Refining the standard of care in immune thrombotic thrombocytopenic purpura.

Acute immune thrombotic thrombocytopenic purpura (iTTP) is a medical emergency. In the setting of any thrombotic microangiopathy (TMA), blood should be drawn to measure ADAMTS13 activity and inhibitor levels, and an assessment should be made of TTP risk before receiving ADAMTS13 results. This can include the use of PLASMIC and French scores. Plasma exchange (PE) is then initiated. Upon confirmation of iTTP, with ADAMTS13 less than 10% in the presence of an inhibitor, interventions targeting all facets of iTTP pathophysiology should be instituted: replenishing ADAMTS13 via continued PE; suppressing anti-ADAMTS13 autoantibodies with glucocorticoids and rituximab; and inhibiting the thrombotic process-uncontrolled formation of platelet/Von Willebrand factor (VWF) microthrombi-with caplacizumab. The latter, an addition to existing standards of care, is based on International Society on Thrombosis and Haemostasis guidelines and emphasizes tracking of ADAMTS13 activity. In HERCULES, a pivotal randomized controlled trial, caplacizumab use resulted in fewer recurrent iTTP episodes, decreased PE, and shortened hospital stay. In settings of high suspicion for iTTP, clinicians should consider the administration of caplacizumab before receiving ADAMTS13 results because the greatest benefits of caplacizumab accrued starting it within 3 days of TMA recognition. In HERCULES, serious bleeding events occurred among 11% of those in the caplacizumab group vs 1% in the placebo group, but all resolved, most without intervention. iTTP survivors receiving PE and immunosuppression alone are at a heightened risk for stroke, other cardiovascular disorders, neurocognitive impairment, and kidney disease. Whether rapid prevention of VWF multimer/platelet formation with caplacizumab can suppress such long-term sequelae, and whether caplacizumab can replace PE in initial therapy, are under investigation.

Structure elucidation of a human melanocortin-4 receptor specific orthosteric nanobody agonist.

The melanocortin receptor 4 (MC4R) belongs to the melanocortin receptor family of G-protein coupled receptors and is a key switch in the leptin-melanocortin molecular axis that controls hunger and satiety. Brain-produced hormones such as α-melanocyte-stimulating hormone (agonist) and agouti-related peptide (inverse agonist) regulate the molecular communication of the MC4R axis but are promiscuous for melanocortin receptor subtypes and induce a wide array of biological effects. Here, we use a chimeric construct of conformation-selective, nanobody-based binding domain (a ConfoBody Cb80) and active state-stabilized MC4R-ß2AR hybrid for efficient de novo discovery of a sequence diverse panel of MC4R-specific, potent and full agonistic nanobodies. We solve the active state MC4R structure in complex with the full agonistic nanobody pN162 at 3.4 Å resolution. The structure shows a distinct interaction with pN162 binding deeply in the orthosteric pocket. MC4R peptide agonists, such as the marketed setmelanotide, lack receptor selectivity and show off-target effects. In contrast, the agonistic nanobody is highly specific and hence can be a more suitable agent for anti-obesity therapeutic intervention via MC4R.

Recombinant Lactococcus lactis secreting FliC protein nanobodies for resistance against Salmonella enteritidis invasion in the intestinal tract.

Salmonella Enteritidis is a major foodborne pathogen throughout the world and the increase in antibiotic resistance of Salmonella poses a significant threat to public safety. Natural nanobodies exhibit high affinity, thermal stability, ease of production, and notably higher diversity, making them widely applicable for the treatment of viral and bacterial infections. Recombinant expression using Lactococcus lactis leverages both acid resistance and mucosal colonization properties of these bacteria, allowing the effective expression of exogenous proteins for therapeutic effects. In this study, nine specific nanobodies against the flagellar protein FliC were identified and expressed. In vitro experiments demonstrated that FliC-Nb-76 effectively inhibited the motility of S. Enteritidis and inhibited its adhesion to and invasion of HIEC-6, RAW264.7, and chicken intestinal epithelial cells. Additionally, a recombinant L. lactis strain secreting the nanobody, L. lactis-Nb76, was obtained. Animal experiments confirmed that it could significantly reduce the mortality rates of chickens infected with S. Enteritidis, together with alleviating the inflammatory response caused by the pathogen. These results provide a novel strategy for the treatment of antibiotic-resistant S. Enteritidis infection in the intestinal tract.

