RESUMO
mAbs have been instrumental for targeted cancer therapies. However, their relatively large size and physicochemical properties result in a heterogenous distribution in the tumor microenvironment, usually restricted to the first cell layers surrounding blood vessels, and a limited ability to penetrate the brain. Nanobodies are tenfold smaller, resulting in a deeper tumor penetration and the ability to reach cells in poorly perfused tumor areas. Nanobodies are rapidly cleared from the circulation, which generates a fast target-to-background contrast that is ideally suited for molecular imaging purposes but may be less optimal for therapy. To circumvent this problem, nanobodies have been formatted to noncovalently bind albumin, increasing their serum half-life without majorly increasing their size. Finally, nanobodies have shown superior qualities to infiltrate brain tumors as compared to mAbs. In this review, we discuss why these features make nanobodies prime candidates for targeted therapy of cancer.
Assuntos
Neoplasias Encefálicas , Anticorpos de Domínio Único , Humanos , Anticorpos de Domínio Único/uso terapêutico , Anticorpos Monoclonais , Microambiente TumoralRESUMO
Invasive fungal disease accounts for about 3.8â million deaths annually, an unacceptable rate that urgently prompts the discovery of new knowledge-driven treatments. We report the use of camelid single-domain nanobodies (Nbs) against fungal ß-1,3-glucanosyltransferases (Gel) involved in ß-1,3-glucan transglycosylation. Crystal structures of two Nbs with Gel4 from Aspergillus fumigatus revealed binding to a dissimilar CBM43 domain and a highly conserved catalytic domain across fungal species, respectively. Anti-Gel4 active site Nb3 showed significant antifungal efficacy in vitro and in vivo prophylactically and therapeutically against different A. fumigatus and Cryptococcus neoformans isolates, reducing the fungal burden and disease severity, thus significantly improving immunocompromised animal survival. Notably, C. deneoformans (serotypeâ D) strains were more susceptible to Nb3 and genetic Gel deletion than C. neoformans (serotype A) strains, indicating a key role for ß-1,3-glucan remodelling in C. deneoformans survival. These findings add new insight about the role of ß-1,3-glucan in fungal biology and demonstrate the potential of nanobodies in targeting fungal enzymes to combat invasive fungal diseases.
Assuntos
Aspergillus fumigatus , Domínio Catalítico , Anticorpos de Domínio Único , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/farmacologia , Aspergillus fumigatus/imunologia , Aspergillus fumigatus/enzimologia , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/imunologia , Antifúngicos/química , Antifúngicos/farmacologia , Animais , Camundongos , Glucana Endo-1,3-beta-D-GlucosidaseRESUMO
High-quality affinity probes are critical for sensitive and specific protein detection, in particular for detection of protein biomarkers in the early phases of disease development. Proximity extension assays (PEAs) have been used for high-throughput multiplexed protein detection of up to a few thousand different proteins in one or a few microliters of plasma. Clonal affinity reagents can offer advantages over the commonly used polyclonal antibodies (pAbs) in terms of reproducibility and standardization of such assays. Here, we explore nanobodies (Nbs) as an alternative to pAbs as affinity reagents for PEA. We describe an efficient site-specific approach for preparing high-quality oligo-conjugated Nb probes via enzyme coupling using Sortase A (SrtA). The procedure allows convenient removal of unconjugated affinity reagents after conjugation. The purified high-grade Nb probes were used in PEA, and the reactions provided an efficient means to select optimal pairs of binding reagents from a group of affinity reagents. We demonstrate that Nb-based PEA (nano-PEA) for interleukin-6 (IL6) detection can augment assay performance, compared to the use of pAb probes. We identify and validate Nb combinations capable of binding in pairs without competition for IL6 antigen detection by PEA.
