Bioconjugate Chemistry

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.

Fluorescence imaging in the second near-infrared window (NIR-II, 950-1700 nm) offers reduced scattering, lower autofluorescence, and deeper tissue penetration than NIR-I imaging, but its adoption is limited by the need for custom-synthesized fluorophores. Here, we identify commercially available dyes that exhibit usable NIR-II emission. Eleven visible, far-red, and NIR-I fluorophores were screened under twelve acquisition configurations combining 670, 760, and 808 nm excitation with band-pass (950 nm, 1400 nm) or long-pass (1000 nm, 1250 nm) emission filters. Output varied markedly with fluorophore identity and excitation/emission configuration. Among hydrophobic dyes, DiR exhibited strong emission across almost all excitation and emission filters. Among hydrophilic dyes, strong NIR-II fluorescence was observed for IRDye 680RD (excitation at 670 nm), sulfo-cyanine 7 (excitation at 670 nm and 760 nm), and indocyanine green (excitation at 808 nm). DiR showed a linear concentration-response under 760 nm excitation with BP1400 detection. Upon encapsulation in PEGylated liposomes, strong NIR-II fluorescence was retained. In an in vivo study in mice, NIR-II resolved vasculature that NIR-I could not consistently delineate, and enabled pharmacokinetic analysis. Both windows returned similar ex vivo organ distributions. NIR-II imaging is therefore accessible using commercial off-the-shelf fluorophores, provided the dye is matched to the intended excitation/emission configuration.

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.

The chemical synthesis of well-defined, self-assembling semi-synthetic proteins (SSPs) has attracted growing interest in recent years. Approaches such as micelle-assisted protein-labeling technology (MAPLabTech) and supramolecule-assisted protein-labeling technology (SAPLabTech) have been used to generate a wide range of SSPs. The central challenge in synthesizing SSPs is solubilizing a hydrophobic chemical probe in aqueous medium prior to bioconjugation. Both MAPLabTech and SAPLabTech rely on non-covalent interactions to solubilize hydrophobic probes and present certain limitations. The present study introduces a complementary chemical strategy in which a hydrophobic chemical probe is covalently tagged with a cleavable, water-soluble dendritic domain. This covalent tagging renders the probe fully water-soluble, enabling quantitative bioconjugation to yield monomeric semi-synthetic proteins. Subsequent, selective removal of the solubility tag converts the hydrophilic semi-synthetic proteins into facially amphiphilic, semi-synthetic proteins.

Reporter mRNAs are widely used to screen lipid nanoparticle (LNP) formulations and characterize functional mRNA expression in vivo, yet conventional firefly luciferase (FLuc) imaging fails to detect low-level expression. Consequently, weak or absent FLuc signal can be misinterpreted as failed LNP delivery or translation, potentially excluding formulations and overlooking extrahepatic or off-target expression. Here, we compared FLuc/D-luciferin with an ultrasensitive NLuc-GFP bioluminescence resonance energy transfer luciferase reporter detected with fluorofurimazine (FFz), using SM-102 and U105 LNPs following intravenous administration in mice. Both reporters showed predominantly hepatic and splenic expression. However, NLuc-GFP/FFz produced substantially greater photon output and detected reporter activity in the brain, heart, kidney, lymph nodes and other tissue where FLuc activity was weak or undetectable. Collectively, we show traditional FLuc imaging can underappreciate mRNA delivery, not necessarily because LNPs fail to reach or deliver mRNA to the tissue but because low-level FLuc expression may fall below its assay sensitivity. This limitation can underappreciate mRNA delivery and unintended protein expression in non-target organs. Therefore, we describe a reproducible workflow encompassing reporter design, LNP formulation and characterization, controlled reporter comparison, ex vivo organ imaging, and a cost-effective DSPE-PEG2000-micelle FFz formulation. This provides a more sensitive framework that improves detection and interpretation of low-level functional expression during preclinical mRNA-LNP screening and safety assessment.

The initial attachment of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) to host cell sialosides is critical for infection, yet its precise receptor specificity remains poorly understood. Here, we describe a chemoenzymatic methodology to synthesize a comprehensive panel of 6-sulfo sialyl Lewisx (6-sulfo-SLex) containing glycans. Our approach entails the enzymatic assembly of an oligo-lactosamine chain modified at specific positions with N-trifluoroacetyl-glucosamine (GlcNTFA) moieties. Mild base treatment removes the TFA group to yield glucosamine, which effectively blocks enzymatic fucosylation. By leveraging this approach alongside the unique substrate selectivity of GlcNAc-6-O-sulfotransferases 2 (CHST-2), we achieved the selective preparation of fucosylated 6-sulfo-SLex glycans. Microarray screening of these printed glycans revealed that a 6-sulfo-SLex derivative presented on an extended LacNAc chain is the preferred host receptor for MERS-CoV. Conjugation of this lead compound to a polyglycerol-based dendrimer generated a multivalent inhibitor that potently blocks hemagglutination of human red blood cells by the MERS-CoV spike protein N-terminal domain (NTD). Furthermore, computational modeling demonstrated that the fucose moiety does not directly contact the viral spike protein. Instead, it pre-organizes the ligand into a favorable conformation, enabling a critical salt bridge between the glycans sulfate group and the guanidinium side chain of viral residue Arg307.

