RESUMEN
Natural backbone-cyclized proteins have an increased thermostability and resistance towards proteases, characteristics that have sparked interest in head-to-tail cyclization as a method to stability-enhance proteins used in diagnostics and therapeutic applications, for example. In this proof-of principle study, we have produced and investigated a head-to-tail cyclized and HER2-specific ZHER2:342 Affibody dimer. The sortase A-mediated cyclization reaction is highly efficient (>95%) under optimized conditions, and renders a cyclic ZHER3:342-dimer with an apparent melting temperature, Tm, of 68 °C, which is 3 °C higher than that of its linear counterpart. Circular dichroism spectra of the linear and cyclic dimers looked very similar in the far-UV range, both before and after thermal unfolding to 90 °C, which suggests that cyclization does not negatively impact the helicity or folding of the cyclic protein. The cyclic dimer had an apparent sub-nanomolar affinity (Kd ~750 pM) to the HER2-receptor, which is a ~150-fold reduction in affinity relative to the linear dimer (Kd ~5 pM), but the anti-HER2 Affibody dimer remained a high-affinity binder even after cyclization. No apparent difference in proteolytic stability was detected in an endopeptidase degradation assay for the cyclic and linear dimers. In contrast, in an exopeptidase degradation assay, the linear dimer was shown to be completely degraded after 5 min, while the cyclic dimer showed no detectable degradation even after 60 min. We further demonstrate that a site-specifically DyLight 594-labeled cyclic dimer shows specific binding to HER2-overexpressing cells. Taken together, the results presented here demonstrate that head-to-tail cyclization can be an effective strategy to increase the stability of an Affibody dimer.
Asunto(s)
Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Biocatálisis , Neoplasias de la Mama/metabolismo , Cisteína Endopeptidasas/metabolismo , Multimerización de Proteína , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Neoplasias de la Mama/patología , Dicroismo Circular , Ciclización , Femenino , Humanos , Cinética , Células MCF-7 , Microscopía Fluorescente , Péptido Hidrolasas/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Resonancia por Plasmón de SuperficieRESUMEN
Photoisomerization of the trans and cis isomers of azobenzene derivatives has been used to control the function of biomolecules in a reversible and nondestructive manner. In this study, affibody molecules, representing a class of small, helical proteins that can be engineered for binding to a wide range of target proteins, have been investigated by the incorporation of a photoswitchable azobenzene derivative in the molecule. Three different Z domain variants were produced by solid phase peptide synthesis and conjugated by thiol-directed chemistry to an azobenzene-based photoswitch. The proteins were screened for binding to and light elution from an IgG-sepharose affinity column. One of the tested Z variants, ZC3, showed efficient binding to the column and could be eluted by irradiation with light at 400 nm. In a reverse affinity chromatography assay, where the ZC3 variant was coupled to sepharose, human IgG1 could be captured to the column and partially eluted by light. Further studies of the azobenzene-conjugated ZC3 domain by surface plasmon resonance (SPR) confirmed the high affinity binding to IgG, and circular dichroism (CD) spectroscopy showed that the protein has a high α-helical secondary structure content.
Asunto(s)
Inmunoglobulina G/metabolismo , Luz , Proteína Estafilocócica A/química , Proteína Estafilocócica A/metabolismo , Modelos Moleculares , Unión Proteica/efectos de la radiación , Dominios Proteicos , Resonancia por Plasmón de SuperficieRESUMEN
Antibody-DNA conjugates are powerful tools for DNA-assisted protein analysis. Growing usage of these methods demands efficient production of high-quality conjugates. We developed an easy and fast synthesis route yielding covalent antibody-DNA conjugates with a defined conjugation site and low batch-to-batch variability. We utilize the Z domain from protein A, containing the unnatural amino acid 4-benzoylphenylalanine (BPA) for photoaffinity labeling of the antibodies' Fc region. Z(xBPA) domains are C-terminally modified with triple-glycine (G3)-modified DNA-oligonucleotides via enzymatic Sortase A coupling. We show reliable modification of the most commonly used IgG's. To prove our conjugates' functionality, we detected antibody-antigen binding events in an assay called Droplet Barcode Sequencing for Protein analysis (DBS-Pro). It confirms not only retained functionality for both conjugate parts but also the potential of using DBS-Pro for quantifying protein abundances. As intermediates are easily storable and our approach is modular, it offers a convenient strategy for screening various antibody-DNA conjugates using the same starting material.
