Search

Simple Summary: Mitochondrial DNA evolves rapidly, is maternally inherited, and is relatively small and compact in most animal species, making it a popular marker in molecular studies. In genomics, the study of mitochondrial DNA is important for understanding the origins, history, and adaptation of domesticated species. This review examines the D-loop region of mitochondrial DNA in Arabian camels to explore maternal inheritance, genetic diversity, and evolutionary history. It highlights significant genetic differences and adaptive traits in the mitogenome of Arabian camels. The D-loop region contains extensive polymorphisms and haplotypes, which provide insights into camel domestication and breeding processes. Comparative analyses with other camelid species reveal unique genetic signatures in the Arabian camel. Finally, this review integrates recent advancements in mitochondrial genomics, demonstrating potential applications in conservation and breeding programs to enhance the understanding and preservation of Camelidae genetics. The Camelidae family, ranging from southwest Asia to north Africa, South America, and Australia, includes key domesticated species adapted to diverse environments. Among these, the Arabian camel (Camelus dromedarius) is vital to the cultural and economic landscape of the Arabian Peninsula. This review explores the mitochondrial DNA of the dromedary camel, focusing on the D-loop region to understand its genetic diversity, maternal inheritance, and evolutionary history. We aim to investigate the unique characteristics of Arabian camel mtDNA, analyze the D-loop for genetic diversity and maternal lineage patterns, and explore the implications of mitochondrial genomic studies for camel domestication and adaptation. Key findings on mtDNA structure and variation highlight significant genetic differences and adaptive traits. The D-loop, essential for mtDNA replication and transcription, reveals extensive polymorphisms and haplotypes, providing insights into dromedary camel domestication and breeding history. Comparative analyses with other camelid species reveal unique genetic signatures in the Arabian camel, reflecting its evolutionary and adaptive pathways. Finally, this review integrates recent advancements in mitochondrial genomics, demonstrating camel genetic diversity and potential applications in conservation and breeding programs. Through comprehensive mitochondrial genome analysis, we aim to enhance the understanding of Camelidae genetics and contribute to the preservation and improvement of these vital animals. [ABSTRACT FROM AUTHOR]

The injudicious usage of antibiotics during infections caused by Gram-negative bacteria leads to the emergence of β-lactamases. Among them, the NDM-1 enzyme poses a serious threat to human health. Developing new antibiotics or inhibiting β-lactamases might become essential to reduce and prevent bacterial infections. Nanobodies (Nbs), the smallest antigen-binding single-domain fragments derived from Camelidae heavy-chain-only antibodies, targeting enzymes, are innovative alternatives to develop effective inhibitors. The biopanning of an immune VHH library after phage display has helped to retrieve recombinant antibody fragments with high inhibitory activity against recombinant-NDM-1 enzyme. Nb02NDM-1, Nb12NDM-1, and Nb17NDM-1 behaved as uncompetitive inhibitors against NDM-1 with Ki values in the nM range. Remarkably, IC50 values of 25.0 nM and 8.5 nM were noted for Nb02NDM-1 and Nb17NDM-1, respectively. The promising inhibition of NDM-1 by Nbs highlights their potential application in combating particular Gram-negative infections. [ABSTRACT FROM AUTHOR]

Nanobodies, also referred to as single domain-based VHHs, are antibody fragments derived from heavy-chain only IgG antibodies found in the Camelidae family. Due to their small size, simple structure, high antigen binding affinity, and remarkable stability in extreme conditions, nanobodies possess the potential to overcome several of the limitations of conventional monoclonal antibodies. For many years, nanobodies have been of great interest in a wide variety of research fields, particularly in the diagnosis and treatment of diseases. This culminated in the approval of the world's first nanobody based drug (Caplacizumab) in 2018 with others following soon thereafter. This review will provide an overview, with examples, of (i) the structure and advantages of nanobodies compared to conventional monoclonal antibodies, (ii) methods used to generate and produce antigen-specific nanobodies, (iii) applications for diagnostics, and (iv) ongoing clinical trials for nanobody therapeutics as well as promising candidates for clinical development. [ABSTRACT FROM AUTHOR]

