic and thermodynamic analysis for the hybridization of HIF1 antisense DNA strands to its immobilized Intron2-Exon3 splice site sense DNA strand to provide insight, in general, on the optimal length and insight into optimal design of DNA ASOs. We provide a process on how to design experiments to: 1.) obtain oligonucleotide-length dependent kinetics, 2.) analyze reactions to obtain association and dissociation rate kinetics (ka, kd), assess if hybridization follows a 2-state model and to obtain kinetic dissociation constants (Kd), 3.) perform temperature-dependent hybridization kinetics to obtain thermodynamic values ({Delta}H{degrees}, {Delta}S{degrees} and {Delta}G{degrees}) that can give insight into the molecular interactions driving hybridization, 4.) compare experimental thermodynamic values to values derived from nearest-neighbor prediction models to identify atypical reactions and importantly 5.) enable calculations to predict oligomer hybridization affinity at the physiological 37 {degrees}C temperature to asses if the design of the oligomer will have the required cellular activity for a therapeutic effect. The strategy and results presented throughout the paper are compared to previous SPR reports and suggestions made to optimize kinetic studies.

Hybridoma screening in semi-solid medium typically employs antigens labeled with visible fluorophores (e.g., FITC, AF488) to enable single-step identification of antibody-secreting clones. However, conventional chemical conjugation via NHS-esters or isothiocyanate groups frequently modifies lysine residues located within epitopes, potentially abrogating antibody recognition of these critical regions. Here, we describe a SpyTag SpyCatcher-based site-specific labeling strategy that circumvents epitope damage during semi-solid medium screening. A 16-amino-acid SpyTag was genetically fused to the C-terminus of the target antigen, enabling covalent conjugation to an sfGFP SpyCatcher fluorescent probe. In semi-solid medium supplemented with SpyTag-antigen and sfGFPSpyCatcher, positive hybridoma clones were readily identified by distinct fluorescent halos, whereas negative clones showed no detectable signal. Notably, the site-specific method yielded a significantly higher frequency of fluorescence-positive clones compared to the conventional AF488-labeled antigen method, suggesting that epitope preservation enhances screening recovery. Furthermore, this approach did not impair hybridoma growth or final clone positivity, offering a simple, rapid, and epitope-compatible method for monoclonal antibody screening.

We present a simple and efficient way to add cis dialdehydes, phosphoryl groups, or cis diols to the 3 prime end of oligonucleotides using periodate oxidized nucleotides (oNTPs) and terminal deoxynucleotidyl transferase (TdT). The 3 prime end cis dialdehyde-modified oligos are generated by incubating TdT with an oligo for several minutes followed by addition of a oNTP and incubated at 30 degrees C for 30 minutes to an hour. After allowing the addition of the cis dialdehydes, heating the reaction mixture at 90 to 95 degrees C for 10 minutes yields oligonucleotides with 3 prime phosphoryl groups. The 3 prime cis diol modified oligos are synthesized by starting with 3 prime cis diol nucleotides (HO-NTPs). The cis dialdehyde modified oligonucleotides and cis diols may then be used for a variety of investigations such as studying the interaction of proteins with the 3 prime end of DNA, and possibly RNA. As an example, we demonstrate the efficacy of using an oligonucleotide modified with oGMP at the 3 prime end as an affinity label for TdT and identified a peptide fragment that has been shown to contain two of three aspartate residues found to be in the TdT active site.

