A selection and optimization strategy for single-domain antibodies targeting the PHF6 linear peptide within the tau intrinsically disordered protein.

The use of variable domain of the heavy-chain of the heavy-chain-only antibodies (VHHs) as disease-modifying biomolecules in neurodegenerative disorders holds promises, including targeting of aggregation-sensitive proteins. Exploitation of their clinical values depends however on the capacity to deliver VHHs with optimal physico-chemical properties for their specific context of use. We described previously a VHH with high therapeutic potential in a family of neurodegenerative diseases called tauopathies. The activity of this promising parent VHH named Z70 relies on its binding within the central region of the tau protein. Accordingly, we carried out random mutagenesis followed by yeast two-hybrid screening to obtain optimized variants. The VHHs selected from this initial screen targeted the same epitope as VHH Z70 as shown using NMR spectroscopy and had indeed improved binding affinities according to dissociation constant values obtained by surface plasmon resonance spectroscopy. The improved affinities can be partially rationalized based on three-dimensional structures and NMR data of three complexes consisting of an optimized VHH and a peptide containing the tau epitope. Interestingly, the ability of the VHH variants to inhibit tau aggregation and seeding could not be predicted from their affinity alone. We indeed showed that the in vitro and in cellulo VHH stabilities are other limiting key factors to their efficacy. Our results demonstrate that only a complete pipeline of experiments, here described, permits a rational selection of optimized VHH variants, resulting in the selection of VHH variants with higher affinities and/or acting against tau seeding in cell models.

Sequence tolerance of immunoglobulin variable domain framework regions to noncanonical intradomain disulfide linkages.

Most immunoglobulin (Ig) domains bear only a single highly conserved canonical intradomain, inter-ß-sheet disulfide linkage formed between Cys23-Cys104, and incorporation of rare noncanonical disulfide linkages at other locations can enhance Ig domain stability. Here, we exhaustively surveyed the sequence tolerance of Ig variable (V) domain framework regions (FRs) to noncanonical disulfide linkages. Starting from a destabilized VH domain lacking a Cys23-Cys104 disulfide linkage, we generated and screened phage-displayed libraries of engineered VHs, bearing all possible pairwise combinations of Cys residues in neighboring ß-strands of the Ig fold FRs. This approach identified seven novel Cys pairs in VH FRs (Cys4-Cys25, Cys4-Cys118, Cys5-Cys120, Cys6-Cys119, Cys22-Cys88, Cys24-Cys86, and Cys45-Cys100; the international ImMunoGeneTics information system numbering), whose presence rescued domain folding and stability. Introduction of a subset of these noncanonical disulfide linkages (three intra-ß-sheet: Cys4-Cys25, Cys22-Cys88, and Cys24-Cys86, and one inter-ß-sheet: Cys6-Cys119) into a diverse panel of VH, VL, and VHH domains enhanced their thermostability and protease resistance without significantly impacting expression, solubility, or binding to cognate antigens. None of the noncanonical disulfide linkages identified were present in the natural human VH repertoire. These data reveal an unexpected permissiveness of Ig V domains to noncanonical disulfide linkages at diverse locations in FRs, absent in the human repertoire, whose presence is compatible with antigen recognition and improves domain stability. Our work represents the most complete assessment to date of the role of engineered noncanonical disulfide bonding within FRs in Ig V domain structure and function.

Quantitative flow cytometric selection of tau conformational nanobodies specific for pathological aggregates.

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 Alzheimer's and Parkinson's 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 Alzheimer's 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 a transgenic mouse model 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.

Synthetic receptor platform to identify loss-of-function single nucleotide variants and designed mutants in the death receptor Fas/CD95.