Asunto(s)
Anticuerpos Monoclonales/química , ADN/química , Inmunoconjugados/química , Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/química , Fenilalanina/análogos & derivados , Etiquetas de Fotoafinidad/química , Aminoaciltransferasas/inmunología , Aminoaciltransferasas/metabolismo , Anticuerpos Monoclonales/inmunología , Reacciones Antígeno-Anticuerpo/inmunología , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/inmunología , Cisteína Endopeptidasas/metabolismo , ADN/inmunología , Humanos , Inmunoconjugados/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Fenilalanina/químicaRESUMEN
Site-specific labeling of antibodies can be performed using the immunoglobulin-binding Z domain, derived from staphylococcal protein A (SpA), which has a well-characterized binding site in the Fc region of antibodies. By introducing a photoactivable probe in the Z domain, a covalent bond can be formed between the Z domain and the antibody by irradiation with UV light. The aim of this study was to improve the conjugation yield for labeling of different subclasses of IgG having different sequence composition, using a photoactivated Z domain variant. Four different variants of the Z domain (Z5BPA, Z5BBA, Z32BPA, and Z32BBA) were synthesized to investigate the influence of the position of the photoactivable probe and the presence of a flexible linker between the probe and the protein. For two of the variants, the photoreactive benzophenone group was introduced as part of an amino acid side chain by incorporation of the unnatural amino acid benzoylphenylalanine (BPA) during peptide synthesis. For the other two variants, the photoreactive benzophenone group was attached via a flexible linker by coupling of benzoylbenzoic acid (BBA) to the ε-amino group of a selectively deprotected lysine residue. Photoconjugation experiments using human IgG1, mouse IgG1, and mouse IgG2A demonstrated efficient conjugation for all antibodies. It was shown that differences in linker length had a large impact on the conjugation efficiency for labeling of mouse IgG1, whereas the positioning of the photoactivable probe in the sequence of the protein had a larger effect for mouse IgG2A. Conjugation to human IgG1 was only to a minor extent affected by position or linker length. For each subclass of antibody, the best variant tested using a standard conjugation protocol resulted in conjugation efficiencies of 41-66%, which corresponds to on average approximately one Z domain attached to each antibody. As a combination of the two best performing variants, Z5BBA and Z32BPA, a Z domain variant with two photoactivable probes (Z5BBA32BPA) was also synthesized with the aim of targeting a wider panel of antibody subclasses and species. This new reagent could efficiently couple to all antibody subclasses that were targeted by the single benzophenone-labeled Z domain variants, with conjugation efficiencies of 26-41%.
Asunto(s)
Anticuerpos Monoclonales/química , Inmunoglobulina G/química , Animales , Humanos , Ratones , Modelos Moleculares , Estructura Molecular , Péptidos/síntesis química , Péptidos/química , Péptidos/aislamiento & purificación , Fenilalanina/análogos & derivados , Fenilalanina/química , Rayos UltravioletaRESUMEN
Glucagon-like peptide 1 (GLP-1) and related peptide agonists have been extensively investigated for glycaemic control in Type 2 diabetes, and may also have therapeutic applications for other diseases. Due to the short half-life (t1/2 < 2 min) of the endogenous peptide, caused by proteolytic degradation and renal clearance, different strategies for half-life extension and sustained release have been explored. In the present study, conjugates between a GLP-1 analogue and a 5 kDa albumin-binding domain (ABD) derived from streptococcal protein G have been chemically synthesized and evaluated. ABD binds with high affinity to human serum albumin, which is highly abundant in plasma and functions as a drug carrier in the circulation. Three different GLP-1-ABD conjugates, with the two peptides connected by linkers of two, four, and six PEG units, respectively, were synthesized and tested in mouse pancreatic islets at high (11 mM) and low (3 mM) glucose concentration. Insulin release upon stimulation was shown to be glucose-dependent, showing no significant difference between the three different GLP-1-ABD conjugates and unconjugated GLP-1 analogue. The biological activity, in combination with the high affinity binding to albumin, make the GLP-1-ABD conjugates promising GLP-1 receptor agonists expected to show extended in vivo half-life.