Assuntos
Anticorpos de Domínio Único , Anticorpos , Indicadores e Reagentes , Interleucina-6 , Oligonucleotídeos , Reprodutibilidade dos TestesRESUMO
Ovarian cancer ranks fifth in cancer-related deaths among women. Since ovarian cancer patients are often asymptomatic, most patients are diagnosed only at an advanced stage of disease. This results in a 5-year survival rate below 50%, which is in strong contrast to a survival rate as high as 94% if detected and treated at an early stage. Monitoring serum biomarkers offers new possibilities to diagnose ovarian cancer at an early stage. In this study, nanobodies targeting the ovarian cancer biomarkers human epididymis protein 4 (HE4), secretory leukocyte protease inhibitor (SLPI), and progranulin (PGRN) were evaluated regarding their expression levels in bacterial systems, epitope binning, and antigen-binding affinity by enzyme-linked immunosorbent assay and surface plasmon resonance. The selected nanobodies possess strong binding affinities for their cognate antigens (KD~0.1-10 nM) and therefore have a pronounced potential to detect ovarian cancer at an early stage. Moreover, it is of utmost importance that the limits of detection (LOD) for these biomarkers are in the pM range, implying high specificity and sensitivity, as demonstrated by values in human serum of 37 pM for HE4, 163 pM for SLPI, and 195 pM for PGRN. These nanobody candidates could thus pave the way towards multiplexed biosensors.
Assuntos
Neoplasias Ovarianas , Anticorpos de Domínio Único , Humanos , Feminino , Detecção Precoce de Câncer , Carcinoma Epitelial do Ovário , Neoplasias Ovarianas/diagnóstico , Biomarcadores Tumorais , ProgranulinasRESUMO
Uncontrolled growth of solid tumors will result in a hallmark hypoxic condition, whereby the key transcriptional regulator of hypoxia inducible factor-1α (HIF-1α) will be stabilized to activate the transcription of target genes that are responsible for the metabolism, proliferation, and metastasis of tumor cells. Targeting and inhibiting the transcriptional activity of HIF-1 may provide an interesting strategy for cancer therapy. In the present study, an immune library and a synthetic library were constructed for the phage display selection of Nbs against recombinant PAS B domain protein (rPasB) of HIF-1α. After panning and screening, seven different nanobodies (Nbs) were selected, of which five were confirmed via immunoprecipitation to target the native HIF-1α subunit. The inhibitory effect of the selected Nbs on HIF-1 induced activation of target genes has been evaluated after intracellular expression of these Nbs in HeLa cells. The dramatic inhibition of both intrabody formats on the expression of HIF-1-related target genes has been confirmed, which indicated the inhibitory efficacy of selected Nbs on the transcriptional activity of HIF-1.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Anticorpos de Domínio Único/farmacologia , Transcrição Gênica/efeitos dos fármacos , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Hipóxia Celular/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Domínios Proteicos/genética , Domínios Proteicos/imunologia , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/metabolismo , Transfecção , Neoplasias do Colo do Útero/patologiaRESUMO
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.
Assuntos
Leucemia Mieloide Aguda/imunologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Anticorpos de Domínio Único/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Epitopos/imunologia , Feminino , Humanos , Cinética , Camundongos , Camundongos SCID , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Anticorpos de Domínio Único/genética , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único , Temperatura de TransiçãoRESUMO
One of the hallmarks of cancer is the overproduction of growth factors such as EGF. Despite the clinical success achieved by EGFR-targeted therapies, their long-term efficacy is compromised by the onset of drug-resistant mutations. To address this issue, a family of camelid-derived single-domain antibodies (Nbs) were generated, obtaining the first direct EGF inhibitors that prevent EGFR phosphorylation and pathway activation through this new mechanism of action. The two best Nbs were subjected to a detailed investigation of their interaction mechanism that revealed important differences in their binding kinetics and equilibrium thermodynamics. These distinct behaviors at the biophysical level translate into an equally efficient inhibition of the cellular EGFR phosphorylation, thus proving the efficacy of these Nbs to turn off the initiation of this key oncogenic pathway in cancer cells.