GB18-06, a nanobody targeting GDF15, effectively alleviates weight loss and restores physical function in cachexia models.

Cachexia is a complicated metabolic syndrome mainly associated with cancers, characterized by extreme weight loss and muscle wasting. It is a debilitating condition that negatively affects prognosis and survival. However, there is currently no effective pharmacological intervention that can reverse body weight loss and improve physical performance in patients with cachexia. Growth differentiation factor 15 (GDF15) can suppress appetite and regulate energy balance through binding to glial cell-derived neurotrophic factor receptor alpha-like (GFRAL). In order to develop a novel, effective treatment for cachexia, we generated a GDF15-targeting VHH nanobody, GB18-06, that was able to bind GDF15 with high affinity. In vitro, GB18-06 potently inhibited the GDF15-GFRAL signaling pathway, leading to a reduction of downstream ERK and AKT phosphorylation levels; in vivo, GB18-06 alleviated weight loss (>20%) in cancer and chemotherapy-induced cachexia models in mice. Compared with the control (phosphate-buffered saline) group, the ambulatory activity of mice in the GB18-06-treated group also increased 77%. Furthermore, GB18-06 exhibited desirable pharmacokinetic properties and an excellent developability profile. Our study has demonstrated a means of developing targeted treatment for cachexia with high efficacy, potentially leading to improved clinical outcomes and quality of life for patients with cachexia.

Structural basis of µ-opioid receptor targeting by a nanobody antagonist.

The µ-opioid receptor (µOR), a prototypical G protein-coupled receptor (GPCR), is the target of opioid analgesics such as morphine and fentanyl. Due to the severe side effects of current opioid drugs, there is considerable interest in developing novel modulators of µOR function. Most GPCR ligands today are small molecules, however biologics, including antibodies and nanobodies, represent alternative therapeutics with clear advantages such as affinity and target selectivity. Here, we describe the nanobody NbE, which selectively binds to the µOR and acts as an antagonist. We functionally characterize NbE as an extracellular and genetically encoded µOR ligand and uncover the molecular basis for µOR antagonism by determining the cryo-EM structure of the NbE-µOR complex. NbE displays a unique ligand binding mode and achieves µOR selectivity by interactions with the orthosteric pocket and extracellular receptor loops. Based on a ß-hairpin loop formed by NbE that deeply protrudes into the µOR, we design linear and cyclic peptide analogs that recapitulate NbE's antagonism. The work illustrates the potential of nanobodies to uniquely engage with GPCRs and describes lower molecular weight µOR ligands that can serve as a basis for therapeutic developments.

Evaluation of an Affinity-Enhanced Anti-SARS-CoV2 Nanobody Design Workflow Using Machine Learning and Molecular Dynamics.

In silico optimization of protein binding has received a great deal of attention in the recent years. Since in silico prefiltering of strong binders is fast and cheap compared to in vitro library screening methods, the advent of powerful hardware and advanced machine learning algorithms has made this strategy more accessible and preferred. These advances have already impacted the global response to pandemic threats. In this study, we proposed and tested a workflow for designing nanobodies targeting the SARS-CoV-2 spike protein receptor binding domain (S-RBD) using machine learning techniques complemented by molecular dynamics simulations. We evaluated the feasibility of this workflow using a test set of 3 different nanobodies and 2 different S-RBD variants, from in silico design and bacterial expression to binding assays of the designed nanobody mutants. We successfully designed nanobodies that were subsequently tested against both the wild-type (Wuhan type) and the delta variant S-RBD and found 2 of them to be stronger binders compared to the wild-type nanobody. We use this case study to describe both the strengths and weaknesses of this in silico assisted nanobody design strategy.

