Bioconjugate Chemistry

Tools to interrogate glycoconjugate-protein interactions in the context living cells are highly attractive for the identification of critically important functional binding partners of glycan-binding proteins. These interactions are challenging to interrogate due to low affinity and rapid dissociation rates of glycan-protein binding events. The use of photo-crosslinkers to capture glycan-protein interaction complexes has shown great promise for identifying binding partners involved in these interactions. Current methodologies use metabolic oligosaccharide engineering (MOE) to incorporate photo-crosslinking sugars. However, these MOE strategies are not amenable to all cell types and can result in low incorporation and cell-surface display of the photo-crosslinking probe, limiting their utility for studying many types of interactions. We describe here an exo-enzymatic strategy for selectively introducing photo-crosslinking probes into cell-surface glycoconjugates using the recombinant human sialyltransferase ST6GAL1 and a diazirine-linked CMP-Neu5Ac derivative. Probe introduction is highly efficient, amenable to different cell types and resulted in improved crosslinking when compared to MOE. This exo-enzymatic labeling approach can selectively introduce the photo-crosslinking sugar on to specific glycan epitopes and subclasses by harnessing the specificity of the sialyltransferase employed, underscoring its potential as a tool to interrogate and identify glycoconjugate ligands for diverse glycan-binding proteins.

Like sialylation, fucose usually locates at the non-reducing ends of various glycans on glycoproteins and constitutes important glycan epitopes. Detecting the substrate glycans of fucosyltransferases is important for understanding how these glycan epitopes are regulated in response to different growth conditions and external stimuli. Here we report the detection of these glycans via enzymatic incorporation of fluorescent tagged fucose using fucosyltransferases including FUT2, FUT6, FUT7, and FUT8 and FUT9. More specifically, we describe the detection of substrate glycans of FUT8 and FUT9 on therapeutic antibodies and the detection of high mannose glycans on glycoproteins by enzymatic conversion of high mannose glycans to the substrate glycans of FUT8. By establishing a series of precursor glycans, we also demonstrate the substrate specificities of FUT8. Furthermore, using simultaneous enzymatic incorporation of both fluorescent sialic acids and fluorescent fucoses, we demonstrate the interplay between fucosylation and sialylation.

Novel antibodies have been created for targeted degradation of extracellular and membrane proteins in the lysosome. The mechanism of degradation of target proteins for these antibodies has involved either chemical conjugation of synthetic mannose 6-phosphate (M6P) or engineered bispecific antibodies. Currently, recombinant antibodies cannot be produced with naturally phosphorylated N-glycans. Here, we report the development of a novel platform technology for producing bifunctional therapeutic antibodies with high levels of M6P-bearing glycans directly from producing cells. The antibodies designated as phosphorylated N-glycosylated peptide chimeric antibodies (PNCA) maintain their affinity for antigens with concurrent high affinity binding to cell surface cation-independent mannose-6-phosphate receptors that facilitate internalization and delivery of antibody/antigen complexes to lysosomes for efficient degradation of both target extracellular soluble and membrane proteins. This PNCA approach provides a simple, scalable, and viable approach for producing naturally phosphorylated bifunctional antibodies from production cell lines for targeted protein degradation in lysosomes.