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

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.

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.

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 describe the concept for a new modality to create multi-specific therapeutics that utilizes modified nucleic acids as a scaffold to concatenate multiple warheads (hence called 'catamers'). Catamers are assembled via Watson-Crick interactions into multi-specific molecules from individual components such as ligand-binding modules, protease-cleavage sites, and toxins. Each warhead is covalently bound to an oligonucleotide, which in turn is connected to the catamer scaffold. Post-synthesis of the individual catamer components, they are annealed together such that Watson-Crick base pairs on the oligonucleotides dictate the organization of the warheads on the complete catamer. Notably, catamers are not aptamers, though they can contain aptamers as warheads, along with small molecules and peptides. By design, catamers are intended to address three key problems that have hamstrung the field of multi-specifics: (i) optimization rate; (ii) manufacturing cost; and (iii) unpredictable immunogenicity (Amash et al., 2024 PMID: 39189686). Catamers are purely synthetic and do not require cultured cells to produce. These advantages are based on the underlying design of the catamer and will require experimental validation in future studies. As a proof-of-concept catamer, we focus on a prostate-specific membrane antigen (PSMA)-targeted toxin conjugate. In molecular simulations, this catamer displays structural characteristics to support its intended pharmacology. Importantly, the design addresses one of the key shortcomings of antibody drug-conjugates (ADCs): off-target toxicity caused by release of the toxin after pinocytosis by healthy cells in contact with the blood.

Compact fluorescent proteins (FPs) with red-shifted emission are needed for deep-tissue short-wavelength infrared (SWIR) imaging. We engineered a GAF domain from the JSC1 cyanobacteriochrome of thermophilic Leptolyngbya sp. into three monomeric, biliverdin-binding FPs of 19.1 kDa: miRFP729nano, miRFP732nano and miRFP735nano, with excitation/emission maxima of 714/729, 716/732 and 719/735 nm, respectively. Their off-peak fluorescence beyond 1,000 nm was several-fold higher than that of miRFP718nano previously used for SWIR imaging. miRFP732nano functioned as a fusion tag, a component of target-stabilized nanobodies, and a reporter of NF-{kappa}B and AP-1 transcriptional activities. It enabled single-laser, dual-color three-photon imaging with EGFP to depths of [~]950 m in cortex and [~]300 m in spinal cord. In mice, miRFP732nano supported SWIR imaging of skeletal muscle, inflammatory signaling and intracellular targets. Combining SWIR detection with biocompatible 4-aminoantipyrine-based in vivo tissue clearing enhanced signal and image sharpness. These red-shifted NIR FPs expand the genetically encoded toolkit for deep-tissue imaging.

The current treatment regimen for neuroblastoma involves immunotherapy, including a monoclonal antibody that recognizes disialoganglioside (GD2), expressed at high levels on neuroblastoma. GD2 is not present on most normal tissues except for nerves. Thus, anti-GD2 antibody treatment causes substantial, dose-limiting neuropathic pain. B7-H3 is overexpressed on multiple tumor types, including neuroblastoma, and is largely absent on nerves and other normal tissues. We designed a bispecific antibody (bsAb) that requires simultaneous binding of these two tumor antigens to achieve tight binding of tumor cells. Our preclinical research shows that, compared with a monospecific anti-GD2 antibody, the GD2xB7-H3 bsAb has improved tumor specificity, comparable antitumor efficacy, and reduced nerve binding and pain-associated toxicity. Since this bsAb does not bind to nerves, it may permit more tolerable and sustained treatment schedules than are currently feasible with monospecific anti-GD2 antibodies. In addition, its enhanced tumor specificity may support future development as a targeted delivery platform for antibody-drug conjugates or other payload-based therapies, potentially improving both efficacy and quality of life for patients with neuroblastoma.