PirA- or PirB-binding nanobodies can protect whiteleg shrimp from the acute hepatopancreatic necrosis disease toxin.

Acute hepatopancreatic necrosis disease (AHPND) is a devastating shrimp disease caused by a binary toxin, PirAB, produced by Vibrio parahaemolyticus and other closely related bacteria. To address AHPND, over 300 unique single-domain antibodies (also known as nanobodies) derived from the VHH domains of Lama glama heavy-chain-only antibodies were raised against either PirA or PirB and characterized. Nanobodies were shortlisted based on their affinities for either PirA or PirB, their relative stability in intestinal fluids, and their ability to reduce PirAB-induced death in brine shrimp Artemia salina. From these data, a subset of nanobodies was tested for their ability to reduce AHPND in whiteleg shrimp Penaeus vannamei, and nanobodies targeting either PirA or PirB provided significant disease protection to whiteleg shrimp. These results show that nanobodies can be a new option for shrimp farmers to reduce or eliminate the impact of AHPND on their operations.

Foot-and-mouth disease virus antigenic landscape and reduced immunogenicity elucidated in atomic detail.

Unlike most other picornaviruses, foot-and-mouth disease (FMD) intact virions (146S) dissociate easily into small pentameric subunits (12S). This causes a dramatically decreased immunogenicity by a mechanism that remains elusive. Here, we present the high-resolution structures of 12S (3.2 Å) and its immune complex of a single-domain antibody (VHH) targeting the particle interior (3.2 Å), as well as two 146S-specific VHHs complexed to distinct sites on the 146S capsid surface (3.6 Å and 2.9 Å). The antigenic landscape of 146S is depicted using 13 known FMD virus-antibody complexes. Comparison of the immunogenicity of 146S and 12S in pigs, focusing on the resulting antigenic sites and incorporating structural analysis, reveals that dissociation of 146S leads to structural alteration and destruction of multiple epitopes, resulting in significant differences in antibody profiles/lineages induced by 12S and 146S. Furthermore, 146S generates higher synergistic neutralizing antibody titers compared to 12S, whereas both particles induce similar total FMD virus specific antibody titers. This study can guide the structure-based rational design of novel multivalent and broad-spectrum recombinant vaccines for protection against FMD.

Imaging the immune sequelae of infection with SARS-CoV-2 in nonhuman primates by using two nanobody PET-tracers.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) impacts multiple anatomical sites. Whether this is due to the virus itself or is a secondary effect caused by the influx and activation of immune cells is not known. Positron emission tomography (PET) with immunoglobulins can provide insights into which sites and cells are activated in a living animal. Our aim is to use two nanobodies as tools to monitor (1) the distribution of antigen presenting cells (APC) by virtue of their Mafa-DR expression profile, (2) virus-infected cells and viral particles using a nanobody against the SARS-CoV-2 spike protein. Two [89Zr]-labeled nanobodies that target the SARS-CoV-2 spike protein and major histocompatability complex (MHC) class II antigens (Mafa-DR), respectively, are used to monitor their distribution during an experimental SARS-CoV-2 infection in a nonhuman primate model. Scans are obtained before infection and on Day 3 and 10 post infection (pi) in two macaques each. The [89Zr]anti-SARS-CoV-2 spike nanobody localized to SARS-CoV-2-associated lung lesions and the nasal mucosa, while the [89Zr]anti-human leukocyte antigen (HLA)-DR nanobody was predominantly found in non-affected lung tissue after infection. We also detected, pi, upregulation of the Mafa-DR signal, indicative of recruitment of professional APCs, in the superior sagittal sinus. [89Zr]-labeled nanobodies show recruitment of macrophages/monocytes in non-lesional lung tissue in cynomolgus macaques after experimental infection with SARS-CoV-2, as well as accumulation of the spike protein in both lung lesions and the nasal mucosa during infection. These results show the possibility of in vivo monitoring the quality and quantity of immune responses during the initial stages of an infection.