Glycan microarrays of sequence-defined glycans are the most widely used approach for high-throughput studies of interactions of glycans with glycan binding proteins. Currently nitrocellulose (NC) coated or N-hydroxysuccinimide (NHS) functionalized glass slides are the two most commonly used surfaces for immobilizing glycan probes as noncovalent and covalent arrays, respectively. The mode of glycan presentation can have an influence on the microarray readouts and is an important consideration in the glycan recognition knowledgebase. Here we present the development of glycan probes, tagged with a novel type of tri-functional Fmoc-amino-azido (FAA) linker, which can be readily converted into lipid-tagged or amino-terminating glycan probes for generating neoglycolipid (NGL)-based noncovalent arrays as well as covalent arrays. The azido functionality in the FAA glycan probes provides a means to light up the glycans via biorthogonal Click chemistry so that they become scanner-readable on the covalent arrays, which represents an advance in array quality control. Here analyses were carried out with a diverse set of 36 glycan binding proteins (GBPs) to compare the performance of 44 glycans presented in a liposomal formulation as noncovalent arrays on NC coated slides and on two types of NHS slides as covalent arrays. With most of anti-glycan antibodies and plant lectins investigated, there were negligible or subtle differences in the binding detected in different arrays. However, there were some striking differences observed between the covalent and noncovalent arrays. These include binding of Lotus Tetragonolobus Lectin (LTL) on the covalent but not the noncovalent arrays, and a clear preference observed for glycans on the noncovalent array in the binding of adhesins (VP1 proteins) of two human polyomaviruses JCPyV and BKPyV, and the human immune lectins Siglecs 7 and 9. Subtle yet significant differences in the binding to low affinity glycan ligands were also observed with three bacterial toxins in different arrays. These results revealed interesting insights into the binding behaviour of different GBPs on the noncovalent and covalent arrays and highlight the importance to consider different array platforms in elucidating glycan mediated interactions. The FAA glycan probes can be readily rendered fluorescent via Click chemistry in solution, enabling the detection of GBPs at the surface of cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/548266v1_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@8f2a49org.highwire.dtl.DTLVardef@1212409org.highwire.dtl.DTLVardef@c4ea39org.highwire.dtl.DTLVardef@1ed6a88_HPS_FORMAT_FIGEXP M_FIG C_FIG A new trifunctional linker can present glycans on different array surfaces for analyses of soluble glycan binding proteins or as fluorescent probes for binding by proteins at the surface of cells.

Of the various conjugation strategies for cellular biomolecules, Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is the preferred click chemistry approach due to its fast reaction rate and the commercial availability of a wide range of conjugates. While extracellular labeling of biomolecules using CuAAC has been widely adopted, intracellular labeling in live cells has been challenging as the high copper concentrations required for CuAAC reaction is toxic to biological systems. As a critical first step towards CuAAC-mediated intracellular labeling, an ultrasensitive CuAAC ligand is needed to reduce cytosolic copper concentrations while maintaining fast reaction kinetics. Here, we developed BTT-DNA, a new DNA oligomer-conjugated CuAAC ligand for click reaction biomolecular labeling. The DNA oligo attachment serves several purposes, including: 1. Increased localization of copper atoms near the ligand, which enables ligation of azide tags with much lower copper concentrations than commercially available CuAAC ligands and without the addition of exogenous copper salt; 2. Allows nucleic acid template-driven proximity ligation by choosing the attached DNA sequence, 3. Enables the liposome encapsulation and delivery of the ligand into live cells, and 4. Facilitates intracellular labeling of nascent phospholipids in live cells. We demonstrate that BTT-DNA mediated labeling has little to no effect on the overall cell health.