Conformational flexibility plays an essential role in antibodies' functional and structural stability. They facilitate and determine the strength of antigen–antibody interactions. Camelidae express an interesting subtype of single-chain antibody, named Heavy Chain only Antibody. They have only one N-terminal Variable domain (VHH) per chain, composed of Frameworks (FRs) and Complementarity Determining regions (CDRs) like their VH and VL counterparts in IgG. Even when expressed independently, VHH domains display excellent solubility and (thermo)stability, which helps them to retain their impressive interaction capabilities. Sequence and structural features of VHH domains contributing to these abilities have already been studied compared to classical antibodies. To have the broadest view and understand the changes in dynamics of these macromolecules, large-scale molecular dynamics simulations for a large number of non-redundant VHH structures have been performed for the first time. This analysis reveals the most prevalent movements in these domains. It reveals the four main classes of VHHs dynamics. Diverse local changes were observed in CDRs with various intensities. Similarly, different types of constraints were observed in CDRs, while FRs close to CDRs were sometimes primarily impacted. This study sheds light on the changes in flexibility in different regions of VHH that may impact their in silico design. [ABSTRACT FROM AUTHOR]

Simple Summary: In this study, for the first time, we performed a detailed histological analysis of the cornea and palisades of Vogt in the different wild ruminant species. The material for the research was taken from 49 adult wild ruminants (Bovidae, Camelidae, Cervidae, Giraffidae and Tragulidae) constituting 13 species coming from the Wroclaw Zoological Garden (Poland), the Warsaw Zoological Garden (Poland) and own collection of the Division of Animal Anatomy (Wroclaw). Our results showed that the number of layers of the cornea, i.e., five layers (anterior corneal epithelium, anterior limiting membrane (Bowman's layer), the proper substance of the cornea, the posterior limiting membrane (Descemet's membrane) and posterior corneal epithelium) or four layers (no Bowman's layer) is not constant within even the same genus that includes the given species of the tested animal. The results of this study can form the basis for further research on the immunohistochemistry of the cornea in the maintenance of structural integrity and fluid balance in wild ruminants that can be used in veterinary ophthalmology diagnostics. In the study, we data concerning the histological and morphometrical examination of the cornea and palisades of Vogt in the different species of ruminants from the families Bovidae, Camelidae, Cervidae, Giraffidae and Tragulidae, coming from the Warsaw Zoological Garden, the Wroclaw Zoological Garden and the Division of Animal Anatomy. The following ruminant species were investigated: common wildebeest, Kirk's dik-dik, Natal red duiker, scimitar oryx, sitatunga, Philippine spotted deer, Père David's deer, moose, reindeer, reticulated giraffe, okapi, Balabac mouse-deer and alpaca. The cornea of ruminant species such as the common wildebeest, Kirk's dik-dik, Natal red duiker, scimitar oryx, reindeer and Balabac mouse-deer consisted of four layers (not found in the Bowman's layer): the anterior corneal epithelium, the proper substance of the cornea, the posterior limiting membrane (Descemet's membrane) and the posterior corneal epithelium (endothelium). The anterior corneal epithelium was composed of a multilayer keratinizing squamous epithelium, which was characterized in the studied ruminants with a variable number of cell layers but also with a different thickness both in the central epithelium part and in the peripheral part. Moreover, the proper substance of cornea was thinnest in Balabac mouse-deer, Kirk's dik-dik, Natal red duiker, scimitar oryx, Philippine spotted deer, alpaca, reindeer and sitatunga and was thickest in the reticulated giraffe. The thickest Descemet's membrane was observed in the Père David's deer. The corneal limbus is characterized by a large number of pigment cell clusters in Kirk's dik-dik, scimitar oryx, moose, Balabac mouse-deer and alpaca. In the common wildebeest, Père David's deer, moose, reticulated giraffe, okapi and alpaca, the palisades of Vogt were marked in the form of a crypt-like structure. The corneal limbus epithelium in the examined ruminants was characterized by a variable number of cell layers but also a variable number of melanocytes located in different layers of this epithelium. The detailed knowledge of the corneal structure of domestic and wild animals can contribute to the even better development of methods for treating eye diseases in veterinary medicine. [ABSTRACT FROM AUTHOR]