Antibody-oligonucleotide conjugates (AOCs) offer a promising solution to delivery challenges of therapeutic oligonucleotides. However, the relationship between their complex chemical architectures and biological activity remains poorly understood, limiting the development of important structure-function relationships. For siRNA-containing AOCs, increasing the siRNA-to-antibody ratio beyond one (drug-to-antibody ratio, DAR>1) reduces potency, attributed to altered pharmacokinetics arising from increased negative charge density. Here, we investigated whether simple chemical modifications could improve AOC efficacy and mitigate limitations associated with higher DAR. Introduction of a single C16 lipid modification to the siRNA significantly enhanced target gene silencing compared to the unmodified AOC. Extending this modification to a DAR2 architecture, in which two siRNAs are conjugated per antibody, restored the loss of activity associated with increasing DAR from 1 to 2. Notably, at an equivalent siRNA dose (1 mg/kg), the DAR2-C16 AOC requires half the amount of antibody while achieving knockdown comparable to the DAR1-C16 AOC. We also developed a charge-balancing ionizable linker (CBIL) designed to partially compensate for the negative charge of siRNA. Incorporation of the CBIL enhanced target gene silencing in heart and skeletal muscle without a corresponding increase in hepatic activity, resulting in a shift toward greater extrahepatic activity relative to liver. Together, these findings demonstrate that chemical modification of both the siRNA payload and antibody-siRNA linker can be used to tune AOC potency and tissue activity, while enabling higher payload loading without compromising efficacy. These results establish chemical design as an important strategy for expanding the architecture and therapeutic potential of AOCs.

mRNA therapeutics have demonstrated transformative potential in infectious disease, oncology and genetic disorders. Parallel to the rapid clinical advancement of mRNA therapies, research into in vitro transcription (IVT), the pivotal process of mRNA manufacturing, has intensified significantly. However, the majority of these studies omitted cap analog, an essential substrate for co-transcriptional capped IVT (co-IVT), likely to simplify experimental systems. Nevertheless, co-IVT remains the dominant industrial standard for the production of mRNA therapeutics. Here, we report an optimized co-IVT process that achieves a yield of more than 26 g/L while maintaining capping efficiency of over 99.5%, and achieving a 30-fold reduction in double-stranded RNA (dsRNA), the most critical process-related impurity. These performance gains substantially improve mRNA production efficiency and enhance mRNA quality, thereby accelerating the transition to commercial-scale manufacturing of mRNA therapeutics. Moreover, the performance was attained using either of two mechanistically distinct engineered T7 RNA polymerase (RNAP) mutants. Both mutants enabled robust mRNA synthesis under process conditions that have not been previously reported. These observations provide mechanistic insights into IVT and advance our fundamental understanding of this critical bio-manufacturing step.

A platform method to associate native proteins and peptides onto a liposome membrane using site-specific N-terminal alkylation based on 1H-1,2,3-triazole-4-carbaldehyde (TA4C) is developed. The TA4C reagent reacts with the N-terminal -amino group of native proteins under mild aqueous conditions in a single step, without genetic engineering or protecting group strategies. Equipping TA4C with hexyl and nonyl chains provides a direct handle for tuning the association between the protein and membrane. N-terminal alkylation of green fluorescent protein (GFP) as a model proceeds in high yield (93% for the hexyl group and 70% for the nonyl group), and tethering the N-terminal alkyl group on GFP efficiently associates the protein with the liposomal membrane, as confirmed by confocal laser scanning microscopy and dynamic light scattering. We extended this strategy to an investigation of the GE11 peptide, a ligand for the epidermal growth factor receptor (EGFR). The liposome immobilized with GE11 peptide possessing an N-terminal alkyl group enables active targeting with EGFR-overexpressing A431 cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/742023v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1f4095org.highwire.dtl.DTLVardef@1c91509org.highwire.dtl.DTLVardef@841647org.highwire.dtl.DTLVardef@1d2909b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Graphene oxide (GO) has primarily been investigated as a carrier for intracellular delivery of therapeutic molecules. In previous work, we identified a cell type-dependent interaction pattern in which GO remained predominantly associated with the plasma membrane of cancer cells but was internalised by non-cancerous epithelial cells. Here, we explored whether plasma membrane-associated GO can be used as a platform to present bioactive ligands and influence cell-surface receptor signalling in cancer cells. To test this hypothesis, we targeted integrin receptors at the plasma membrane in glioblastoma cell models using an RGD-containing peptide non-covalently complexed with GO. We assessed GO-peptide interactions, cellular interactions/uptake, motility, and focal adhesion signalling readouts. Peptide association was quantified using a 2,4,6-trinitrobenzene sulfonic acid (TNBSA) assay, and GO was characterised by atomic force microscopy, X-ray photoelectron spectroscopy, X-ray diffraction, and colloidal measurements. Immediately after complexation, ~70% of RGD was associated with GO. Peptide association increased the nitrogen signal and shifted the principal GO XRD peak while retaining nanosheet morphology. Biological responses were examined in U87 and U251 glioblastoma cells with different integrin-positive fractions, and in non-cancerous BEAS-2B bronchial epithelial cells. Confocal microscopy showed that GO and GO:RGD remained predominantly localised on the plasma membrane in U87 and U251 cells, whereas greater intracellular localisation was observed in BEAS-2B cells. Importantly, GO:RGD significantly reduced key indicators of cell motility: cell velocity in U87 and U251 cells, with trajectory and mean-square-displacement analyses supporting restricted cellular movement. Free RGD had no significant effect, while GO alone produced a smaller reduction in motility only in U251 cells. No treatment significantly altered BEAS-2B motility. Flow cytometry also showed a reduced pFAK-associated signal in GO:RGD-treated U87 cells. These findings establish a proof of concept that the cell-line-dependent plasma membrane localisation of GO can be exploited as a membrane-associated nano-bio interface for cell-surface-active ligands, opening the way for the development of GO-based platforms that modulate receptor-mediated signalling and cell behaviour.