Synthetic biology has emerged as a useful technology for studying cytokine signal transduction. Recently, we described fully synthetic cytokine receptors to phenocopy trimeric receptors such as the death receptor Fas/CD95. Using a nanobody as an extracellular-binding domain for mCherry fused to the natural receptor's transmembrane and intracellular domain, trimeric mCherry ligands were able to induce cell death. Among the 17,889 single nucleotide variants in the SNP database for Fas, 337 represent missense mutations that functionally remained largely uncharacterized. Here, we developed a workflow for the Fas synthetic cytokine receptor system to functionally characterize missense SNPs within the transmembrane and intracellular domain of Fas. To validate our system, we selected five functionally assigned loss-of-function (LOF) polymorphisms and included 15 additional unassigned SNPs. Moreover, based on structural data, 15 gain-of-function or LOF candidate mutations were additionally selected. All 35 nucleotide variants were functionally investigated through cellular proliferation, apoptosis and caspases 3 and 7 cleavage assays. Collectively, our results showed that 30 variants resulted in partial or complete LOF, while five lead to a gain-of-function. In conclusion, we demonstrated that synthetic cytokine receptors are a suitable tool for functional SNPs/mutations characterization in a structured workflow.

Development of a V5-tag-directed nanobody and its implementation as an intracellular biosensor of GPCR signaling.

Protein-protein interactions (PPIs) form the foundation of any cell signaling network. Considering that PPIs are highly dynamic processes, cellular assays are often essential for their study because they closely mimic the biological complexities of cellular environments. However, incongruity may be observed across different PPI assays when investigating a protein partner of interest; these discrepancies can be partially attributed to the fusion of different large functional moieties, such as fluorescent proteins or enzymes, which can yield disparate perturbations to the protein's stability, subcellular localization, and interaction partners depending on the given cellular assay. Owing to their smaller size, epitope tags may exhibit a diminished susceptibility to instigate such perturbations. However, while they have been widely used for detecting or manipulating proteins in vitro, epitope tags lack the in vivo traceability and functionality needed for intracellular biosensors. Herein, we develop NbV5, an intracellular nanobody binding the V5-tag, which is suitable for use in cellular assays commonly used to study PPIs such as BRET, NanoBiT, and Tango. The NbV5:V5 tag system has been applied to interrogate G protein-coupled receptor signaling, specifically by replacing larger functional moieties attached to the protein interactors, such as fluorescent or luminescent proteins (∼30 kDa), by the significantly smaller V5-tag peptide (1.4 kDa), and for microscopy imaging which is successfully detected by NbV5-based biosensors. Therefore, the NbV5:V5 tag system presents itself as a versatile tool for live-cell imaging and a befitting adaptation to existing cellular assays dedicated to probing PPIs.

Inhibition of cleavage of human complement component C5 and the R885H C5 variant by two distinct high affinity anti-C5 nanobodies.

The human complement system plays a crucial role in immune defense. However, its erroneous activation contributes to many serious inflammatory diseases. Since most unwanted complement effector functions result from C5 cleavage into C5a and C5b, development of C5 inhibitors, such as clinically approved monoclonal antibody eculizumab, are of great interest. Here, we developed and characterized two anti-C5 nanobodies, UNbC5-1 and UNbC5-2. Using surface plasmon resonance, we determined a binding affinity of 119.9 pM for UNbC5-1 and 7.7 pM for UNbC5-2. Competition experiments determined that the two nanobodies recognize distinct epitopes on C5. Both nanobodies efficiently interfered with C5 cleavage in a human serum environment, as they prevented red blood cell lysis via membrane attack complexes (C5b-9) and the formation of chemoattractant C5a. The cryo-EM structure of UNbC5-1 and UNbC5-2 in complex with C5 (3.6 Å resolution) revealed that the binding interfaces of UNbC5-1 and UNbC5-2 overlap with known complement inhibitors eculizumab and RaCI3, respectively. UNbC5-1 binds to the MG7 domain of C5, facilitated by a hydrophobic core and polar interactions, and UNbC5-2 interacts with the C5d domain mostly by salt bridges and hydrogen bonds. Interestingly, UNbC5-1 potently binds and inhibits C5 R885H, a genetic variant of C5 that is not recognized by eculizumab. Altogether, we identified and characterized two different, high affinity nanobodies against human C5. Both nanobodies could serve as diagnostic and/or research tools to detect C5 or inhibit C5 cleavage. Furthermore, the residues targeted by UNbC5-1 hold important information for therapeutic inhibition of different polymorphic variants of C5.