While radiopharmaceutical therapy (RPT) has become part of the standard-of-care for patients with advanced prostate cancers and neuroendocrine tumors (NETs), cures are elusive and normal tissue toxicity remain a challenge. Chemical groups susceptible to cleavage by enzymes present in tumors, tumor microenvironment or in normal tissues, have the potential to improve the therapeutic index for RPT. Using DOTA-TATE as an example and drawing from strategies used to develop antibody-drug conjugates, we designed, and synthesized, a chemically diverse series of linkers between the chelator (DOTA) and the targeting vector (TATE). Of the 10 agents we tested, two with cleavable linker domains reduced kidney retention compared to DOTA-TATE: the previously reported DOTA-MVK({varepsilon})-TATE, and a novel agent bearing cleavable beta-galactose ({beta}-Gal) unit, DOTA-{beta}-Gal-TATE. In murine models of NETs, positron emission tomography (PET) was used to image yttrium-86 (86Y)-labeled variants and show that, while the 86Y-DOTA-MVK({varepsilon})-TATE exhibits similar tumor uptake to the parent non-cleavable 86Y-DOTA-TATE, 86Y-DOTA-{beta}-Gal-TATE shows enhanced tumor uptake, resulting in up to 10-fold improvement in the tumor-to-kidney ratios compared to 86Y-DOTA-TATE. In vitro and in vivo studies confirm high efficiency, enzyme-specific cleavage of 86Y-DOTA-MVK({varepsilon})-TATE and 86Y-DOTA-{beta}-Gal-TATE, supporting a key role for cleavable linker chemistry in the observed outcomes. RPT studies using actinium-225 (225Ac)-labeled variants confirm that all agents are therapeutically effective and well tolerated. While both cleavable variants exhibit superior local control, overall survival, and more favorable toxicity profile when compared with 225Ac-DOTA-TATE, 225Ac-DOTA-{beta}-Gal-TATE demonstrated lower nephrotoxicity. Our findings suggest a potentially generalizable strategy for improving the pharmacokinetics of radiopharmaceutical therapy agents. One Sentence SummaryA {beta}-galactose-cleavable linker reduces kidney toxicity, enhances tumor targeting and therapeutic efficacy in radiopharmaceutical therapy.

1Chemical Exchange Saturation Transfer (CEST) magnetic resonance imaging (MRI) has been identified as a novel alternative to classical diagnostic imaging. Over the last several decades, many studies have been conducted to determine possible CEST agents, such as endogenously expressed compounds or proteins, that can be utilized to produce contrast with minimally invasive procedures and reduced or non-existent levels of toxicity. In recent years there has been an increased interest in the generation of genetically engineered CEST contrast agents, typically based on existing proteins with CEST contrast or modified to produce CEST contrast. We have developed an in-silico method for the evolution of peptide sequences to optimize CEST contrast and showed that these peptides could be combined to create de novo biosensors for CEST MRI. A single protein, superCESTide 2.0, was designed to be 198 amino acids. SuperCESTide 2.0 was expressed in E. coli and purified with size-exclusion chromatography. The magnetic transfer ratio asymmetry (MTRasym) generated by superCESTide 2.0 was comparable to levels seen in previous CEST reporters, such as protamine sulfate (salmon protamine, SP), Poly-L-Lysine (PLL), and human protamine (hPRM1). This data shows that novel peptides with sequences optimized in silico for CEST contrast that utilizes a more comprehensive range of amino acids can still produce contrast when assembled into protein units expressed in complex living environments.

Resistance to chimeric antigen receptor (CAR) T cell therapy develops through multiple mechanisms including antigen-loss escape and tumor-induced immune suppression. Expression of multiple CARs may overcome multi-antigen-loss escape. Similarly, expression of switch receptors that convert inhibitory immune checkpoint signals into positive costimulatory signals may enhance CAR T cell activity in the tumor microenvironment. Engineering multiple features into one cell product, however, is limited by transgene packaging constraints of current vector systems. Here, we describe a leucine zipper-based cell sorting methodology that enables selective single-step immunomagnetic purification of cells co-transduced with two vectors, designed to potentially double the number of incorporated transgenes. This "Zip-sorting" system facilitated generation of T cells simultaneously expressing up to four CARs and co-expressing up to three switch receptors. These multi-CAR multi-Switch receptor arrays enabled T cells to eliminate antigenically heterogeneous syngeneic leukemia populations co-expressing multiple inhibitory ligands. Zip-sorted multi-CAR multi-Switch receptor T cells represent a potent therapeutic strategy to overcome multiple mechanisms of CAR T cell resistance.