Assuntos
Anticorpos de Domínio Único/imunologia , Sequência de Aminoácidos , Ativação Enzimática , Fator de Crescimento Epidérmico/administração & dosagem , Receptores ErbB/imunologia , Humanos , Fosforilação , Anticorpos de Domínio Único/químicaRESUMO
Site-specific functionalization of nanobodies after introducing bioorthogonal groups offers the possibility to biofunctionalize surfaces with a uniformly oriented layer of nanobodies. In this paper, expressed protein ligation (EPL) was used for site-specific alkynation of the model nanobody NbBcII10. In contrast to EPL constructs, which are typically expressed in the cytoplasm, nanobodies are expressed in the periplasm where its oxidizing environment ensures a correct folding and disulfide bond formation. Different pathways were explored to express the EPL constructs in the periplasm but simultaneously, the effect of cytoplasmic expression on the functionality of NbBcII10 was also evaluated. By using Escherichia coli SHuffle®T7 cells, it was demonstrated that expression of the EPL complex in the cytoplasm was readily established and that site-specifically mono-alkynated nanobodies can be produced with the same binding properties as the non-modified NbBcII10 expressed in the periplasm. In conclusion, this paper shows that periplasmic expression of the EPL complex is quite challenging, but cytoplasmic expression has proven to be a valuable alternative.
Assuntos
Citoplasma/metabolismo , Escherichia coli/metabolismo , Expressão Gênica , Periplasma/metabolismo , Anticorpos de Domínio Único , Citoplasma/genética , Escherichia coli/genética , Periplasma/genética , Anticorpos de Domínio Único/biossíntese , Anticorpos de Domínio Único/genéticaRESUMO
The gene for a protein domain, derived from a tumor marker, fused to His tag codons and under control of a T7 promotor was expressed in E. coli strain BL21 (DE3). The recombinant protein was purified from cell lysates through immobilized metal affinity chromatography and size-exclusion chromatography. A contaminating bacterial protein was consistently co-purified, even using stringent washing solutions containing 50 or 100 mM imidazole. Immunization of a dromedary with this contaminated protein preparation, and the subsequent generation and panning of the immune Nanobody library yielded several Nanobodies of which 2/3 were directed against the bacterial contaminant, reflecting the immunodominance of this protein to steer the dromedary immune response. Affinity adsorption of this contaminant using one of our specific Nanobodies followed by mass spectrometry identified the bacterial contaminant as FKBP-type peptidyl-prolyl cis-trans isomerase (SlyD) from E. coli. This SlyD protein contains in its C-terminal region 14 histidines in a stretch of 31 amino acids, which explains its co-purification on Ni-NTA resin. This protein is most likely present to varying extents in all recombinant protein preparations after immobilized metal affinity chromatography. Using our SlyD-specific Nb 5 we generated an immune-complex that could be removed either by immunocapturing or by size exclusion chromatography. Both methods allow us to prepare a recombinant protein sample where the SlyD contaminant was quantitatively eliminated.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli , Peptidilprolil Isomerase/química , Anticorpos de Domínio Único , Animais , Camelus , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Peptidilprolil Isomerase/antagonistas & inibidores , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Anticorpos de Domínio Único/biossíntese , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/isolamento & purificaçãoRESUMO
BACKGROUND: Variable domains of camelid heavy-chain antibodies, commonly named nanobodies, have high biotechnological potential. In view of their broad range of applications in research, diagnostics and therapy, engineering their stability is of particular interest. One important aspect is the improvement of thermostability, because it can have immediate effects on conformational stability, protease resistance and aggregation propensity of the protein. METHODS: We analyzed the sequences and thermostabilities of 78 purified nanobody binders. From this data, potentially stabilizing amino acid variations were identified and studied experimentally. RESULTS: Some mutations improved the stability of nanobodies by up to 6.1°C, with an average of 2.3°C across eight modified nanobodies. The stabilizing mechanism involves an improvement of both conformational stability and aggregation behavior, explaining the variable degree of stabilization in individual molecules. In some instances, variations predicted to be stabilizing actually led to thermal destabilization of the proteins. The reasons for this contradiction between prediction and experiment were investigated. CONCLUSIONS: The results reveal a mutational strategy to improve the biophysical behavior of nanobody binders and indicate a species-specificity of nanobody architecture. GENERAL SIGNIFICANCE: This study illustrates the potential and limitations of engineering nanobody thermostability by merging sequence information with stability data, an aspect that is becoming increasingly important with the recent development of high-throughput biophysical methods.