Characterization of the bispecific VHH antibody tarperprumig (ALXN1820) specific for properdin and designed for low-volume administration.

The bispecific antibody tarperprumig (ALXN1820) was developed as a treatment option for diseases involving dysregulated complement alternative pathway (AP) activity that could be administered in small volumes, either subcutaneously or intravenously. Tarperprumig incorporates a C-terminal variable domain of a heavy chain only antibody (VHH) that binds properdin (FP) connected via a flexible linker to an N-terminal VHH that binds human serum albumin (HSA). The purified bispecific VHH antibody exhibits an experimental molecular weight average of 27.4 kDa and can be formulated at > 100 mg/mL. Tarperprumig binds tightly to FP and HSA with sub-nanomolar affinity at pH 7.4 and can associate simultaneously with FP and HSA to form a ternary complex. Tarperprumig potently and dose-dependently inhibits to completion in vitro AP-dependent complement C5b-9 formation, AP-dependent hemolysis, and the AP deposition of C3, FP and C9. X-ray crystallography revealed that the isolated FP-binding VHH recognizes the thrombospondin repeat 5 domain of FP, thereby preventing FP from binding to the AP convertase owing to severe steric hindrance. Tarperprumig cross-reacts with cynomolgus monkey FP and serum albumin. In summary, tarperprumig exhibits properties tailored for subcutaneous administration and is currently in clinical development for the treatment of complement AP-related disorders.

NanoPlex: a universal strategy for fluorescence microscopy multiplexing using nanobodies with erasable signals.

Fluorescence microscopy has long been a transformative technique in biological sciences. Nevertheless, most implementations are limited to a few targets, which have been revealed using primary antibodies and fluorescently conjugated secondary antibodies. Super-resolution techniques such as Exchange-PAINT and, more recently, SUM-PAINT have increased multiplexing capabilities, but they require specialized equipment, software, and knowledge. To enable multiplexing for any imaging technique in any laboratory, we developed NanoPlex, a streamlined method based on conventional antibodies revealed by engineered secondary nanobodies that allow the selective removal of fluorescence signals. We develop three complementary signal removal strategies: OptoPlex (light-induced), EnzyPlex (enzymatic), and ChemiPlex (chemical). We showcase NanoPlex reaching 21 targets for 3D confocal analyses and 5-8 targets for dSTORM and STED super-resolution imaging. NanoPlex has the potential to revolutionize multi-target fluorescent imaging methods, potentially redefining the multiplexing capabilities of antibody-based assays.

Single domain antibody-scFv conjugate targeting amyloid ß and TfR penetrates the blood-brain barrier and interacts with amyloid ß.

Neurodegenerative diseases such as Alzheimer's disease (AD) pose substantial challenges to patients and health-care systems, particularly in countries with aging populations. Immunotherapies, including the marketed antibodies lecanemab (Leqembi®) and donanemab (KisunlaTM), offer promise but face hurdles due to limited delivery across the blood-brain barrier (BBB). This limitation necessitates high doses, resulting in increased costs and a higher risk of side effects. This study explores transferrin receptor (TfR)-binding camelid single-domain antibodies (VHHs) for facilitated brain delivery. We developed and evaluated fusion proteins (FPs) combining VHHs with human IgG Fc domains or single-chain variable fragments (scFvs) of the anti-amyloid-beta (Aß) antibody 3D6. In vitro assessments showed varying affinities of the FPs for TfR. In vivo evaluations indicated that specific VHH-Fc and VHH-scFv fusions reached significant brain concentrations, emphasizing the importance of optimal TfR binding affinities. The VHH-scFv fusions were further investigated in mouse models with Aß pathology, showing higher retention compared to wild-type mice without Aß pathology. Our findings suggest that these novel VHH-based FPs hold potential for therapeutic and diagnostic applications in AD, providing a strategy to overcome BBB limitations and enhance brain targeting of antibody-based treatments. Furthermore, our results suggest that a given bispecific TfR-binding fusion format has a window of "optimal" affinity where parenchymal delivery is adequate, while blood pharmacokinetics aligns with the desired application of the fusion protein.