Single-domain antibodies, known as nanobodies (Nbs), are widely used in structural biology, therapeutics, and as molecular probes in biology and biotechnology. Nbs towards soluble proteins are routinely developed via alpaca immunization or directed evolution in yeast cell-surface display. However, for membrane proteins, the targets are generally detergent-solubilized, and there remains a need for Nb development methods against membrane proteins in a native-like membrane environment. To address this need, we present a protocol for Nb selection via extraction of membrane proteins into amphiphilic polymers such as styrene-maleic acid to produce purified membrane proteins in stable liponanoparticles. Proof of generality is demonstrated by applying the pipeline to four membrane-resident enzymes of differing fold, oligomerization state, and membrane topology (reentrant membrane helix, transmembrane, membrane-associated). Following screening for optimal stabilization into liponanoparticles, Nbs were selected against four target proteins from glycoconjugate biosynthesis pathways. The selected Nbs showed high affinity and selectivity towards their target proteins with KD apparent values ranging from 15 nM to 200 nM, depending on the Nb-protein conjugate. In accordance with their tight binding, various Nb-protein complexes were found to be stable to size-exclusion chromatography purification. The Nbs were also amenable to sortase-mediated ligation, enabling their conversion into molecular probes for the target membrane protein. The ability to select for such high-affinity Nb against membrane proteins in SMALP will facilitate their widespread application in cell biology and biomedical applications.

Molecular fluorescence-guided surgery has shown promise for tumor margin delineation but is limited by its depth profiling capability. Interestingly, most fluorophores, either clinically approved or in clinical trials, can also be used as photoacoustic contrast agents, yet their use is limited due to the low light fluence permitted for clinical use and the limited sensitivity of current photoacoustic imaging systems. There is therefore an urgent unmet need to establish methods for enhancing contrast in molecular targeted PA imaging which could potentially complement and overcome limitations in molecular fluorescence guided therapies. In this study, we compare the photoacoustic (PA) and fluorescence imaging capabilities of a cetuximab-IRDye800 conjugate in a subcutaneous tumor xenograft model. We demonstrate that while the fluorescence signal increases steadily over time after administration of cetuximab-IRDye800, PA signal peaks early ([~]2 fold higher at 6-hour as compared to pre-injection controls) and then decreases ([~]1.3 fold higher at 24-hour as compared to pre-injection controls). This pattern aligns with previous findings using other antibody-conjugated PA contrast agents. Mechanistically, we demonstrate that the formation of H-aggregates upon antibody conjugation enhances PA contrast of the IRDye800. The disruption of these H-aggregates, as the antibody-dye conjugate is degraded post receptor-mediated endocytosis, decreases PA signal intensity. The timeframe of maximum PA signal and decrease thereafter is consistent with the time frame of receptor-mediated endocytosis of cetuximab-IRDye800. Our data suggests that tumor cell surface binding results in peak PA signal while lysosomal localization and degradation results in a significant drop in PA signal. Our study sheds light on the distinct temporal dynamics of PA and fluorescence signals of Cetuximab-IRDye800 conjugate and we propose that optimizing IRDye800 conjugation to antibodies can further enhance PA signal intensity when timed to precisely to capture IRDye800 in an H-aggregate form.

Nucleic acid crosslinkers that covalently join commentary strands have applications as both pharmaceuticals and biochemical probes. Psoralen is a popular crosslinker moiety that reacts with double stranded DNA and RNA upon irradiation with long wave UV light. A commercially available compound EZ-Link Psoralen-PEG3-Biotin has been used in many studies to crosslink DNA and double strand RNA for genome-wide investigations. Here we present a novel probe, AP3B, which uses a psoralen derivative, 4-aminomethyltrioxsalen, to biotinylate nucleic acids. We show that this compound is 8-fold more effective at labeling DNA in cells and several hundred-fold more effective at crosslinking two strands of DNA in vitro than the commercially available compound EZ-Link Psoralen-PEG3-Biotin.