Assuntos
Engenharia de Proteínas , Anticorpos de Domínio Único/química , Sequência de Aminoácidos , Agregados Proteicos , Conformação Proteica , Estabilidade ProteicaRESUMO
To quantify and characterize the potentially toxic protein aggregates associated with neurodegenerative diseases, a high-throughput assay based on measuring the extent of aggregate-induced Ca2+ entry into individual lipid vesicles has been developed. This approach was implemented by tethering vesicles containing a Ca2+ sensitive fluorescent dye to a passivated surface and measuring changes in the fluorescence as a result of membrane disruption using total internal reflection microscopy. Picomolar concentrations of Aß42 oligomers could be observed to induce Ca2+ influx, which could be inhibited by the addition of a naturally occurring chaperone and a nanobody designed to bind to the Aß peptide. We show that the assay can be used to study aggregates from other proteins, such as α-synuclein, and to probe the effects of complex biofluids, such as cerebrospinal fluid, and thus has wide applicability.
Assuntos
Cálcio/metabolismo , Bicamadas Lipídicas/metabolismo , Agregados Proteicos/fisiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/imunologia , Peptídeos beta-Amiloides/metabolismo , Cálcio/química , Clusterina/química , Clusterina/metabolismo , Corantes Fluorescentes/química , Humanos , Cinética , Bicamadas Lipídicas/química , Imagem Óptica , Ligação Proteica , Anticorpos de Domínio Único/imunologiaRESUMO
GAK (cyclin G-associated kinase) is a key regulator of clathrin-coated vesicle trafficking and plays a central role during development. Additionally, due to the unusually high plasticity of its catalytic domain, it is a frequent 'off-target' of clinical kinase inhibitors associated with respiratory side effects of these drugs. In the present paper, we determined the crystal structure of the GAK catalytic domain alone and in complex with specific single-chain antibodies (nanobodies). GAK is constitutively active and weakly associates in solution. The GAK apo structure revealed a dimeric inactive state of the catalytic domain mediated by an unusual activation segment interaction. Co-crystallization with the nanobody NbGAK_4 trapped GAK in a dimeric arrangement similar to the one observed in the apo structure, whereas NbGAK_1 captured the activation segment of monomeric GAK in a well-ordered conformation, representing features of the active kinase. The presented structural and biochemical data provide insight into the domain plasticity of GAK and demonstrate the utility of nanobodies to gain insight into conformational changes of dynamic molecules. In addition, we present structural data on the binding mode of ATP mimetic inhibitors and enzyme kinetic data, which will support rational inhibitor design of inhibitors to reduce the off-target effect on GAK.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Multimerização Proteica/fisiologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/fisiologia , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/fisiologia , Animais , Apoproteínas/química , Apoproteínas/fisiologia , Camelus , Domínio Catalítico/fisiologia , Cristalização/métodos , Ativação Enzimática/fisiologia , Humanos , Conformação ProteicaRESUMO
Fluorescence molecular imaging plays a vital role in image-guided surgery. In this context, the urokinase plasminogen activator receptor (uPAR) is an interesting biomarker enabling the detection and delineation of various tumor types due to its elevated expression on both tumor cells and the tumor microenvironment. In this study, anti-uPAR Nanobodies (Nbs) are generated through llama immunization with human and murine uPAR protein. Extensive in vitro characterization and in vivo testing with radiolabeled variants are conducted to assess their pharmacokinetics and select lead compounds. Subsequently, the selected Nbs are converted into fluorescent agents, and their application for fluorescence-guided surgery is evaluated in various subcutaneous and orthotopic tumor models. The study yields a panel of high-affinity anti-uPAR Nbs, showing specific binding across multiple types of cancer cells in vitro and in vivo. Lead fluorescently-labeled compounds exhibit high tumor uptake with high contrast at 1 h after intravenous injection across all assessed uPAR-expressing tumor models, outperforming a non-targeting control Nb. Additionally, rapid and accurate tumor localization and demarcation are demonstrated in an orthotopic human glioma model. Utilizing these Nbs can potentially enhance the precision of surgical tumor resection and, consequently, improve survival rates in the clinic.