Asunto(s)
Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/química , Albúmina Sérica/química , Animales , Técnicas Biosensibles , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , Semivida , Humanos , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos , Ratones Obesos , Péptidos/síntesis química , Péptidos/química , Estructura Terciaria de Proteína , RatasRESUMEN
PURPOSE: Site-specific approaches to bioconjugation produce well-defined and homogeneous immunoconjugates with potential for superior in vivo behavior compared to analogs synthesized using traditional, stochastic methods. The possibility of incorporating photoaffinity chemistry into a site-specific bioconjugation strategy is particularly enticing, as it could simplify and accelerate the preparation of homogeneous immunoconjugates for the clinic. In this investigation, we report the synthesis, in vitro characterization, and in vivo evaluation of a site-specifically modified, 89Zr-labeled radioimmunoconjugate created via the reaction between an mAb and an Fc-binding protein bearing a photoactivatable 4-benzoylphenylalanine residue. PROCEDURES: A variant of the Fc-binding Z domain of protein A containing a photoactivatable, 4-benzoylphenylalanine residue - Z(35BPA) - was modified with desferrioxamine (DFO), combined with the A33 antigen-targeting mAb huA33, and irradiated with UV light. The resulting immunoconjugate - DFOZ(35BPA)-huA33 - was purified and characterized via SDS-PAGE, MALDI-ToF mass spectrometry, surface plasmon resonance, and flow cytometry. The radiolabeling of DFOZ(35BPA)-huA33 was optimized to produce [89Zr]Zr-DFOZ(35BPA)-huA33, and the immunoreactivity of the radioimmunoconjugate was determined with SW1222 human colorectal cancer cells. Finally, the in vivo performance of [89Zr]Zr-DFOZ(35BPA)-huA33 in mice bearing subcutaneous SW1222 xenografts was interrogated via PET imaging and biodistribution experiments and compared to that of a stochastically labeled control radioimmunoconjugate, [89Zr]Zr-DFO-huA33. RESULTS: HuA33 was site-specifically modified with Z(35BPA)-DFO, producing an immunoconjugate with on average 1 DFO/mAb, high in vitro stability, and high affinity for its target. [89Zr]Zr-DFOZ(35BPA)-huA33 was synthesized in 95% radiochemical yield and exhibited a specific activity of 2 mCi/mg and an immunoreactive fraction of ~ 0.85. PET imaging and biodistribution experiments revealed that high concentrations of the radioimmunoconjugate accumulated in tumor tissue (i.e., ~ 40%ID/g at 120 h p.i.) but also that the Z(35BPA)-bearing immunoPET probe produced higher uptake in the liver, spleen, and kidneys than its stochastically modified cousin, [89Zr]Zr-DFO-huA33. CONCLUSIONS: Photoaffinity chemistry and an Fc-binding variant of the Z domain were successfully leveraged to create a novel site-specific strategy for the synthesis of radioimmunoconjugates. The probe synthesized using this method - DFOZ(35BPA)-huA33 - was well-defined and homogeneous, and the resulting radioimmunoconjugate ([89Zr]Zr-DFOZ(35BPA)-huA33) boasted high specific activity, stability, and immunoreactivity. While the site-specifically modified radioimmunoconjugate produced high activity concentrations in tumor tissue, it also yielded higher uptake in healthy organs than a stochastically modified analog, suggesting that optimization of this system is necessary prior to clinical translation.
Asunto(s)
Inmunoconjugados , Neoplasias , Humanos , Animales , Ratones , Inmunoconjugados/química , Distribución Tisular , Tomografía de Emisión de Positrones/métodos , Circonio/química , Línea Celular Tumoral , Deferoxamina/químicaRESUMEN
Affibody molecules are a class of affinity proteins. Their small size (7 kDa) in combination with the high (subnanomolar) affinity for a number of cancer-associated molecular targets makes them suitable for molecular imaging. Earlier studies demonstrated that the selection of radionuclide and chelator may substantially influence the tumor-targeting properties of affibody molecules. Moreover, the placement of chelators for labeling of affibody molecules with (99m)Tc at different positions in affibody molecules influenced both blood clearance rate and uptake in healthy tissues. This introduces an opportunity to improve the contrast of affibody-mediated imaging. In this comparative study, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the synthetic affibody molecule Z(HER2:S1) at three different positions: DOTA-A1-Z(HER2:S1) (N-terminus), DOTA-K58-Z(HER2:S1) (C-terminus), and DOTA-K50-Z(HER2:S1) (middle of helix 3). The affinity for HER2 differed slightly among the variants and the K(D) values were determined to be 133 pM, 107 pM and 94 pM for DOTA-A1-Z(HER2:S1), DOTA-K50-Z(HER2:S1), and DOTA-K58-Z(HER2:S1), respectively. Z(HER2:S1)-K50-DOTA showed a slightly lower melting point (57 °C) compared to DOTA-A1-Z(HER2:S1) (64 °C) and DOTA-K58-Z(HER2:S1) (62 °C), but all variants showed good refolding properties after heat treatment. All conjugates were successfully labeled with (111)In resulting in a radiochemical yield of 99% with preserved binding capacity. In vitro specificity studies using SKOV-3 and LS174T cell lines showed that the binding of the radiolabeled compounds was HER2 receptor-mediated, which also was verified in vivo using BALB/C nu/nu mice with LS174T and Ramos lymphoma xenografts. The three conjugates all showed specific uptake in LS174T xenografts in nude mice, where DOTA-A1-Z(HER2:S1)and DOTA-K58-Z(HER2:S1) showed the highest uptake. Overall, DOTA-K58-Z(HER2:S1) provided the highest tumor-to-blood ratio, which is important for a high-contrast imaging. In conclusion, the positioning of the DOTA chelator influences the cellular processing and the biodistribution pattern of radiolabeled affibody molecules, creating preconditions for imaging optimization.