Detecting protein-protein interactions within cells is challenging. Transgenic approaches risk altering protein function via fluorescent tagging, while in situ methods lack in vivo compatibility. Here, we introduce fluorogenic probes with dual-tetrazine pegylated branched arms linked to xanthene dye. Activation requires both tetrazine arms to interact simultaneously with target proteins, enabling dual-substrate recognition. We applied our method to detect protein-protein interactions in both fixed and living cells, utilizing antibody conjugation for fixed cells and genetic code expansion for real-time detection in living cells. Our strategy ensures versatile applicability and seamless transition between fixed and living systems.

Preclinical evidence has suggested that the Glucocorticoid-Induced TNFR-related protein (GITR) may be valuable a target for the development of anticancer therapeutics, but clinical studies with GITR ligand (GITRL) have been disappointing. Here, we report the development of a fusion protein featuring GITR ligand (GITRL) fused to the F8 antibody which targets the alternatively-spliced EDA domain of fibronectin, a tumor-associated antigen often found around the tumor neovasculature. Five different formats for F8-GITRL fusion proteins were cloned and characterized, but quantitative biodistribution studies failed to evidence a preferential accumulation at the tumor site. The in vivo tumor targeting properties of F8-GITRL could be substantially improved by enzymatic deglycosylation or site-directed mutagenesis of the N-glycosylation consensus sequence. However, therapy studies in a murine model of cancer with the glycoengineered F8-GITRL N74S and N157T variant failed to elicit a durable anti-tumor response, both in monotherapy and in combination with PD-1 blockade. HIGHLIGHTSO_LIDifferent formats of fusion proteins featuring Glucocorticoid-induced TNFR-related protein ligand (GITRL) fused to a tumor-targeting antibody were produced. C_LIO_LIThe tumor uptake of the fusion proteins could be increased by enzymatic deglycosylation of the fusion protein or by site-directed mutagenesis of the N-glycosylation consensus sequences. C_LIO_LIThe fusion protein developed in this study failed to show any anti-tumor activity either alone or in combination with PD-1 inhibition. C_LI

Recently developed secondary nanobodies or single-domain antibodies present a powerful tool for immunodetection. Unlike traditional antibodies, their monovalence enables pre-association with primary antibodies prior to sample staining, presenting a straightforward affinity-based antibody labeling solution. This not only simplifies and streamlines immunoassays, it also supports multiplexed techniques where conflicts in the species of the desired primary antibodies preclude standard indirect immunostaining. Despite these advantages, the use of secondary nanobodies remains sparse, due perhaps to a lack of evaluation on their suitability for assays requiring quantification and an assessment of optimal protocols for their use. Here, we present a set of experiments spanning single-molecule detection to cell imaging that can be used to validate secondary nanobody binding, specificity, and their propensity for mis-targeted binding in multiplex assays. Using these tools, we analyzed the binding properties of commercially available secondary nanobodies and outline optimized protocols for their robust use.

Interactions between glycans and glycan-binding proteins (GBPs) mediate diverse cellular functions, and therefore are of diagnostic and therapeutic significance. Current leading strategies for studying glycan-GBP interactions require specialized knowledge and instrumentation. In this study, we report a strategy for studying glycan-GBP interactions that uses PCR, qPCR and next-generation sequencing (NGS) technologies that are more routinely accessible. Our headpiece conjugation-code ligation (HCCL) strategy couples glycans with unique DNA codes that specify glycan sugar moieties and glycosidic linkages when sequenced. We demonstrate the technology by synthesizing a DNA encoded glycan library of 50 biologically relevant glycans (DEGL-50) and probing interactions against 25 target proteins including lectins and antibodies. Data show glycan-GPB interactions in solution that are consistent with lower content, lower throughput ELISA assays. Data further demonstrate how monovalent and multivalent headpieces can be used to increase glycan-GPB interactions and enrich signals while using smaller sample sizes. The flexibility of our modular HCCL strategy has potential for producing large glycan libraries, facilitating high content-high throughput glycan binding studies, and increasing access to lower cost glyco-analyses. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC="FIGDIR/small/017012v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@fb1c1dorg.highwire.dtl.DTLVardef@1f2c3ceorg.highwire.dtl.DTLVardef@11546e7org.highwire.dtl.DTLVardef@1e21a83_HPS_FORMAT_FIGEXP M_FIG C_FIG