Assuntos
Receptores de Ativador de Plasminogênio Tipo Uroquinase , Anticorpos de Domínio Único , Cirurgia Assistida por Computador , Animais , Anticorpos de Domínio Único/imunologia , Camundongos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Cirurgia Assistida por Computador/métodos , Humanos , Modelos Animais de Doenças , Linhagem Celular Tumoral , Imagem Óptica/métodos , Neoplasias/imunologia , Neoplasias/diagnóstico por imagem , Neoplasias/cirurgia , Corantes Fluorescentes , Camelídeos AmericanosRESUMO
Human African trypanosomiasis or sleeping sickness, caused by the protozoan parasite Trypanosoma brucei, is characterized by the manipulation of the host's immune response to ensure parasite invasion and persistence. Uncovering key molecules that support parasite establishment is a prerequisite to interfere with this process. We identified Q586B2 as a T. brucei protein that induces IL-10 in myeloid cells, which promotes parasite infection invasiveness. Q586B2 is expressed during all T. brucei life stages and is conserved in all Trypanosomatidae. Deleting the Q586B2-encoding Tb927.6.4140 gene in T. brucei results in a decreased peak parasitemia and prolonged survival, without affecting parasite fitness in vitro, yet promoting short stumpy differentiation in vivo. Accordingly, neutralization of Q586B2 with newly generated nanobodies could hamper myeloid-derived IL-10 production and reduce parasitemia. In addition, immunization with Q586B2 delays mortality upon a challenge with various trypanosomes, including Trypanosoma cruzi. Collectively, we uncovered a conserved protein playing an important regulatory role in Trypanosomatid infection establishment.
Assuntos
Trypanosoma brucei brucei , Trypanosoma cruzi , Tripanossomíase Africana , Animais , Humanos , Trypanosoma brucei brucei/genética , Interleucina-10/genética , Fatores de Virulência , Parasitemia/parasitologia , Tripanossomíase Africana/parasitologiaRESUMO
The antigen-binding fragment of functional heavy chain antibodies (HCAbs) in camelids comprises a single domain, named the variable domain of heavy chain of HCAbs (VHH). The VHH harbors remarkable amino acid substitutions in the framework region-2 to generate an antigen-binding domain that functions in the absence of a light chain partner. The substitutions provide a more hydrophilic, hence more soluble, character to the VHH but decrease the intrinsic stability of the domain. Here we investigate the functional role of an additional hallmark of dromedary VHHs, i.e. the extra disulfide bond between the first and third antigen-binding loops. After substituting the cysteines forming this interloop cystine by all 20 amino acids, we selected and characterized several VHHs that retain antigen binding capacity. Although VHH domains can function in the absence of an interloop disulfide bond, we demonstrate that its presence constitutes a net advantage. First, the disulfide bond stabilizes the domain and counteracts the destabilization by the framework region-2 hallmark amino acids. Second, the disulfide bond rigidifies the long third antigen-binding loop, leading to a stronger antigen interaction. This dual beneficial effect explains the in vivo antibody maturation process favoring VHH domains with an interloop disulfide bond.