Antagonistic nanobodies implicate mechanism of GSDMD pore formation and potential therapeutic application.

Inflammasome activation results in the cleavage of gasdermin D (GSDMD) by pro-inflammatory caspases. The N-terminal domains (GSDMDNT) oligomerize and assemble pores penetrating the target membrane. As methods to study pore formation in living cells are insufficient, the order of conformational changes, oligomerization, and membrane insertion remained unclear. We have raised nanobodies (VHHs) against human GSDMD and find that cytosolic expression of VHHGSDMD-1 and VHHGSDMD-2 prevents oligomerization of GSDMDNT and pyroptosis. The nanobody-stabilized GSDMDNT monomers partition into the plasma membrane, suggesting that membrane insertion precedes oligomerization. Inhibition of GSDMD pore formation switches cell death from pyroptosis to apoptosis, likely driven by the enhanced caspase-1 activity required to activate caspase-3. Recombinant antagonistic nanobodies added to the extracellular space prevent pyroptosis and exhibit unexpected therapeutic potential. They may thus be suitable to treat the ever-growing list of diseases caused by activation of (non-) canonical inflammasomes.

Neutralizing Nanobodies against Venoms from Naja haje Species Captured in North Africa.

Snakebite envenoming (SBE) remains a severely neglected public health issue, particularly affecting tropical and subtropical regions, with Africa experiencing an estimated 435,000 to 580,000 snakebites annually, leading to high morbidity and mortality rates, especially across Africa and Asia. Recognized as a Neglected Tropical Disease, SBE management is further complicated by the inadequate efficacy of current antivenom treatments. Of particular concern are cobras (Naja sp.), whose neurotoxins can induce rapid fatal respiratory paralysis. In this study, we investigate the potential of nanobodies as a promising next-generation of immunotherapeutics against cobra venoms. Through a dual strategy of the characterization of venom toxic fractions from cobras captured for the first time in Algeria and Tunisia biotopes, coupled with in vitro assays to evaluate their interactions with acetylcholine receptors, and subsequent immunization of dromedaries to produce specific nanobodies, we identified two lethal fractions, F5 and F6, from each venom, and selected five nanobodies with significant binding and neutralizing of 3DL50 (0.74 mg/kg). The combination of these nanobodies demonstrated a synergistic effect, reaching 100% neutralizing efficacy of 2DL50 lethal venom fraction (0.88 mg/kg) doses in mice. Additionally, our findings highlighted the complex mechanism of cobra venom action through the lethal synergism among its major toxins.

Spatiotemporal Delivery of Dual Nanobodies by Engineered Probiotics to Reverse Tumor Immunosuppression via Targeting Tumor-Derived Exosomes.

The anti-PD-L1 and its bispecific antibodies have exhibited durable antitumor immunity but still elicit immunosuppression mainly caused by tumor-derived exosomes (TDEs), leading to difficulty in clinical transformation. Herein, engineered Escherichia coli Nissle 1917 (EcN) coexpressing anti-PD-L1 and anti-CD9 nanobodies (EcN-Nb) are developed and decorated with zinc-based metal-organic frameworks (MOFs) loaded with indocyanine green (ICG), to generate EcN-Nb-ZIF-8CHO-ICG (ENZC) for spatiotemporal lysis of bacteria for immunotherapy. The tumor-homing hybrid system can specifically release nanobodies in response to near-infrared (NIR) radiation, thereby targeting TDEs and changing their biological distribution, remodeling tumor-associated macrophages to M1 states, activating more effective and cytotoxic T lymphocytes, and finally, leading to the inhibition of tumor proliferation and metastasis. Altogether, the microfluidic-enabled MOF-modified engineered probiotics target TDEs and activate the antitumor immune response in a spatiotemporally manipulated manner, offering promising TDE-targeted immune therapy.

A Nanobody of PEDV S1 Protein: Screening and Expression in Escherichia coli.