Asunto(s)
Quelantes/química , Compuestos Heterocíclicos con 1 Anillo/química , Radioisótopos de Indio , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacocinética , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Diseño de Fármacos , Estabilidad de Medicamentos , Femenino , Humanos , Marcaje Isotópico , Ratones , Datos de Secuencia Molecular , Unión Proteica , Receptor ErbB-2/inmunología , Proteínas Recombinantes de Fusión/química , Relación Estructura-Actividad , Distribución TisularRESUMEN
PURPOSE: In disseminated prostate cancer, expression of human epidermal growth factor receptor type 2 (HER2) is one of the pathways to androgen independence. Radionuclide molecular imaging of HER2 expression in disseminated prostate cancer might identify patients for HER2-targeted therapy. Affibody molecules are small (7 kDa) targeting proteins with high potential as tracers for radionuclide imaging. The goal of this study was to develop an optimal Affibody-based tracer for visualization of HER2 expression in prostate cancer. METHODS: A synthetic variant of the anti-HER2 Z(HER2:342) Affibody molecule, Z(HER2:S1), was N-terminally conjugated with the chelators DOTA, NOTA and NODAGA. The conjugated proteins were biophysically characterized by electrospray ionization mass spectroscopy (ESI-MS), circular dichroism (CD) spectroscopy and surface plasmon resonance (SPR)-based biosensor analysis. After labelling with (111)In, the biodistribution was assessed in normal mice and the two most promising conjugates were further evaluated for tumour targeting in mice bearing DU-145 prostate cancer xenografts. RESULTS: The HER2-binding equilibrium dissociation constants were 130, 140 and 90 pM for DOTA-Z(HER2:S1), NOTA-Z(HER2:S1) and NODAGA-Z(HER2:S1), respectively. A comparative study of (111)In-labelled DOTA-Z(HER2:S1), NOTA-Z(HER2:S1) and NODAGA-Z(HER2:S1) in normal mice demonstrated a substantial influence of the chelators on the biodistribution properties of the conjugates. (111)In-NODAGA-Z(HER2:S1) had the most rapid clearance from blood and healthy tissues. (111)In-NOTA-Z(HER2:S1) showed high hepatic uptake and was excluded from further evaluation. (111)In-DOTA-Z(HER2:S1) and (111)In-NODAGA-Z(HER2:S1) demonstrated specific uptake in DU-145 prostate cancer xenografts in nude mice. The tumour uptake of (111)In-NODAGA-Z(HER2:S1), 5.6 ± 0.4%ID/g, was significantly lower than the uptake of (111)In-DOTA-Z(HER2:S1), 7.4 ± 0.5%ID/g, presumably because of lower bioavailability due to more rapid clearance. (111)In-NODAGA-Z(HER2:S1) provided higher tumour-to-blood ratio, but somewhat lower tumour-to-liver, tumour-to-spleen and tumour-to-bone ratios. CONCLUSION: Since distant prostate cancer metastases are situated in bone or bone marrow, the higher tumour-to-bone ratio is the most important. This renders (111)In-DOTA-Z(HER2:S1) a preferable agent for imaging of HER2 expression in disseminated prostate cancer.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Transformación Celular Neoplásica , Quelantes/química , Radioisótopos de Indio/química , Neoplasias de la Próstata/patología , Receptor ErbB-2/inmunología , Acetatos/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/farmacocinética , Sitios de Unión , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos con 1 Anillo/química , Humanos , Marcaje Isotópico , Masculino , Ratones , Imagen Molecular , Datos de Secuencia Molecular , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Especificidad por SustratoRESUMEN