Assuntos
Cisteína/química , Dissulfetos/química , Cadeias Pesadas de Imunoglobulinas/química , Anticorpos de Cadeia Única/química , Animais , Camelus , Cisteína/genética , Cisteína/metabolismo , Dissulfetos/metabolismo , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Estabilidade Proteica , Estrutura Terciária de Proteína , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismoRESUMO
ß-Amyloid (Aß) immunotherapy has become a promising strategy for reducing the level of Aß in brain. New immunological approaches have been recently proposed for rapid, early diagnosis, and molecular treatment of neurodegenerative diseases related to Alzheimer's Disease (AD). The combination of proteolytic epitope excision and extraction and mass spectrometry using digestion with various proteases has been shown to be an efficient tool for the identification and molecular characterization of antigenic determinants. Here, we report the identification of the Aß epitope recognized by the variable domain of single chain llama anti-Aß-antibodies, termed Aß-nanobodies, that have been discovered in the blood of camelids and found to be promising candidates for immunotherapy of AD. The epitope recognized by two Aß-specific nanobodies was identified by proteolytic epitope extraction- and excision-mass spectrometry using a series of proteases (trypsin, chymotrypsin, GluC-protease, and LysC-protease). Matrix-assisted laser desorption ionization--mass spectrometric analysis of the affinity--elution fraction provided the epitope, Aß(17-28), in the mid- to carboxy-terminal domain of Aß, which has been shown to exert an Aß-fibril inhibiting effect. Affinity studies of the synthetic epitope confirmed that the Aß(17-28) peptide is the minimal fragment that binds to the nanobodies. The interactions between the nanobodies and full length Aß(1-40) or Aß-peptides containing or lacking the epitope sequence were further characterized by enzyme linked immunosorbent assay and bioaffinity analysis. Determinations of binding affinities between the Aß-nanobodies and Aß(1-40) and the Aß(17-28) epitope provided K(D) values of approximately 150 and 700 nmol, respectively. Thus, the knowledge of the epitope may be highly useful for future studies of Aß-aggregation (oligomerization and fibril formation) and for designing new aggregation inhibitors.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/imunologia , Camelídeos Americanos/imunologia , Epitopos/química , Epitopos/imunologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologia , Doença de Alzheimer/imunologia , Doença de Alzheimer/terapia , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Peptídeo Hidrolases/química , Peptídeo Hidrolases/imunologia , Proteólise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Reduced levels of the Sortilin-related receptor with A-type repeats (SORLA) in different brain regions as well as in the cerebrospinal fluid have been associated with Alzheimer's disease. Methods and reagents to develop reliable detection assays to quantify SORLA and its specific isoforms are therefore much needed. Nanobodies (Nbs) are unique biomolecules derived from the blood of camelids that display advantageous physicochemical and antigen affinity properties, making them attractive tools with great relevance to both diagnostic and therapeutic applications. Here, we purified and characterized eight Nbs that were isolated from the blood of an alpaca immunized with the recombinant extracellular domain of SORLA. The selected Nbs showed high affinity to SORLA in the low nanomolar range as observed by surface plasmon resonance. For mapping of the Nbs' epitopes within the antigen, we transiently transfected HEK293 cells with a panel of SORLA deletion constructs, and developed a protocol of immunostaining by applying fluorescent dye conjugated Nbs. With this method, we showed that the selected Nbs specifically recognize a part of SORLA containing Fibronectin-type III domains, representing promising tools not only for disease clarifying research, but also for translational medicine as candidates for clinical diagnostic purposes.