Inter-rater and intra-rater agreement of [99mTc]-labelled NM-01, a single-domain programmed death-ligand 1 (PD-L1) antibody, using quantitative SPECT/CT in non-small cell lung cancer.

BACKGROUND: Immune checkpoint inhibitors, including those against programmed cell death protein-1 (PD-1) or its ligand (PD-L1), are routinely used to treat non-small cell lung cancer (NSCLC). PD-L1 is a validated prognostic and predictive immunohistochemical biomarker of anti-PD-1/PD-L1 therapy but displays temporospatial heterogeneity of expression. Non-invasive radiopharmaceutical techniques, including technetium-99m [99mTc]-labelled anti-PD-L1 single-domain antibody (NM-01) SPECT/CT, have the potential to improve the predictive value of PD-L1 assessment. This study aims to determine the inter- and intra-rater agreement of the quantitative measurement of [99mTc]NM-01 SPECT/CT in NSCLC. METHODS: Participants (n = 14) with untreated advanced NSCLC underwent [99mTc]NM-01 SPECT/CT at baseline (n = 3) or at baseline plus 9-week follow-up (n = 11). [99mTc]NM-01 uptake (of primary lung, lymph node, thoracic and distant metastases, and healthy reference tissues) was measured using SUVmax and malignant lesion-to-blood pool ratios with Siemens xSPECT Broad Quantification software by three independent raters. Intraclass correlation coefficients (ICC) were calculated and Bland-Altman plot analysis performed to determine inter- and intra-rater agreement. RESULTS: There was excellent inter-rater agreement of manual freehand SUVmax scores of primary lung tumour (T; n = 25; ICC 1.00; 95% CI 0.99-1.00), individual lymph node metastases (LN; n = 56; ICC 0.97; 95% CI 0.95-0.98), thoracic metastases (ThMet; n = 9; ICC 0.94; 95% CI 0.83-0.99) and distant metastases (DisMet; n = 21; ICC 0.91; 95% CI 0.83-0.96). The inter-rater ICCs of tumour-to-blood pool (T:BP), LN:BP, ThMet:BP and DisMet:BP measures of [99mTc]NM-01 uptake also demonstrated good or excellent agreement. Manual freehand scoring of T, LN, ThMet, DisMet and their ratios using [99mTc]NM-01 SPECT/CT following a 28-day interval was consistent for all raters with good or excellent intra-rater agreement demonstrated (ICCs range 0.86-1.00). CONCLUSION: Quantitative assessment of [99mTc]NM-01 SPECT/CT in NSCLC, using SUVmax of malignant primary or metastatic lesions and their ratios with healthy reference tissues, demonstrated good or excellent inter- and intra-rater agreement in this study. Further validation with ongoing and future larger cohort studies is now warranted. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov identifier no. NCT04436406 (registered 18th June 2020; available at https://clinicaltrials.gov/ct2/show/NCT04436406 ) and NCT04992715 (registered 5th August 2021; available at https://clinicaltrials.gov/ct2/show/NCT04992715 ).

Nanobody inhibitors of Plexin-B1 identify allostery in plexin-semaphorin interactions and signaling.

Plexin-B1 is a receptor for the cell surface semaphorin, Sema4D. This signaling system has been implicated in a variety of human diseases, including cancer, multiple sclerosis and osteoporosis. While inhibitors of the Plexin-B1:Sema4D interaction have been previously reported, understanding their mechanism has been hindered by an incomplete structural view of Plexin-B1. In this study, we have raised and characterized a pair of nanobodies that are specific for mouse Plexin-B1 and which inhibit the binding of Sema4D to mouse Plexin-B1 and its biological activity. Structural studies of these nanobodies reveal that they inhibit the binding of Sema4D in an allosteric manner, binding to epitopes not previously reported. In addition, we report the first unbound structure of human Plexin-B1, which reveals that Plexin-B1 undergoes a conformational change on Sema4D binding. These changes mirror those seen upon binding of allosteric peptide modulators, which suggests a new model for understanding Plexin-B1 signaling and provides a potential innovative route for therapeutic modulation of Plexin-B1.