Single-domain antibodies (sdAbs), derived from the heavy chain only antibodies found in camelids such as llamas have the potential to provide rugged detection reagents with high affinities, and the ability to refold after denaturation. We have isolated and characterized sdAbs specific to staphylococcal enterotoxin B (SEB) which bind to two distinct epitopes and are able to function in a sandwich immunoassay for toxin detection. Characterization of these sdAbs revealed that each exhibited nanomolar binding affinities or better. Melting temperatures for the sdAbs ranged from approximately 60 °C to over 70 °C, with each demonstrating at least partial refolding after denaturation and several were able to completely refold. A first set of sdAbs was isolated by panning the library using adsorbed antigen, all of which recognized the same epitope on SEB. Epitope mapping suggested that these sdAbs bind to a particular fragment of SEB (VKSIDQFLYFDLIYSI) containing position L45 (underlined), which is involved in binding to the major histocompatibility complex (MHC). Differences in the binding affinities of the sdAbs to SEB and a less-toxic vaccine immunogen, SEBv (L45R/Y89A/Y94A) were also consistent with binding to this epitope. A sandwich panning strategy was utilized to isolate sdAbs which bind a second epitope. This epitope differed from the initial one obtained or from that recognized by previously isolated anti-SEB sdAb A3. Using SEB-toxin spiked milk we demonstrated that these newly isolated sdAbs could be utilized in sandwich-assays with each other, A3, and with various monoclonal antibodies. [ABSTRACT FROM AUTHOR]

Simple Summary: Camelidae are induced ovulators whose ovulation is tightly regulated by multiple factors. Understanding the biological mechanisms underlying follicular development, hormone secretion, and ovulation requires investigating the potential molecular pathways involved in these mechanisms. However, little is known about these molecular pathways in Bactrian camels. To screen and identify candidate biomarkers after seminal plasma (SP)-induced ovulation in the ovaries, we performed comprehensive proteomic and molecular biological analyses of the ovaries from camels that were intramuscularly injected with either seminal plasma or phosphate-buffered saline. Identification of these candidate biomarkers will enable a better understanding of reproduction in Bactrian camels. Our findings suggest candidate proteins for further studies on the molecular mechanisms of induced ovulation. Camelidae are induced ovulators whose ovulation is tightly regulated by multiple factors. Understanding the biological mechanisms underlying follicular development, hormone secretion, and ovulation requires investigating the potential molecular pathways involved. However, little is known about these pathways in Bactrian camels. To screen and identify candidate biomarkers after inducing ovulation, this study performed comprehensive proteomic and molecular biological analyses of the ovaries from two camel groups (n = 6). We identified 5075 expressed ovarian proteins, of which 404 were differentially expressed (264 upregulated, 140 downregulated) (p < 0.05 or p < 0.01), in samples from plasma-induced versus control camels. Gene ontology annotation identified the potential functions of the differentially expressed proteins (DEPs). These results validated the differential expression for a subset of these proteins using Western blot (p < 0.05) and immunofluorescence staining. Three DEPs (FST, NR5A1, and PRL) were involved in neurochemical signal transduction, as well as endocrine and reproductive hormone regulatory processes. The Kyoto Encyclopedia of Genes and Genomes analysis indicated the involvement of several pathways, including the calcium, cAMP, gonadotropin-releasing hormone, MAPK, and neuroactive ligand–receptor signaling pathways, suggesting that induced ovulation depends on the hypothalamic–pituitary–ovarian axis. Identifying these candidate biomarkers enables a better understanding of Bactrian camel reproduction. Ovarian proteomic profiling and the measurement of selected proteins using more targeted methods is a promising approach for studying induced-ovulation mechanisms. [ABSTRACT FROM AUTHOR]

In the particular case of the Camelidae family, immunoglobulin proteins have evolved into a unique and more simplified architecture with only heavy chains. The variable domains of these chains, named VHHs, have a number of Complementary Determining Regions (CDRs) reduced by half, and can function as single domains making them good candidates for molecular tools. 3D structure prediction of these domains is a beneficial and advantageous step to advance their developability as molecular tools. Nonetheless, the conformations of CDRs loops in these domains remain difficult to predict due to their higher conformational diversity. In addition to CDRs loop diversity, our earlier study has established that Framework Regions (FRs) are also not entirely conformationally conserved which establishes a need for more rigorous analyses of these regions that could assist in template selection. In the current study, VHHs models using different template selection strategies for comparative modeling using Modeller have been extensively assessed. This study analyses the conformational changes in both CDRs and FRs using an original strategy of conformational discretization based on a structural alphabet. Conformational sampling in selected cases is precisely reported. Some interesting outcomes of the structural analyses of models also draw attention towards the distinct difficulty in 3D structure prediction of VHH domains. [ABSTRACT FROM AUTHOR]