D-Ribitol-5-phosphate (Rbo5P) plays vital roles in bacterial and mammalian development. In mammals, Rbo5P is an essential component of the O-mannosyl glycan on -dystroglycan, and its defective biosynthesis causes a group of dystroglycanopathies. Supplementation with cytidine diphosphate ribitol (CDP-ribitol, CDP-Rbo) and its analogues has shown therapeutic potential for certain dystroglycanopathies. However, facile and scalable strategies to prepare CDP-Rbo remain unavailable. Here, we report a practical one-pot enzymatic strategy for gram-scale CDP-Rbo production. Starting from inexpensive ribitol, Rbo5P is first generated by L-ribulokinase (AraB) and then converted to CDP-Rbo by CDP-ribitol pyrophosphorylase (TarI) in 90% yield. To streamline the process, AraB and TarI were fused into a single bifunctional biocatalyst, AraB-TarI, which converts ribitol directly into CDP-Rbo on a gram-scale in a one-step reaction. Furthermore, a nucleotide recycling strategy was developed to lower the cost and increase the atom economy from 47% to 75%. This work provides a green and scalable route to CDP-Rbo and a reliable material supply for developing therapeutics against dystroglycanopathies.

Protein-based fluorescent sensors are a powerful addition to the biology toolbox for their ability to be stably expressed within living organisms, tissues, cells, and subcellular compartments, with the capacity to report on the presence of specific target molecules or other analytes. At the same time, sensor components will unavoidably present opportunities for unintended interaction with endogenous cellular machinery, potentially confounding both sensor function and cell health. Interactions with host components may not be readily predictable during the sensor design process, especially when simultaneously optimizing many other sensor parameters such as fluorescence response, dynamic range, and kinetics. Characterizing effects of sensor expression on cells is currently a laborious ad hoc process; new methods to characterize the cell expression effects of sensors and their variants could dramatically improve sensor design pipelines, laying the groundwork to recognize potentially problematic expression side effects earlier in the iterative design and testing workflow. Here, we take a dual high-content imaging-based and biochemical approach to examine sensor interactions with native cell biology, focusing on the widely used GCaMP calcium sensor. We identify a morphology-based signature of the cellular effects of high sensor expression in a neuroblastoma cell line. Subsequently, we identify biochemical interactions between GCaMP and a component of the mammalian cytoskeleton and track morphological features in sensor-expressing cells that lack these structural components. Our findings present an entry point for engineering new minimally cross-reactive sensor versions given a contextual biological understanding of sensor overexpression. We anticipate that as this and related workflows are incorporated into sensor engineering pipelines, bioorthogonality can be more systematically assessed and prioritized in diverse sensor scaffolds.