Identification, binding, and structural characterization of single domain anti-PD-L1 antibodies inhibitory of immune regulatory proteins PD-1 and CD80.

Programmed death-ligand 1 (PD-L1) is a key immune regulatory protein that interacts with programmed cell death protein 1 (PD-1), leading to T-cell suppression. Whilst this interaction is key in self-tolerance, cancer cells evade the immune system by overexpressing PD-L1. Inhibition of the PD-1/PD-L1 pathway with standard monoclonal antibodies has proven a highly effective cancer treatment; however, single domain antibodies (VHH) may offer numerous potential benefits. Here, we report the identification and characterization of a diverse panel of 16 novel VHHs specific to PD-L1. The panel of VHHs demonstrate affinities of 0.7 nM to 5.1 µM and were able to completely inhibit PD-1 binding to PD-L1. The binding site for each VHH on PD-L1 was determined using NMR chemical shift perturbation mapping and revealed a common binding surface encompassing the PD-1-binding site. Additionally, we solved crystal structures of two representative VHHs in complex with PD-L1, which revealed unique binding modes. Similar NMR experiments were used to identify the binding site of CD80 on PD-L1, which is another immune response regulatory element and interacts with PD-L1 localized on the same cell surface. CD80 and PD-1 were revealed to share a highly overlapping binding site on PD-L1, with the panel of VHHs identified expected to inhibit CD80 binding. Comparison of the CD80 and PD-1 binding sites on PD-L1 enabled the identification of a potential antibody binding region able to confer specificity for the inhibition of PD-1 binding only, which may offer therapeutic benefits to counteract cancer cell evasion of the immune system.

Tripartite split-GFP assay to identify selective intracellular nanobody that suppresses GTPase RHOA subfamily downstream signaling.

Strategies based on intracellular expression of artificial binding domains present several advantages over manipulating nucleic acid expression or the use of small molecule inhibitors. Intracellularly-functional nanobodies can be considered as promising macrodrugs to study key signaling pathways by interfering with protein-protein interactions. With the aim of studying the RAS-related small GTPase RHOA family, we previously isolated, from a synthetic phage display library, nanobodies selective towards the GTP-bound conformation of RHOA subfamily proteins that lack selectivity between the highly conserved RHOA-like and RAC subfamilies of GTPases. To identify RHOA/ROCK pathway inhibitory intracellular nanobodies, we implemented a stringent, subtractive phage display selection towards RHOA-GTP followed by a phenotypic screen based on F-actin fiber loss. Intracellular interaction and intracellular selectivity between RHOA and RAC1 proteins was demonstrated by adapting the sensitive intracellular protein-protein interaction reporter based on the tripartite split-GFP method. This strategy led us to identify a functional intracellular nanobody, hereafter named RH28, that does not cross-react with the close RAC subfamily and blocks/disrupts the RHOA/ROCK signaling pathway in several cell lines without further engineering or functionalization. We confirmed these results by showing, using SPR assays, the high specificity of the RH28 nanobody towards the GTP-bound conformation of RHOA subfamily GTPases. In the metastatic melanoma cell line WM266-4, RH28 expression triggered an elongated cellular phenotype associated with a loss of cellular contraction properties, demonstrating the efficient intracellular blocking of RHOA/B/C proteins downstream interactions without the need of manipulating endogenous gene expression. This work paves the way for future therapeutic strategies based on protein-protein interaction disruption with intracellular antibodies.

High-efficiency recombinant protein purification using mCherry and YFP nanobody affinity matrices.