Despite progress made in the development of anticancer therapeutics, traditional small-molecule chemotherapeutics often struggle to overcome toxicity, efficacy, and off-target effects. These specific issues can be overcome by either encapsulating the drug or by targeting it directly to the tumor cell. Here, we describe a novel targeted nanoparticle (referred to as a nano-antibody-drug conjugate Targeted Nanosphere or nADC/TNS), based cancer therapeutic platform that can improve the efficacy of a broad range of existing therapeutics. Targeting is antibody-directed, as with antibody-drug conjugates (ADCs). Still, the payload per antibody is vastly greater by orders of magnitude (a thousand for nADC/TNS versus two to eight for ADCs). The nADC/TNS consists of an approximately 80 nm drug-filled nanoparticle composed of phospholipids, cholesterol, and UV cross-linkable diacetylene lipids. We describe the preparation, characterization, and evaluation of nADC/TNS as a novel, versatile, and effective treatment modality for cancer and potentially other diseases. This report focuses on data with nADC/TNS variants NV101 (anti-CD99 targeted, doxorubicin-filled), NV102 (anti-CD19 targeted, doxorubicin-filled), and NV103 (anti-CD99 targeted, irinotecan-filled). We investigated NV101 and NV103 in a mouse model with implanted and metastatic Ewing tumors (ES). NV101 demonstrated significant tumor burden reduction while NV103 induced complete ablation of ES tumors. NV102 demonstrated complete ablation of chemotherapy-resistant relapsed adult lymphocytic leukemia (ALL). These results document the potential superior efficacy of antibody-targeted nanoparticles containing a variety of small-molecule payloads, compared to their free molecule equivalents.

1.Single-domain antibodies, also known as nanobodies, are broadly important for studying the structure and conformational states of several classes of proteins, including membrane proteins, enzymes, and amyloidogenic proteins. Conformational nanobodies specific for aggregated conformations of amyloidogenic proteins are particularly needed to better target and study aggregates associated with a growing class of associated diseases, especially neurodegenerative disorders such as Alzheimers and Parkinsons diseases. However, there are few reported nanobodies with both conformational and sequence specificity for amyloid aggregates, especially for large and complex proteins such as the tau protein associated with Alzheimers disease, due to difficulties in selecting nanobodies that bind to complex aggregated proteins. Here, we report the selection of conformational nanobodies that selectively recognize aggregated (fibrillar) tau relative to soluble (monomeric) tau. Notably, we demonstrate that these nanobodies can be directly isolated from immune libraries using quantitative flow cytometric sorting of yeast-displayed libraries against tau aggregates conjugated to quantum dots, and this process eliminates the need for secondary nanobody screening. The isolated nanobodies demonstrate conformational specificity for tau aggregates in brain samples from both transgenic tau mouse models and human tauopathies. We expect that our facile approach will be broadly useful for isolating conformational nanobodies against diverse amyloid aggregates and other complex antigens.

Genetic tags are transformative tools for investigating the function, localization, and interactions of cellular proteins. Most studies today are reliant on selective labeling of more than one protein to obtain comprehensive information on a proteins behavior in situ. Some proteins can be analyzed by fusion to protein tag, such as green fluorescent protein, HaloTag, or SNAP-Tag. Other proteins benefit from labeling via small peptide tags, such as the recently reported versatile interacting peptide (VIP) tags. VIP tags enable observations of protein localization and trafficking with bright fluorophores or nanoparticles. Here we expand the VIP toolkit by presenting two new tags: TinyVIPER and PunyVIPER. These two tags were designed for use with MiniVIPER for labeling up to three distinct proteins at once in living cells. Labeling is mediated by the formation of a high affinity, biocompatible heterodimeric coiled coil. Each tag was validated by fluorescence microscopy, including observation of transferrin receptor 1 trafficking in live cells. We verified that labeling via each tag is highly specific, with no cross-reactivity between the three VIP tags under cellular conditions. Lastly, the self-sorting tags were used for simultaneous labeling of three protein targets (i.e., TOMM20, histone 2B, and actin), highlighting their utility for multicolor microscopy. MiniVIPER, TinyVIPER, and PunyVIPER are small and robust peptide tags for selective labeling of cellular proteins.