FluPep is a 16 amino-acid peptide with potent anti-viral activity against influenza A virus, including the H1N1 subtype. To understand how FluPep exerts its effects, gold nanoparticles were functionalised with FluPep, since gold nanoparticles provide a very sensitive probe that can be detected optically. Our results demonstrate that nanoparticle-FluPep conjugates did not bind to influenza virus particles in vitro. However, the nanoparticle-FluPep conjugates bound to polyanions on the cell surface, and after washing cells they remained cell-associated and were able to inhibit flu virus infectivity. Nanoparticle-FluPep conjugates bound to heparin affinity columns and their binding to cells could be reduced by pre-treatment of the cells with heparinase and neuraminidase, but not with chondroitinase ABC. These data indicated that FluPep bound to heparan sulfate and sialic acid-containing glycans in the pericellular matrix and in doing so prevented viral infectivity. The importance of the interaction of FluPep with heparan sulfate (HS) was examined by extending FluPep at its C-terminus with amino acid sequences responsible for the interactions of growth factors with heparin. The resultant two new engineered peptides, termed superFluPep1 and superFluPep2, were more potent than parental FluPep. The results demonstrate that FluPep likely exerts its antiviral activity by interfering with virus-cell interactions and that enhancing the binding of such peptides to heparan sulfate is an effective strategy to increase their anti viral potency.

Rationale: Neuroblastoma is a devastating pediatric malignancy, for which surgical resection is a key factor in long-term survival. However, there are significant challenges in its resection, particularly in high-risk disease, as neuroblastoma encases surrounding critical structures, is often difficult to distinguish from desmoplastic or scar tissue, and can carry occult deposits of disease not readily identified on preoperative imaging or intraoperative visualization. Building on the principles of fluorescent and radio-guided surgery, in combination with the known overexpression of GD2 in neuroblastoma, we sought to develop and optimize 111In-Dinutuximab-IRDye800, a dual-modality GD2-targeted intraoperative molecular imaging agent, for use in pediatric neuroblastoma to help enhance patient safety while facilitating a more complete resection. Methods: Dinutuximab was conjugated to IRDye800 and DTPA, then radiolabeled with Indium-111 to yield 111In-Dinutuximab-IRDye800. Optimization occurred through ELISA assay to assess binding affinity, fluorescence intensity analysis to determine the optimal fluorescent degree of labeling, and phototoxicity testing through flow cytometry. Rodent models of neuroblastoma were then generated through injection of SK-N-BE(2) human neuroblastoma cells into the left adrenal glands of nude mice or RNU rats. A series of fluorescent and gamma biodistributions was performed, varying the dose, timing, and specific activity of the tracer. Tumor and organ uptake of the tracer was compared with one- or two-way ANOVA as appropriate, with Sidaks multiple comparison test to compare tumor uptake to individual organs. Once optimization was complete, a clinically significant events study modeled after human clinical trials was performed to evaluate the in vivo capabilities of 111In-Dinutuximab-IRDye800. Results: Increased ratios of IRDye800 per antibody led to decreased binding affinity for GD2 and was associated with formulation instability without significant return on fluorescence intensity. Specific activity of the tracer was not found to impact overall biodistribution of the tracer. A 45-50 microgram dose of 111In-Dinutuximab-IRDye800 with ratios around 1 DTPA and 1-1.5 IRDye800 per antibody imaged 4 days after tracer administration was found to be the optimal combination that maximized detectable tumor-specific signal. In the clinically significant events study mirroring human IMI clinical trials, fluorescent guidance identified additional malignant lesions not originally detected under white light in 64% of rodents. Conclusions: 111In-Dinutuximab-IRDye800 is a dual-modality GD2-targeted intraoperative imaging agent that is well-poised for clinical translation. As it preserves tumor specificity, yields clinically meaningful radiofluorescent signal, and is well-tolerated without adverse events after optimization was completed, it carries the potential to positively impact the safety and completeness of neuroblastoma resection.

Site-specific protein modification allows diverse functionalities to be introduced while minimizing perturbations to the protein structure and activity. Considerable efforts have been made to achieve site-specific modification of native proteins to overcome the heterogeneity resulting from conventional stochastic Lys or Cys modification. We have previously achieved the selective modification of Lys65 in a native immunoglobulin G1 (IgG1) antibody (trastuzumab) using EzMTG-pG(Fab), which is an engineered zymogen of microbial transglutaminase (EzMTG) fused to a Fab-binding protein G [pG(Fab)]. However, this approach cannot be widely applied to different types of IgG antibodies. Here, we designed pG(Fab)-EzMTG by fusing pG(Fab) to the N-terminus of EzMTG. Notably, switching the fusion partners dramatically altered the IgG modification site from Lys65 to Lys225, which is located in the hinge site of native IgG1 antibodies. This Lys225-selective labeling was applicable to different IgG1 antibodies. As a functional application, the cytotoxic drug monomethyl auristatin E (MMAE) was conjugated to Lys225 of trastuzumab, and the resulting antibody-drug conjugate exhibited antigen-specific cytotoxicity. These findings demonstrate that fusion-protein architecture determines site selectivity in proximity-directed enzymatic modification, providing a strategy for the site-specific functionalization of native antibodies.