Bacterial sortases are widely used in sortase-mediated ligation (SML) experiments for various protein engineering applications. The power of these enzymes to bind and cleave a specific recognition motif, followed by ligation to another substrate using a ping-pong reaction mechanism has numerous applications in vaccine and antibody/nanobody drug conjugate development, as a diagnostic and therapeutic tool, in creating novel insulin derivatives, etc. The most widely used sortase for SML is the class A sortase (SrtA) from Staphylococcus aureus (saSrtA), and its engineered derivatives. Despite its utility, saSrtA and other endogenous sortases are relatively inefficient enzymes and use can be limited by the need for specific recognition of the Cell Wall Sorting Signal (CWSS), sequence Leu-Pro-X-Thr-Gly, where X=any amino acid. Therefore, there is a need to continue to identify new tools for SML and to develop screening assays towards these endeavors. Here, we present optimization procedures for a FRET-based assay utilizing the GFP derivatives mTurquoise2 and SYFP2 to directly monitor formation of ligation products generated via SML. Similar to related assays, our recombinant substrates can be easily manipulated to screen either the substrate recognition motif, second substrate nucleophile, and/or sortase variants themselves. We believe continued optimization of this assay for a variety of high throughput uses in sortase screening strategies is possible, providing a proof-of-concept approach for continued SML reagent development.

Accurate determination of kinase inhibitor binding modes could provide essential information for understanding resistance mechanisms and accelerating drug discovery. While conventional structural methods such as X-ray crystallography, cryo-EM and NMR provide high-resolution information but are low-throughput and capture largely static snapshots of dynamic protein-ligand interactions Here, we introduce a single-molecule nanopore tweezer platform that functionally subtypes ATP-competitive Abl kinase inhibitors by resolving distinct ionic current signatures of Abl-inhibitor complexes. This approach distinguishes Type I, Type IIA, and Type IIB inhibitors without structural determination. We further show how clinically relevant Abl variants (T315I and E255V) reshape inhibitor engagement and binding modes. By combining baseline probability features with wavelet-based time-frequency descriptors, ensemble machine-learning models achieved 97.5% classification accuracy across seven kinase inhibitor binding modes at sub-angstrom resolution and enabled deconvolution of mixed-inhibitor samples at nanomolar concentrations. These results establish nanopore tweezers as a label-free, super-resolution platform for profiling kinase conformational states and inhibitor binding modes, complementing structural approaches and supporting precision oncology.

The methylation of cytosines at the 5th position (d5mC) is one of the most common epigenetic modifications, and alterations in methylation profile of cells are known to be involved in progression of many diseases including cancer. Increased stability of DNA accompanied by decreased flexibility upon methylation is thought to be a reason behind methylation profiles. The effects of d5mC on DNA stability and structure were investigated via systematic changes in the number and position of d5mCs in DDD. Our results revealed that d5mC substitutions changed DNA conformation and increased the stability only slightly. Next, the effect of DNA methylation on DNA-small molecule interactions was investigated using DDD and fully methylated analogue, DDD8. All the molecules examined (EtBr, Dox, Net and Hoe) had slightly higher affinity to DDD8 compared to DDD. Conversely, their effect, especially Doxs, on DDD structure was more pronounced. Further investigations via MD simulations revealed high selectivity of Dox towards a single intercalation site where the methoxy group of Dox interacts with the methyl group of d5mC and that of two precedent dT to create a highly stable hydrophobic cluster. Hydrophobic cluster formation was not observed upon Dox binding to DDD. Our results rationalize the increased stability of DDD8 over DDD, and open new routes for the design of drugs targeting epigenetic modifications. We suggest, the design of drugs that can engage in hydrophobic interactions with methyl groups in the major groove of a 5-dTdTd5mCdG-3 sequence might lead the way in specific targeting of hypermethylated regions in cancer cells.