Porcine epidemic diarrhea virus (PEDV) has caused significant economic losses to the pig farming industry in various countries for a long time. Currently, there are no highly effective preventive or control measures available. Research into the pathogenic mechanism of PEDV has shown that it primarily causes infection by binding the S protein to the CD13 (APN) receptor on the membrane of porcine intestinal epithelial cells. The S1 region contains three neutralization epitopes and multiple receptor-binding domains, which are closely related to viral antigenicity and ad-sorption invasion. Nanobodies are a type of single-domain antibody that have been discovered in recent years. They can be expressed on a large scale through prokaryotic expression systems, which makes them cost-effective, stable, and less immunogenic. This study used a phage display library of nanobodies against the PEDV S1 protein. After three rounds of selection and enrichment, the DNA sequence of the highly specific nanobody S1Nb1 was successfully obtained. To obtain soluble nanobody S1Nb1, its DNA sequence was inserted into the vector Pcold and a solubility-enhancing SUMO tag was added. The resulting recombinant vector, Pcold-SUMO-S1Nb1, was then transformed into E. coli BL21(DE3) to determine the optimal expression conditions for the nanobody. Following purification using Ni-column affinity chromatography, Western blot analysis confirmed the successful purification of S1Nb1 carrying the solubility-enhancing tag. ELISA results demonstrated a strong affinity between the S1Nb1 nanobody and PEDV S1 protein.

Functional Divergence in the Affinity and Stability of Non-Canonical Cysteines and Non-Canonical Disulfide Bonds: Insights from a VHH and VNAR Study.

Single-domain antibodies, including variable domains of the heavy chains of heavy chain-only antibodies (VHHs) from camelids and variable domains of immunoglobulin new antigen receptors (VNARs) from cartilaginous fish, show the therapeutic potential of targeting antigens in a cytosol reducing environment. A large proportion of single-domain antibodies contain non-canonical cysteines and corresponding non-canonical disulfide bonds situated on the protein surface, rendering them vulnerable to environmental factors. Research on non-canonical disulfide bonds has been limited, with a focus solely on VHHs and utilizing only cysteine mutations rather than the reducing agent treatment. In this study, we examined an anti-lysozyme VNAR and an anti-BC2-tag VHH, including their non-canonical disulfide bond reduced counterparts and non-canonical cysteine mutants. Both the affinity and stability of the VNARs and VHHs decreased in the non-canonical cysteine mutants, whereas the reduced-state samples exhibited decreased thermal stability, with their affinity remaining almost unchanged regardless of the presence of reducing agents. Molecular dynamics simulations suggested that the decrease in affinity of the mutants resulted from increased flexibility of the CDRs, the disappearance of non-canonical cysteine-antigen interactions, and the perturbation of other antigen-interacting residues caused by mutations. These findings highlight the significance of non-canonical cysteines for the affinity of single-domain antibodies and demonstrate that the mutation of non-canonical cysteines is not equivalent to the disruption of non-canonical disulfide bonds with a reducing agent when assessing the function of non-canonical disulfide bonds.

Enhancing the sensitivity of immunomagnetic assay for 3-phenoxybenzonic acid: a novel approach utilizing magnetosome-nanobody complexes and gold nanoparticles probes.

The 3-phenoxybenzoic acid (3-PBA) residues in environment are posing a significant challenge to our daily lives. To establish a more sensitive and rapid detection method, anti-3-PBA nanobodies (Nbs) were immobilized onto magnetosomes (bacterial magnetic nanoparticles, termed as BMPs), forming a robust BMP-Nb complex. The 3-PBA derivative was labeled with horseradish peroxidase (HRP) and further associated with gold nanoparticles (AuNPs) to create a highly sensitive probe (3-PBA-HRP-AuNP). An innovative immunoassay that combined BMP-Nb complex with 3-PBA-HRP-AuNP was developed for determinaton of 3-PBA. This method enabled the determination of 3-PBA with a half-maximum signal inhibition concentration (IC50) of 1.03 ng/mL, which was more sensitive than that of using 3-PBA-HRP as tracer with an IC50 of 2.18 ng/mL. The reliability of the assay was evidenced by the quantitative recovery of 3-PBA from water and soil samples ranging from 76.85 to 95.64%. The 3-PBA residues determined by this assay in actual water samples were between < LOD and 2.54 ng/mL and were between < LOD and 11.25 ng/g (dw) in real soils, respectively, which agreed well with those of liquid chromatography mass spectrometry (LC-MS). Collectively, the BMP-Nb and 3-PBA-HRP-AuNP-based immunoassay provides a powerful tool for the precise detection of 3-PBA residues in environment matrices, reinforcing our capacity to monitor and mitigate potential ecological and health impacts associated with this prevalent pollutant.