Simple Summary: Alpacas (Vicugna pacos) are South American members of the tribe Lamini of the Camelidae family. They are bred for their fiber, which is considered a luxury material. Interest in alpaca breeding is increasing in Poland, but the local alpaca population is relatively young and heterogeneous. The poor quality of alpaca fiber results from uncontrolled crossing with llamas (Lama glama). Hybridization between the two species is a well-known phenomenon among alpaca breeders worldwide and is the cause of poor fiber quality, which leads to economic losses. Microsatellite markers can distinguish alpacas from llamas and indicate the level of admixture. However, it is difficult to determine in which generation the admixture took place. The high genetic diversity of alpacas bred in Poland has emerged as a consequence of their mixed origins. In this context, the microsatellite markers recommended by the International Society for Animal Genetics have been shown to be highly useful for individual identification and parentage testing of alpacas. This study aimed to characterize the population structure and genetic diversity of alpacas maintained in Poland using 17 microsatellite markers recommended by the International Society for Animal Genetics. The classification of llamas, alpacas, and hybrids of both based on phenotype is often difficult due to long-term admixture. Our results showed that microsatellite markers can distinguish alpacas from llamas and provide information about the level of admixture of one species in another. Alpacas admixed with llamas constituted 8.8% of the tested individuals, with the first-generation hybrid displaying only 7.4% of llama admixture. The results showed that Poland hosts a high alpaca genetic diversity as a consequence of their mixed origin. More than 200 different alleles were identified and the average observed heterozygosity and expected heterozygosity values were 0.745 and 0.768, respectively, the average coefficient of inbreeding was 0.034, and the average polymorphism information content value was 0.741. The probability of exclusion for one parent was estimated at 0.99995 and for two parents at 0.99999. [ABSTRACT FROM AUTHOR]

The role of γδ T cells in vertebrate immunity is still an unsolved puzzle. Species such as humans and mice display a low percentage of these T lymphocytes (i.e., "γδ low species") with a restricted diversity of γδ T cell receptors (TR). Conversely, artiodactyl species (i.e., "γδ high species") account for a high proportion of γδ T cells with large γ and δ chain repertoires. The genomic organisation of the TR γ (TRG) and δ (TRD) loci has been determined in sheep and cattle, noting that a wide number of germline genes that encode for γ and δ chains characterise their genomes. Taking advantage of the current improved version of the genome assembly, we have investigated the genomic structure and gene content of the dromedary TRD locus, which, as in the other mammalian species, is nested within the TR α (TRA) genes. The most remarkable finding was the identification of a very limited number of variable germline genes (TRDV) compared to sheep and cattle, which supports our previous expression analyses for which the somatic hypermutation mechanism is able to enlarge and diversify the primary repertoire of dromedary δ chains. Furthermore, the comparison between genomic and expressed sequences reveals that D genes, up to four incorporated in a transcript, greatly contribute to the increased diversity of the dromedary δ chain antigen binding-site. [ABSTRACT FROM AUTHOR]

Nanobody (Nb), a new type of biorecognition element generally from Camelidae, has the characteristics of small molecular weight, high stability, great solubility and high expression level in E. coli. In this study, with 19-nortestosterone (19-NT), an anabolic androgenic steroid as target drug, three specific Nbs against 19-NT were selected from camel immune library by phage display technology. The obtained Nbs showed excellent thermostability and organic solvent tolerance. The nanobody Nb2F7 with the best performance was used to develop a sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for 19-NT detection. Under optimized conditions, the standard curve of ic-ELISA was fitted with a half-maximal inhibitory concentration (IC50) of 1.03 ng/mL and a detection limit (LOD) of 0.10 ng/mL for 19-NT. Meanwhile, the developed assay had low cross- reactivity with analogs and the recoveries of 19-NT ranged from 82.61% to 99.24% in spiked samples. The correlation coefficient between ic-ELISA and the ultra-performance liquid chromatography/mass spectrometry (UPLC-MS/MS) method was 0.9975, which indicated that the nanobody-based ic-ELISA could be a useful tool for a rapid analysis of 19-NT in animal urine samples. [ABSTRACT FROM AUTHOR]

In vivo clinical applications of nanobodies (VHHs) require molecules that induce minimal immunoresponse and therefore possess sequences as similar as possible to the human VH domain. Although the relative sequence variability in llama nanobodies has been used to identify scaffolds with partially humanized signature, the transformation of the Camelidae hallmarks in the framework2 still represents a major problem. We assessed a set of mutants in silico and experimentally to elucidate what is the contribution of single residues to the VHH stability and how their combinations affect the mutant nanobody stability. We described at molecular level how the interaction among residues belonging to different structural elements enabled a model llama nanobody (C8WT, isolated from a naïve library) to be functional and maintain its stability, despite the analysis of its primary sequence would classify it as aggregation-prone. Five chimeras formed by grafting CDRs isolated from different nanobodies into C8WT scaffold were successfully expressed as soluble proteins and both tested clones preserved their antigen binding specificity. We identified a nanobody with human hallmarks that seems suitable for humanizing selected camelid VHHs by grafting heterologous CDRs in its scaffold and could serve for the preparation of a synthetic library of human-like single domains. [ABSTRACT FROM AUTHOR]