Mammalian cell lines are important expression systems for large proteins and protein complexes, particularly when the acquisition of post-translational modifications in the protein's native environment is desired. However, low or variable transfection efficiencies are challenges that must be overcome to use such an expression system. Expression of recombinant proteins as a fluorescent protein fusion enables real-time monitoring of protein expression, and also provides an affinity handle for one-step protein purification using a suitable affinity reagent. Here, we describe a panel of anti-GFP and anti-mCherry nanobody affinity matrices and their efficacy for purification of GFP/YFP or mCherry fusion proteins. We define the molecular basis by which they bind their target proteins using X-ray crystallography. From these analyses, we define an optimal pair of nanobodies for purification of recombinant protein tagged with GFP/YFP or mCherry, and demonstrate these nanobody-sepharose supports are stable to many rounds of cleaning and extended incubation in denaturing conditions. Finally, we demonstrate the utility of the mCherry-tag system by using it to purify recombinant human topoisomerase 2α expressed in HEK293F cells. The mCherry-tag and GFP/YFP-tag expression systems can be utilized for recombinant protein expression individually or in tandem for mammalian protein expression systems where real-time monitoring of protein expression levels and a high-efficiency purification step is needed.

Structures of synthetic nanobody-SARS-CoV-2 receptor-binding domain complexes reveal distinct sites of interaction.

Combating the worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of new variants demands understanding of the structural basis of the interaction of antibodies with the SARS-CoV-2 receptor-binding domain (RBD). Here, we report five X-ray crystal structures of sybodies (synthetic nanobodies) including those of binary and ternary complexes of Sb16-RBD, Sb45-RBD, Sb14-RBD-Sb68, and Sb45-RBD-Sb68, as well as unliganded Sb16. These structures reveal that Sb14, Sb16, and Sb45 bind the RBD at the angiotensin-converting enzyme 2 interface and that the Sb16 interaction is accompanied by a large conformational adjustment of complementarity-determining region 2. In contrast, Sb68 interacts at the periphery of the SARS-CoV-2 RBD-angiotensin-converting enzyme 2 interface. We also determined cryo-EM structures of Sb45 bound to the SARS-CoV-2 spike protein. Superposition of the X-ray structures of sybodies onto the trimeric spike protein cryo-EM map indicates that some sybodies may bind in both "up" and "down" configurations, but others may not. Differences in sybody recognition of several recently identified RBD variants are explained by these structures.

A Rationally Designed Bovine IgA Fc Scaffold Enhances in planta Accumulation of a VHH-Fc Fusion Without Compromising Binding to Enterohemorrhagic E. coli.

We previously isolated a single domain antibody (VHH) that binds Enterohemorrhagic Escherichia coli (EHEC) with the end-goal being the enteromucosal passive immunization of cattle herds. To improve the yield of a chimeric fusion of the VHH with an IgA Fc, we employed two rational design strategies, supercharging and introducing de novo disulfide bonds, on the bovine IgA Fc component of the chimera. After mutagenizing the Fc, we screened for accumulation levels after transient transformation in Nicotiana benthamiana leaves. We identified and characterized five supercharging and one disulfide mutant, termed '(5 + 1)Fc', that improve accumulation in comparison to the native Fc. Combining all these mutations is associated with a 32-fold increase of accumulation for the Fc alone, from 23.9 mg/kg fresh weight (FW) to 599.5 mg/kg FW, as well as a twenty-fold increase when fused to a VHH that binds EHEC, from 12.5 mg/kg FW tissue to 236.2 mg/kg FW. Co-expression of native or mutated VHH-Fc with bovine joining chain (JC) and bovine secretory component (SC) followed by co-immunoprecipitation suggests that the stabilizing mutations do not interfere with the capacity of VHH-Fc to assemble with JC and FC into a secretory IgA. Both the native and the mutated VHH-Fc similarly neutralized the ability of four of the seven most prevalent EHEC strains (O157:H7, O26:H11, O111:Hnm, O145:Hnm, O45:H2, O121:H19 and O103:H2), to adhere to HEp-2 cells as visualized by immunofluorescence microscopy and quantified by fluorometry. These results collectively suggest that supercharging and disulfide bond tethering on a Fc chain can effectively improve accumulation of a VHH-Fc fusion without impacting VHH functionality.