Optimizing the Therapeutic Index of sdAb-Based Radiopharmaceuticals Using Pretargeting.

Single-domain antibodies (sdAbs) demonstrate favorable pharmacokinetic profiles for molecular imaging applications. However, their renal excretion and retention are obstacles for applications in targeted radionuclide therapy (TRT). Methods: Using a click-chemistry-based pretargeting approach, we aimed to reduce kidney retention of a fibroblast activation protein α (FAP)-targeted sdAb, 4AH29, for 177Lu-TRT. Key pretargeting parameters (sdAb-injected mass and lag time) were optimized in healthy mice and U87MG (FAP+) xenografts. A TRT study in a pancreatic ductal adenocarcinoma (PDAC) patient-derived xenograft (PDX) model was performed as a pilot study for sdAb-based pretargeting applications. Results: Modification of 4AH29 with trans-cyclooctene (TCO) moieties did not modify the sdAb pharmacokinetic profile. A 200-µg injected mass of 4AH29-TCO and an 8-h lag time for the injection of [177Lu]Lu-DOTA-PEG7-tetrazine resulted in the highest kidney therapeutic index (2.0 ± 0.4), which was 5-fold higher than that of [177Lu]Lu-DOTA-4AH29 (0.4 ± 0.1). FAP expression in the tumor microenvironment was validated in a PDAC PDX model with both immunohistochemistry and PET/CT imaging. Mice treated with the pretargeting high-activity approach (4AH29-TCO + [177Lu]Lu-DOTA-PEG7-tetrazine; 3 × 88 MBq, 1 injection per week for 3 wk) demonstrated prolonged survival compared with the vehicle control and conventionally treated ([177Lu]Lu-DOTA-4AH29; 3 × 37 MBq, 1 injection per week for 3 wk) mice. Mesangial expansion was reported in 7 of 10 mice in the conventional cohort, suggesting treatment-related kidney morphologic changes, but was not observed in the pretargeting cohort. Conclusion: This study validates pretargeting to mitigate sdAbs' kidney retention with no observation of morphologic changes on therapy regimen at early time points. Clinical translation of click-chemistry-based pre-TRT is warranted on the basis of its ability to alleviate toxicities related to biovectors' intrinsic pharmacokinetic profiles. The absence of representative animal models with extensive stroma and high FAP expression on cancer-associated fibroblasts led to a low mean tumor-absorbed dose even with high injected activity and consequently to modest survival benefit in this PDAC PDX.

Structure-guided in vitro evolution of nanobodies targeting new viral variants.

A major challenge in antiviral antibody therapy is keeping up with the rapid evolution of viruses. Our research shows that nanobodies - single-domain antibodies derived from camelids - can be rapidly re-engineered to combat new viral strains through structure-guided in vitro evolution. Specifically, for viral mutations occurring at nanobody-binding sites, we introduce randomized amino acid sequences into nanobody residues near these mutations. We then select nanobody variants that effectively bind to the mutated viral target from a phage display library. As a proof of concept, we used this approach to adapt Nanosota-3, a nanobody originally identified to target the receptor-binding domain (RBD) of early Omicron subvariants, making it highly effective against recent Omicron subvariants. Remarkably, this adaptation process can be completed in less than two weeks, allowing drug development to keep pace with viral evolution and provide timely protection to humans.