Cytotoxic T cells and natural killer cells can kill target cells based on their expression and release of perforin, granulysin, and granzymes. Genes encoding these molecules have been only poorly annotated in camelids. Based on bioinformatic analyses of genomic resources, sequences corresponding to perforin, granulysin, and granzymes were identified in genomes of camelids and related ungulate species, and annotation of the corresponding genes was performed. A phylogenetic tree was constructed to study evolutionary relationships between the species analyzed. Re-sequencing of all genes in a panel of 10 dromedaries and 10 domestic Bactrian camels allowed analyzing their individual genetic polymorphisms. The data showed that all extant Old World camelids possess functional genes for two pore-forming proteins (PRF1, GNLY) and six granzymes (GZMA, GZMB, GZMH, GZMK, GZMM, and GZMO). All these genes were represented as single copies in the genome except the GZMH gene exhibiting interspecific differences in the number of loci. High protein sequence similarities with other camelid and ungulate species were observed for GZMK and GZMM. The protein variability in dromedaries and Bactrian camels was rather low, except for GNLY and chymotrypsin-like granzymes (GZMB, GZMH). [ABSTRACT FROM AUTHOR]

Single-domain antibodies derive from the heavy-chain-only antibodies of Camelidae (camel, dromedary, llama, alpaca, vicuñas, and guananos; i.e., nanobodies) and cartilaginous fishes (i.e., VNARs). Their small size, antigen specificity, plasticity, and potential to recognize unique conformational epitopes represent a diagnostic and therapeutic opportunity for many central nervous system (CNS) pathologies. However, the blood-brain barrier (BBB) poses a challenge for their delivery into the brain parenchyma. Nevertheless, numerous neurological diseases and brain pathologies, including cancer, result in BBB leakiness favoring single-domain antibodies uptake into the CNS. Some single-domain antibodies have been reported to naturally cross the BBB. In addition, different strategies and methods to deliver both nanobodies and VNARs into the brain parenchyma can be exploited when the BBB is intact. These include device-based and physicochemical disruption of the BBB, receptor and adsorptive-mediated transcytosis, somatic gene transfer, and the use of carriers/shuttles such as cell-penetrating peptides, liposomes, extracellular vesicles, and nanoparticles. Approaches based on single-domain antibodies are reaching the clinic for other diseases. Several tailoring methods can be followed to favor the transport of nanobodies and VNARs to the CNS, avoiding the limitations imposed by the BBB to fulfill their therapeutic, diagnostic, and theragnostic promises for the benefit of patients suffering from CNS pathologies.

Nanobodies (Nbs) are the smallest antigen-binding, single domain fragments derived from heavy-chain-only antibodies from Camelidae. Among the several advantages over conventional monoclonal antibodies, their small size (12–15 kDa) allows them to extravasate rapidly, to show improved tissue penetration, and to clear rapidly from blood, which are important characteristics for cancer imaging and targeted radiotherapy. Herein, we identified Nbs against CD33, a marker for acute myeloid leukemia (AML). A total of 12 Nbs were generated against recombinant CD33 protein, out of which six bound natively CD33 protein, expressed on the surface of acute myeloid leukemia THP-1 cells. The equilibrium dissociation constants (KD) of these six Nbs and CD33 range from 4 to 270 nM, and their melting temperature (Tm) varies between 52.67 and 67.80 °C. None of these Nbs showed leukemogenicity activity in vitro. The selected six candidates were radiolabeled with 99mTc, and their biodistribution was evaluated in THP-1-tumor-bearing mice. The imaging results demonstrated the fast tumor-targeting capacity of the Nbs in vivo. Among the anti-CD33 Nbs, Nb_7 showed the highest tumor uptake (2.53 ± 0.69 % injected activity per gram (IA/g), with low background signal, except in the kidneys and bladder. Overall, Nb_7 exhibits the best characteristics to be used as an anti-CD33 targeting vehicle for future diagnostic or therapeutic applications. [ABSTRACT FROM AUTHOR]