Advanced genetic engineering to achieve in vivo targeting of adenovirus utilizing camelid single domain antibody.

For the developing field of gene therapy the successful address of the basic requirement effective gene delivery has remained a critical barrier. In this regard, the "Holy Grail" vector envisioned by the field's pioneers embodied the ability to achieve efficient and specific in vivo gene delivery. Functional linkage of antibody selectivity with viral vector efficiency represented a logical strategy but has been elusive. Here we have addressed this key issue by developing the technical means to pair antibody-based targeting with adenoviral-mediated gene transfer. Our novel method allows efficient and specific gene delivery. Importantly, our studies validated the achievement of this key vectorology mandate in the context of in vivo gene delivery. Vectors capable of effective in vivo delivery embody the potential to dramatically expand the range of successful gene therapy cures.

Inter- and intraobserver agreement of the quantitative assessment of [99mTc]-labelled anti-programmed death-ligand 1 (PD-L1) SPECT/CT in non-small cell lung cancer.

PURPOSE: Checkpoint inhibition therapy using monoclonal antibodies against programmed cell death protein 1 (PD-1) or its ligand (PD-L1) is now standard management of non-small cell lung cancer (NSCLC). PD-L1 expression is a validated and approved prognostic and predictive biomarker for anti-PD-1/PD-L1 therapy. Technetium-99 m [99mTc]-labelled anti-PD-L1 single-domain antibody (NM-01) SPECT/CT quantification correlates with PD-L1 expression in NSCLC, presenting an opportunity for non-invasive assessment. The aim of this study was to determine the inter- and intraobserver agreement of the quantitative assessment of [99mTc]NM-01 SPECT/CT in NSCLC. METHODS: [99mTc]NM-01 SPECT/CT studies of 21 consecutive NSCLC participants imaged for the evaluation of PD-L1 expression were analysed. Three independent observers measured maximum counts in a tumour region of interest (ROImax) of primary lung, metastatic lesions and normal tissue references of both 1 and 2 h post-injection (n = 42) anonymised studies using a manual technique. Intraclass correlation coefficients (ICC) were calculated, and Bland-Altman plot analysis was performed to determine inter- and intraobserver agreement. RESULTS: Intraclass correlation of primary lung tumour-to-blood pool (T:BP; ICC 0.83, 95% CI 0.73-0.90) and lymph node metastasis-to-blood pool (LN:BP; ICC 0.87, 0.81-0.92) measures of [99mTc]NM-01 uptake was good to excellent between observers. Freehand ROImax of T (ICC 0.94), LN (ICC 0.97), liver (ICC 0.97) and BP (ICC 0.90) reference tissues also demonstrated excellent interobserver agreement. ROImax scoring of healthy lung demonstrated moderate to excellent interobserver agreement (ICC 0.84) and improved when measured consistently at the level of the aortic arch (ICC 0.89). Manual ROImax re-scoring of T, LN, T:BP and LN:BP using [99mTc]NM-01 SPECT/CT following a 42-day interval was consistent with excellent intraobserver agreement (ICC range 0.95-0.97). CONCLUSION: Good to excellent inter- and intraobserver agreement of the quantitative assessment of [99mTc]NM-01 SPECT/CT in NSCLC was demonstrated in this study, including T:BP which has been shown to correlate with PD-L1 status. [99mTc]NM-01 SPECT/CT has the potential to reliably and non-invasively assess PD-L1 expression. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov identifier no. NCT02978196. Registered 30th November 2016.

Exploring cellular biochemistry with nanobodies.

Reagents that bind tightly and specifically to biomolecules of interest remain essential in the exploration of biology and in their ultimate application to medicine. Besides ligands for receptors of known specificity, agents commonly used for this purpose are monoclonal antibodies derived from mice, rabbits, and other animals. However, such antibodies can be expensive to produce, challenging to engineer, and are not necessarily stable in the context of the cellular cytoplasm, a reducing environment. Heavy chain-only antibodies, discovered in camelids, have been truncated to yield single-domain antibody fragments (VHHs or nanobodies) that overcome many of these shortcomings. Whereas they are known as crystallization chaperones for membrane proteins or as simple alternatives to conventional antibodies, nanobodies have been applied in settings where the use of standard antibodies or their derivatives would be impractical or impossible. We review recent examples in which the unique properties of nanobodies have been combined with complementary methods, such as chemical functionalization, to provide tools with unique and useful properties.