The folate receptor (FR) is overexpressed in a range of human tumours including ovarian cancer cells. We propose that the overexpression of the FR on the surface of ovarian tumour cells could be exploited for the selective delivery of a DNA damage response inhibitor (DDRi) in the form of an intact folate drug conjugate (FDC). This approach would improve the therapeutic index of the parent DDRi facilitating combination studies of the DDRi-based FDC with DNA damaging chemotherapy. FR-mediated cellular uptake of the proposed folate drug conjugates is requisite for FDC selective delivery into tumours. In this study, we synthesised a series of olaparib-based folate conjugates that maintained the biochemical PARP1 inhibition associated with olaparib and showed binding affinity for the folate receptor. Significantly, we identified compounds 10b and 11 that selectively enter FR overexpressing tumour cells via folate receptor-mediated endocytosis in their intact form and engage with their target as demonstrated by the potent inhibition of PARylation (KB cells, PARylation IC50 = 5.7 and 3.9 nM; respectively).

Antibody drug conjugates (ADCs) are a burgeoning class of targeted therapies. However, limitations in their synthesis and efficiency of payload delivery restrict their clinical utility. Here we report a supramolecular assembly (SMA) ADC conjugation method, which allows site-specific, uniform drug loading, resulting in an enhanced pharmacokinetic profile and in vivo efficacy. This peptide conjugation strategy relies on spontaneous heterotetrameric coiled-coil formation between a pair of peptides appended on the C-terminus and a drug-loaded complementary pair in aqueous solution. Pairing this SMA conjugation with an antibody that targets the dual-endothlin-1/VEGF signal peptide receptor (DEspR), a pancreatic ductal adenocarcinoma (PDAC) specific receptor, retains antibody binding and plasma stability. When the anti-DEspR monoclonal antibody is conjugated with monomethyl auristatin E (MMAE), the ensuing ADC internalizes following cell surface binding and induces selected cell death in multiple DEspR positive PDAC cell lines. In vivo, the ADC exhibits favorable pharmacokinetics, high tumor specificity, and improves overall survival in a rat orthotopic model of pancreatic peritoneal carcinomatosis, compared to conventional ADC conjugation. A heterotetrameric coiled-coil structure enables the efficient synthesis of a potent ADC, further documenting the versatility of supramolecular scaffolds as key orthogonal building block for site-specific conjugation in biopharmaceutical and biomaterial drug delivery systems. One Sentence SummaryCombining site-specific conjugation of monomethyl auristatin E, via the use of biologically inspired heterotetrameric coiled-coils, with a tumor-selective antibody targeting the dual-endothlin-1/VEGF signal peptide receptor affords a highly effective, ADC, which improves survival in a rat orthotopic model of pancreatic peritoneal carcinomatosis compared to standard cysteine-conjugated analogues with higher drug loading.

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.

The unique cell surface composition of tumor cells forms the molecular basis for many targeting and cell-based therapies. Here, we describe the development of novel peptide-based targeting agents for programmed death ligand 1 (PD-L1). Molecular imaging by peptide agents, coupled with therapeutic intervention using the same modality, represents a critical advancement in cancer management. Whole-body PET imaging of PD-L1 expression offers a superior alternative to traditional immuno-histochemistry, making PD-L1 radiodiagnostic imaging a highly sought-after modality. PD-L1 targeting modalities developed for clinical imaging to date can be divided into antibodies, protein domains, and small macrocyclic peptides with fewer than 20 amino acids. The latter modalities can address many challenges seen in antibody-based targeting vectors. All potent PD-L1 targeting peptide modalities reported to date rely extensively on non-canonical amino acids (ncAAs). Here, we report a comprehensive structure-activity relationship (SAR) analysis of a family of macrocycles discovered from an Sx2Cx8Cx2 phage-display library composed entirely of natural amino acids (x represents 19 natural amino acids excluding Cys). Using >10,000 variants in ''focused'' phage-display libraries, we optimized these macrocycles to achieve single-digit-nanomolar potency in protein- and cell-based assays. En route to this optimization, the activity of 216 synthetic macrocycles towards PD-L1 was measured in five distinct assays; two leads have been evaluated by imaging in tumor xenografts in mice, and the X-ray structure of one advanced lead in complex with PD-L1 has been determined at 2.78 [A] resolution. This publication demonstrates the development potential of PD-L1-targeting macrocycles that do not require extensive incorporation of ncAAs and the democratization of discovery by mapping the optimization path to single-digit-nanomolar assets for targeted radiopharmaceuticals via canonical phage-display technology.