Dialysis-related amyloidosis (DRA), which has been widely recognized to be associated with the accumulation of β2-microglobulin (β2-m) in blood, is one of the most common complications in patients receiving long-term dialysis treatment. The most significant side-effect of existing hemodialysis sorbents for the removal of β2-m from blood is the loss of vital proteins due to non-specific adsorptions. Although the traditional antibodies have the capability to specifically remove β2-m from blood, high cost limits their applications in clinics. Single domain antibodies derived from the Camelidae species serve as a superior choice in the preparation of immunoadsorbents due to their small size, high stability, amenability, simplicity of expression in microbes, and high affinity to recognize and interact with β2-m. In this study, we modified the anti-β2-m VHH by the formylglycine-generating enzyme (FGE), and then directly immobilized the aldehyde-modified VHH to the amino-activated beads. Notably, the fabrication is cost- and time-effective, since all the preparation steps were performed in the crude cell extract without rigorous purification. The accordingly prepared immunoadsorbent with VHHs as ligands exhibited the high capacity of β2-m (0.75 mg/mL). In conclusion, the VHH antibodies were successfully used as affinity ligands in the preparation of novel immunoadsorbents by the site-specific immobilization, and effectively adsorbed β2-m from blood, therefore opening a new avenue for efficient hemodialysis. [ABSTRACT FROM AUTHOR]

Rift Valley fever phlebovirus (RVFV), the causative agent of an emerging zoonotic disease in Africa and Arabia, can infect a variety of species, predominantly ruminants, camelids, and humans. While clinical symptoms are mostly absent in adult ruminants and camelids, RVFV infection may lead to a serious, sometimes fatal disease in humans. Virus transmissions between individuals and between species mainly occur through mosquito bites, but direct or even indirect contact with infectious materials may also result in infection. Although the main reservoir of the virus is not yet identified, small mammals such as rodents and bats may act as amplifying hosts. We therefore inoculated Rousettus aegyptiacus fruit bats that are abundant in northern Africa with the vaccine strain MP-12, in order to elucidate the general competence of this species for virus propagation and transmission. We were able to detect the RVFV genome in the spleen of each of these animals, and re-isolated the virus from the spleen and liver of some animals. Moreover, we were able to identify the Gc RVFV surface antigen in mild subacute multifocal necrotizing hepatic lesions of one bat which was sacrificed 7 days post exposure. These findings demonstrate that Rousettus aegyptiacus fruit bats can propagate RVFV. [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibition (ICI) is a promising cancer therapy, which has progressed rapidly from a preclinical concept to clinical implementation. Commonly considered targets in ICI are CTLA-4, PD-1/PD-L1, and LAG-3, and the list grows. As ICI is generally only beneficial for a subset of patients, there is a need to select patients that are eligible for therapy as well as to monitor therapy response. There is growing interest to do this noninvasively, by molecular imaging with target-specific tracers. To this day, noninvasive imaging has focused on CTLA-4 and PD-1/PD-L1, while there is no noninvasive tool available to accurately assess LAG-3 expression in vivo. In this proof-of-concept study, we developed nanobodies, the smallest functional fragments from camelid heavy chain-only antibodies, to noninvasively evaluate mouse LAG-3 expression using single photon emission computed tomography (SPECT)/CT imaging. The in vitro characterization of 114 nanobodies led to the selection of nine nanobodies binding to mouse LAG-3. The injection of 99m Technetium-labeled nanobodies in healthy mice showed specific uptake in immune peripheral organs like the spleen and lymph nodes, which was not observed in LAG-3 gene knock-out mice. Moreover, nanobody uptake could be visualized using SPECT/CT and correlated to the presence of LAG-3 as assessed in flow cytometry and immunohistochemistry. SPECT/CT scans of tumor bearing mice further confirmed the diagnostic potential of the nanobodies. These findings substantiate the approach to use nanobodies as a tool to image inhibitory immune checkpoints in the tumor environment., Competing Interests: Marleen Keyaerts received travel and accommodation expenses from Bayer; Nick Devoogdt, Sam Massa, Geert Raes are shareholders and consultants of Camel-IDS which develops cancer therapeutics based on radiolabeled nanobodies. Marleen Keyaerts received research funding from Camel-IDS. Quentin Lecocq, Marleen Keyaerts, Karine Breckpot, Geert Raes, Nick Devoogdt has patents on Nanobody imaging and therapy.