A C3-specific nanobody that blocks all three activation pathways in the human and murine complement system.

The complement system is a tightly controlled proteolytic cascade in the innate immune system, which tags intruding pathogens and dying host cells for clearance. An essential protein in this process is complement component C3. Uncontrolled complement activation has been implicated in several human diseases and disorders and has spurred the development of therapeutic approaches that modulate the complement system. Here, using purified proteins and several biochemical assays and surface plasmon resonance, we report that our nanobody, hC3Nb2, inhibits C3 deposition by all complement pathways. We observe that the hC3Nb2 nanobody binds human native C3 and its degradation products with low nanomolar affinity and does not interfere with the endogenous regulation of C3b deposition mediated by Factors H and I. Using negative stain EM analysis and functional assays, we demonstrate that hC3Nb2 inhibits the substrate-convertase interaction by binding to the MG3 and MG4 domains of C3 and C3b. Furthermore, we notice that hC3Nb2 is cross-reactive and inhibits the lectin and alternative pathway in murine serum. We conclude that hC3Nb2 is a potent, general, and versatile inhibitor of the human and murine complement cascades. Its cross-reactivity suggests that this nanobody may be valuable for analysis of complement activation within animal models of both acute and chronic diseases.

Isolation and structural characterization of a Zn2+-bound single-domain antibody against NorC, a putative multidrug efflux transporter in bacteria.

Single-chain antibodies from camelids have served as powerful tools ranging from diagnostics and therapeutics to crystallization chaperones meant to study protein structure and function. In this study, we isolated a single-chain antibody from an Indian dromedary camel (ICab) immunized against a bacterial 14TM helix transporter, NorC, from Staphylococcus aureus We identified this antibody in a yeast display screen built from mononuclear cells isolated from the immunized camel and purified the antibody from Escherichia coli after refolding it from inclusion bodies. The X-ray structure of the antibody at 2.15 Å resolution revealed a unique feature within its CDR3 loop, which harbors a Zn2+-binding site that substitutes for a loop-stabilizing disulfide bond. We performed mutagenesis to compromise the Zn2+-binding site and observed that this change severely hampered antibody stability and its ability to interact with the antigen. The lack of bound Zn2+ also made the CDR3 loop highly flexible, as observed in all-atom simulations. Using confocal imaging of NorC-expressing E. coli spheroplasts, we found that the ICab interacts with the extracellular surface of NorC. This suggests that the ICab could be a valuable tool for detecting methicillin-resistant S. aureus strains that express efflux transporters such as NorC in hospital and community settings.

Early Phase I Study of a 99mTc-Labeled Anti-Programmed Death Ligand-1 (PD-L1) Single-Domain Antibody in SPECT/CT Assessment of PD-L1 Expression in Non-Small Cell Lung Cancer.