Single domain antibodies, often known as nanobodies, are versatile molecules with therapeutic and diagnostic applications, but they are primarily developed through immunization of camelids. This approach is not scalable by automation, not effective for non-immunogenic or toxic antigens, and prevents the use of modified scaffolds for altered pharmacokinetic properties. Synthetic libraries allow for pre-selection of a single domain framework tailored to its intended downstream use. One area of interest for these biologic vectors is radiopharmaceuticals. Ideal radiopharmaceutical pharmacokinetic properties differ from most traditional therapeutics, as short plasma circulation and rapid kidney clearance are necessary to avoid dose-limiting organ radiation. Although there are a growing number of nanobody radiopharmaceuticals in clinical trials, their frameworks and corresponding pharmacokinetic properties vary. One potential method for improving the development of novel single domain antibody radiopharmaceuticals is through synthetic libraries based on nanobodies with proven clinically acceptable pharmacokinetics. We developed a modular synthetic nanobody phage display vector based on the scaffold of the 2Rs15d nanobody that allows for manipulation of the binding and framework regions. Using this vector, we created a library of nanobodies with a randomized CDR2 containing over 1.7x106 unique sequences/{micro}L. As a proof-of-concept, we panned the library for nanobodies binding calreticulin (CALR), a protein critical in immunogenic cell death. One isolated clone, Cal3, has a measured affinity of 140 nM for CALR and is cross-reactive with mouse and human CALR. Using positron emission tomography (PET) imaging, the radiolabeled 64Cu-NOTA-Cal3 demonstrated CALR binding in vivo, representing the first reported synthetic nanobody characterized by PET imaging. This study demonstrates the feasibility of building and panning synthetic libraries for high-affinity radiopharmaceutical nanobodies as an alternative to immunized camelid libraries.

The identification of tumor-specific biomarkers is one of the bottlenecks in the development of cancer therapies. Previous work revealed altered surface levels of reduced/oxidized cysteines in many cancers due to overexpression of redox-controlling proteins such as protein disulfide isomerases on the cell surface. Alterations in surface thiols can promote cell adhesion and metastasis, making thiols attractive targets for treatment. Only a few tools are available to study surface thiols on cancer cells and exploit them for theranostics. Here, we describe a nanobody (CB2) that recognizes B cell lymphoma in a thiol-dependent manner. CB2 binding strictly requires the presence of a non-conserved cysteine in the antigen-binding region and correlates with elevated surface levels of free thiols on B cell lymphoma compared to healthy lymphocytes. Nanobody CB2 can induce complement-dependent cytotoxicity against lymphoma cells when functionalized with synthetic rhamnose trimers. Lymphoma cells internalize CB2 in a thiol-mediated manner such that the nanobody can be used to deliver cytotoxic agents. Hence, surface thiols can be used as lymphoma biomarkers and targeted by thiol-binding nanobodies. Functionalization of internalizable CB2 is the basis for a range of diagnostic and therapeutic applications of this thiol-binding nanobody. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/497993v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1f47ba9org.highwire.dtl.DTLVardef@1e2bd41org.highwire.dtl.DTLVardef@f7151eorg.highwire.dtl.DTLVardef@18b7681_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisNanobody CB2 specifically binds and internalizes into B cell lymphoma via thiol-based interactions. Functionalized CB2 can be used for complement recruitment or drug delivery to lymphoma cells.