Chimeric antigen receptor (CAR)-T cells and CAR-macrophages (or CAR-monocytes) offer complementary antitumor functions. CAR-T cells exert potent cytotoxicity against cancer cells, whereas CAR-macrophages can infiltrate solid tumors, phagocytose cancer cells and promote antigen spreading. Antibody-conjugated lipid nanoparticles (LNPs) have emerged as non-viral mRNA delivery platforms for generating CAR-immune cells. However, these systems have generally been optimized to target a single immune-cell type. Here, to enable the ex vivo generation of CAR-T cells and CAR-monocytes, we developed CLCM08, an anti-CD222 antibody, and co-displayed it with an anti-CD5 antibody on LNPs. CD222 is predominantly expressed by monocytes, macrophages and T cells, and facilitates the internalization of CD222-binding molecules. CD5 is primarily expressed by T cells and has previously been used to enable LNP-mediated mRNA delivery to these cells. Delivery of CAR mRNA by dual CD222/CD5 antibody-conjugated LNPs to primary monocytes and T cells substantially enhanced the ex vivo generation of CAR-monocytes and CAR-T cells compared with non-conjugated LNPs. Moreover, CD19 CAR-T cells and CAR-monocytes generated using dual CD222/CD5 antibody-conjugated LNPs showed enhanced cytotoxic and phagocytic activity, respectively, relative to cells generated using non-conjugated LNPs, with anti-CD222 LNPs producing the largest phagocytic gain in monocytes. These findings establish an adaptable antibody-conjugated LNP-mRNA platform for the ex vivo generation of CAR-T cells and CAR-monocytes.

Mutant-selective and pan-RAS(ON) multi-selective inhibitors have improved clinical outcomes for patients with RAS-driven cancers; however, adaptive resistance limits the depth and durability of response. Using an integrated genetic, transcriptomic, and mass-spectrometry-based quantitative temporal proteomic platform, we profile the adaptive response to KRASG12C inhibitors (KRASi) in KRASG12C-mutant lung adenocarcinoma models. Our analyses reveal remodeling of signaling pathways and the cell surface proteome, including cell surface proteins selectively upregulated across cell lines, tumor models, and treatment durations. Folate receptor alpha (FOLR1) emerged as a therapeutically actionable target that is significantly upregulated with KRASi and is part of a broader acute cellular state transition characterized by acquisition of an epithelial identity. Combining KRASi with mirvetuximab soravtansine-gynx, a clinically approved FOLR1-targeting antibody drug conjugate, enhanced tumor cell cytotoxicity and deepened response and tumor control in cellular and in vivo models. Additionally, this work represents a comprehensive multi-omic resource characterizing non-genetic temporal adaptation to KRASi for future mechanistic and therapeutic investigations.

Metastasis and associated therapy resistance remain the principal drivers of cancer related death, and there is a pressing need for a deeper mechanistic understanding and anti-metastatic therapies. For patients that suffer from hepatocellular carcinomas (HCCs), which are the most common primary liver cancers, frequent systemic metastasis results in bleak 5-year survival prognoses of only 4%. To metastasize, carcinoma cells must acquire an invasive phenotype, which typically requires switching from an epithelial-like apical-basal polarity to the front-rear polarity of mesenchymal-like cells. Signaling cues that originate in the tumor microenvironment can activate cellular morphogenic programs that drive polarity switching, like the epithelial-mesenchymal transition (EMT). Protein kinases control most cell signaling pathways and are highly actionable drug targets; however, systematic studies determining the kinases that underly the epithelial-mesenchymal polarity switch (EMPS) are lacking. We developed an assay platform that integrates mass spectrometry (MS)-based kinome profiling, broadly capturing kinase network activity, with chemical genetic screening using selective kinase inhibitors and quantitative phase imaging (QPI), serving as the phenotypic readout. Applying this approach that we dubbed morphokin-MS, to epithelial-like HCC cell lines that we induced to undergo EMPS identified a conserved network of 12 kinases that contributed to HCC cell polarity switching and directed cell migration; MS-based kinome profiling of 17 HCC patient tumors showed that these kinase are frequently upregulated in human tumors. morphokin-MS also revealed that death-associated protein kinase 3 (DAPK3) is one of the principal drivers of the EMPS and directed HCC cell migration. Thus, our mechanistic studies revealed that DAPK3 forms a complex with DAPK1 and filamin-A inter-acting protein 1-like (FILIP1L), which act as scaffold proteins that recruit DAPK3 to the centrosome. Pharmaco-logical and genetic inhibition of the DAPK1-DAPK3-FILIP1L complex blocked centrosome repositioning and microtubule polarization toward the leading edge of mesenchymal-like HCC cells, directed cell migration, and invasion. Our morphokin-MS method and comprehensive kinome profiling data will serve as a valuable resource for the cancer research community; our discovery of an inducible mesenchymal-like DAPK1-DAPK3-FILIP1L polarity complex that controls centrosome positioning in motile HCC cells may lead to the development of novel therapeutics for combatting cancer metastasis.