Assuntos
Doença de Alzheimer , Anticorpos de Domínio Único , Humanos , Anticorpos de Domínio Único/genética , Mapeamento de Epitopos , Células HEK293 , EpitoposRESUMO
Homeostatic and pathological phenomena often affect multiple organs across the whole organism. Tissue clearing methods, together with recent advances in microscopy, have made holistic examinations of biological samples feasible. Here, we report the detailed protocol for nanobody(VHH)-boosted 3D imaging of solvent-cleared organs (vDISCO), a pressure-driven, nanobody-based whole-body immunolabeling and clearing method that renders whole mice transparent in 3 weeks, consistently enhancing the signal of fluorescent proteins, stabilizing them for years. This allows the reliable detection and quantification of fluorescent signal in intact rodents enabling the analysis of an entire body at cellular resolution. Here, we show the high versatility of vDISCO applied to boost the fluorescence signal of genetically expressed reporters and clear multiple dissected organs and tissues, as well as how to image processed samples using multiple fluorescence microscopy systems. The entire protocol is accessible to laboratories with limited expertise in tissue clearing. In addition to its applications in obtaining a whole-mouse neuronal projection map, detecting single-cell metastases in whole mice and identifying previously undescribed anatomical structures, we further show the visualization of the entire mouse lymphatic system, the application for virus tracing and the visualization of all pericytes in the brain. Taken together, our vDISCO pipeline allows systematic and comprehensive studies of cellular phenomena and connectivity in whole bodies.
Assuntos
Encéfalo , Imageamento Tridimensional , Camundongos , Animais , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Solventes/química , Neuritos , CorantesRESUMO
Rationale: Nanobodies (Nbs) have emerged as an elegant alternative to the use of conventional monoclonal antibodies in cancer therapy, but a detailed microscopic insight into the in vivo pharmacokinetics of different Nb formats in tumor-bearers is lacking. This is especially relevant for the recognition and targeting of pro-tumoral tumor-associated macrophages (TAMs), which may be located in less penetrable tumor regions. Methods: We employed anti-Macrophage Mannose Receptor (MMR) Nbs, in a monovalent (m) or bivalent (biv) format, to assess in vivo TAM targeting. Intravital and confocal microscopy were used to analyse the blood clearance rate and targeting kinetics of anti-MMR Nbs in tumor tissue, healthy muscle tissue and liver. Fluorescence Molecular Tomography was applied to confirm anti-MMR Nb accumulation in the primary tumor and in metastatic lesions. Results: Intravital microscopy demonstrated significant differences in the blood clearance rate and macrophage targeting kinetics of (m) and (biv)anti-MMR Nbs, both in tumoral and extra-tumoral tissue. Importantly, (m)anti-MMR Nbs are superior in reaching tissue macrophages, an advantage that is especially prominent in tumor tissue. The administration of a molar excess of unlabelled (biv)anti-MMR Nbs increased the (m)anti-MMR Nb bioavailability and impacted on its macrophage targeting kinetics, preventing their accumulation in extra-tumoral tissue (especially in the liver) but only partially influencing their interaction with TAMs. Finally, anti-MMR Nb administration not only allowed the visualization of TAMs in primary tumors, but also at a distant metastatic site. Conclusions: These data describe, for the first time, a microscopic analysis of (m) and (biv)anti-MMR Nb pharmacokinetics in tumor and healthy tissues. The concepts proposed in this study provide important knowledge for the future use of Nbs as diagnostic and therapeutic agents, especially for the targeting of tumor-infiltrating immune cells.
Assuntos
Neoplasias , Anticorpos de Domínio Único , Humanos , Receptor de Manose , Lectinas Tipo C , Lectinas de Ligação a Manose , Receptores de Superfície Celular , Macrófagos Associados a Tumor , Neoplasias/tratamento farmacológicoRESUMO
Camel single-domain antibody fragments or Nanobodies, are practical in a wide range of applications. Their unique biochemical and biophysical properties permit an intracellular expression and antigen targeting. The availability of an efficient intracellular selection step would immediately identify the best intracellularly performing functional antibody fragments. Therefore, we assessed a bacterial-two-hybrid system to retrieve such Nanobodies. With GFP as an antigen we demonstrate that antigen-specific Nanobodies of sub-micromolar affinity and stability above 30 kJ/mol, at a titer of 10(-4) can be retrieved in a single-step selection. This was further proven practically by the successful recovery from an 'immune' library of multiple stable, antigen-specific Nanobodies of good affinity for HIV-1 integrase or nucleoside hydrolase. The sequence diversity, intrinsic domain stability, antigen-specificity and affinity of these binders compare favorably to those that were retrieved in parallel by phage display pannings.