Immunotherapy with checkpoint inhibitor programmed cell death 1 (PD-1)/programmed death ligand-1 (PD-L1) antibodies demonstrates improvements in treatment of advanced non-small cell lung cancer. Treatment stratification depends on immunohistochemical PD-L1 measurement of biopsy material, an invasive method that does not account for spatiotemporal heterogeneity. Using a single-domain antibody, NM-01, against PD-L1, radiolabeled site-specifically with 99mTc for SPECT imaging, we aimed to assess the safety, radiation dosimetry, and imaging characteristics of this radiopharmaceutical and correlate tumor uptake with PD-L1 immunohistochemistry results. Methods: Sixteen patients (mean age, 61.7 y; 11 men) with non-small cell lung cancer were recruited. Primary tumor PD-L1 expression was measured by immunohistochemistry. NM-01 was radiolabeled with [99mTc(OH2)3(CO)3]+ complex binding to its C-terminal hexahistidine tag. Administered activity was 3.8-10.4 MBq/kg, corresponding to 100 µg or 400 µg of NM-01. Whole-body planar and thoracic SPECT/CT scans were obtained at 1 and 2 h after injection in all patients, and 5 patients underwent additional imaging at 10 min, 3 h, and 24 h for radiation dosimetry calculations. All patients were monitored for adverse events. Results: No drug-related adverse events occurred in this study. The mean effective dose was 8.84 × 10-3 ± 9.33 × 10-4 mSv/MBq (3.59 ± 0.74 mSv per patient). Tracer uptake was observed in the kidneys, spleen, liver, and bone marrow. SPECT primary tumor-to-blood-pool ratios (T:BP) varied from 1.24 to 2.3 (mean, 1.79) at 1 h and 1.24 to 3.53 (mean, 2.22) at 2 h (P = 0.005). Two-hour primary T:BP ratios correlated with PD-L1 immunohistochemistry results (r = 0.68, P = 0.014). Two-hour T:BP was lower in tumors with ≤1% PD-L1 expression (1.89 vs. 2.49, P = 0.048). Nodal and bone metastases showed tracer uptake. Heterogeneity (>20%) between primary tumor and nodal T:BP was present in 4 of 13 patients. Conclusion: This first-in-human study demonstrates that 99mTc-labeled anti-PD-L1-single-domain antibody SPECT/CT imaging is safe and associated with acceptable dosimetry. Tumor uptake is readily visible against background tissues, particularly at 2 h when the T:BP ratio correlates with PD-L1 immunohistochemistry results.

Clinical Translation of [68Ga]Ga-NOTA-anti-MMR-sdAb for PET/CT Imaging of Protumorigenic Macrophages.

PURPOSE: Macrophage mannose receptor (MMR, CD206) expressing tumor-associated macrophages (TAM) are protumorigenic and was reported to negatively impact therapy responsiveness and is associated with higher chances of tumor relapse following multiple treatment regimens in preclinical tumor models. Since the distribution of immune cells within the tumor is often heterogeneous, sampling "errors" using tissue biopsies will occur. In order to overcome this limitation, we propose positron emission tomography (PET)/X-ray computed tomography (CT) imaging using 68Ga-labeled anti-MMR single-domain antibody fragment (sdAb) to assess the presence of these protumorigenic TAM. PROCEDURES: Cross-reactive anti-MMR-sdAb was produced according to good manufacturing practice (GMP) and conjugated to p-SCN-Bn-NOTA bifunctional chelator for 68Ga-labeling. Biodistribution and PET/CT studies were performed in wild-type and MMR-deficient 3LL-R tumor-bearing mice. Biodistribution data obtained in mice were extrapolated to calculate radiation dose estimates for the human adult using OLINDA software. A 7-day repeated dose toxicity study for NOTA-anti-MMR-sdAb was performed in healthy mice up to a dose of 1.68 mg/kg. RESULTS: [68Ga]Ga-NOTA-anti-MMR-sdAb was obtained with 76 ± 2 % radiochemical yield, 99 ± 1 % radiochemical purity, and apparent molar activity of 57 ± 11 GBq/µmol. In vivo biodistribution analysis showed fast clearance via the kidneys and retention in MMR-expressing organs and tumor, with tumor-to-blood and tumor-to-muscle ratios of 6.80 ± 0.62 and 5.47 ± 1.82, respectively. The calculated effective dose was 0.027 mSv/MBq and 0.034 mSv/MBq for male and female, respectively, which means that a proposed dose of 185 MBq in humans would yield a radiation dose of 5.0 and 6.3 mSv to male and female patients, respectively. In the toxicity study, no adverse effects were observed. CONCLUSIONS: Preclinical validation of [68Ga]Ga-NOTA-anti-MMR-sdAb showed high specific uptake of this tracer in MMR-expressing TAM and organs, with no observed toxicity. [68Ga]Ga-NOTA-anti-MMR-sdAb is ready for a phase I clinical trial.