Glioblastoma (GBM) is the most common primary malignant brain tumor and is typically fatal. GBM therapies are hindered by the impermeability of the blood brain barrier (BBB), the diffuse and infiltrative nature of the tumor, and the high heterogeneity of intratumoral GBM cells. Aptamers are short, synthetic, folded single strands of RNA or DNA or analogs that bind targets with high affinity and specificity. Aptamers are developed via the principles of natural selection, permitting an unbiased approach to therapeutic development. Thus, rather than using rational design to select a target and develop a targeting moiety, cycles of Systematic Evolution of Ligands by Exponential Enrichment (SELEX) are employed in cell culture or in vivo to identify aptamers against unknown targets. Antibody drug conjugates (ADCs) have shown some efficacy for GBM but are limited by their large size and thus depend on leakiness of the BBB. We have recently applied in vivo SELEX to develop anti-GBM aptamers (six-fold smaller in mass than IgG antibodies) and to select aptamer-drug conjugates. Here we report attempts to focus aptamer selection toward internalizing drug-delivery targets and resulting challenges involving loss of tumor specificity in vivo.

Polymer modification of antibodies has attracted attention for enhancing antibody functionality. Modification with polyethylene glycol (PEG) or biopolymers such as antibody-oligonucleotide conjugates has been investigated. Although direct effects at the modification site have been understood, distal effects of modifications on intrinsic antibody functions such as antigen-binding and Fc receptor-binding activities remain poorly characterized. In this study, we prepared site-specifically PEGylated trastuzumab to evaluate the effects of the modification site and the size of PEG chains on antibody functions. Reporter gene assays revealed a significant reduction in antibody-dependent cell cytotoxicity (ADCC) activity in a site- and size-dependent manner. Both the reduction in interaction with the antigen and Fc{gamma}RIIIa were observed in a size-dependent manner. Interestingly, the effect of the modification site was only observed for ADCC activity, which suggests that the higher-order structure of the modified antibody plays a critical role in effector function. These results provide molecular design guidelines for the polymer modification of antibodies.

Chinese hamster ovary (CHO) cells are the dominant platform for recombinant biotherapeutics, yet the impact of producer cell line selection on mammalian cell metabolism remains poorly understood. Here, we performed 41 independent 13C-tracer experiments using uniformly labeled glucose or individual amino acids to comprehensively map carbon utilization in the two principal CHO production platforms: methotrexate-selected CHO-K1 and glutamine synthetase-selected CHO-GS cells. Time-resolved GC-MS analysis revealed distinct metabolic phenotypes spanning central carbon metabolism, amino acid interconversion, lipid biosynthesis, and one-carbon metabolism. CHO-K1 cells exhibited extensive reductive carboxylation and pyruvate carboxylase-mediated anaplerosis, whereas CHO-GS cells redirected glutamate toward glutamine synthesis and relied on asparagine and aspartate to support TCA cycle activity. Isotopomer analysis uncovered substantial intracellular-extracellular cycling of alanine, glycine, glutamate, and serine despite contrasting uptake profiles and quantified differential amino acid contributions to fatty acids and cholesterol. Serine, glycine, and methionine labeling revealed active folate-cycle interconversion in CHO-K1 and enhanced methionine-cycle activity in CHO-GS. Aspartate is identified as a key redox exchange factor and uniquely informative tracer for pathway characterization following glutamine depletion. Together, this exhaustive isotope-tracing framework establishes how producer cell line selection rewires mammalian metabolism and provides a foundation for cell engineering, media optimization, metabolic modeling, and next-generation biomanufacturing.