PURPOSE: Radiolabelled Affibody molecules have demonstrated a potential for visualization of tumour-associated molecular targets. Affibody molecules (7 kDa) are composed of three alpha-helices. Recently, a smaller two-helix variant of Affibody molecules (5.1 kDa) was developed. The aim of this study was to compare two- and three-helix HER2-targeting Affibody molecules directly in vivo. METHODS: The three-helix Affibody molecule ABY-002 and the two-helix Affibody molecule PEP09239 were labelled with (111)In at the N-termini via DOTA chelator. Tumour-targeting properties were directly compared at 1 and 4 h after injection in mice bearing SKOV-3 xenografts with high HER2 expression and LS174T xenografts with low HER2 expression. RESULTS: The dissociation constants (K(D)) for HER2 binding were 78 pM for the three-helix Affibody molecule and 2.1 nM for the two-helix Affibody molecule. (111)In-PEP09239 cleared more rapidly from the blood. In xenografts with high HER2 expression, the uptake of (111)In-ABY-002 was significantly higher than that of (111)In-PEP09239. The tumour-to-blood ratio was higher for (111)In-PEP09239 at 4 h after injection, while there was no significant difference in other tumour-to-organ ratios. The tumour uptake of (111)In-ABY-002 was eightfold higher than that of (111)In-PEP09239 in xenografts with low expression. Tumour-to-blood ratios were equal in this case, but other tumour-to-organ ratios were appreciably higher for the three-helix variant. CONCLUSION: For tumours with high HER2 expression, two-helix HER2-targeting Affibody molecules can provide higher tumour-to-blood ratio at the cost of lower tumour uptake. In the case of low expression, both tumour uptake and tumour-to-organ ratios are appreciably higher for three-helix than for two-helix HER2-targeting Affibody molecules.
Asunto(s)
Radioisótopos de Indio , Imagen Molecular/métodos , Proteínas Recombinantes de Fusión/química , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica , Femenino , Humanos , Marcaje Isotópico , Ratones , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/farmacocinéticaRESUMEN
Treatment of patients with human epidermal growth factor receptor 2 (HER2)-expressing tumors using the monoclonal antibody trastuzumab increases survival. The Affibody-based peptide nucleic acid (PNA)-mediated pretargeted radionuclide therapy has demonstrated efficacy against HER2-expressing xenografts in mice. Structural studies suggest that Affibody molecules and trastuzumab bind to different epitopes on HER2. The aim of this study was to test the hypothesis that a combination of PNA-mediated pretargeted radionuclide therapy and trastuzumab treatment of HER2-expressing xenografts can extend survival compared with monotherapies. Methods: Mutual interference of the primary pretargeting probe ZHER2:342-SR-HP1 and trastuzumab in binding to HER2-expressing cell lines was investigated in vitro. Experimental therapy evaluated the survival of mice bearing HER2-expressing SKOV-3 xenografts after treatment with vehicle, trastuzumab only, pretargeting using Affibody-PNA chimera ZHER2:342-SR-HP1 and complementary probe 177Lu-HP2, and combination of trastuzumab and pretargeting. The ethical permit limited the study to 90 d. The animals' weights were monitored during the study. After study termination, samples of liver and kidneys were evaluated by a veterinary pathologist for toxicity signs. Results: The presence of a large molar excess of trastuzumab had no influence on the affinity of ZHER2:342-SR-HP1 binding to HER2-expressing cells in vitro. The affinity of trastuzumab was not affected by a large excess of ZHER2:342-SR-HP1 The median survival of mice treated with trastuzumab (75.5 d) was significantly longer than the survival of mice treated with a vehicle (59.5 d). Median survival of mice treated with pretargeting was not reached by day 90. Six mice of 10 in this group survived, and 2 had complete remission. All mice in the combination treatment group survived, and tumors in 7 mice had disappeared at study termination. There was no significant difference between animal weights in the different treatment groups. No significant pathologic alterations were detected in livers and kidneys of treated animals. Conclusion: Treatment of mice bearing HER2-expressing xenografts with the combination of trastuzumab and Affibody-mediated PNA-based radionuclide pretargeting significantly increased survival compared with monotherapies. Cotreatment was not toxic for normal tissues.
Asunto(s)
Neoplasias , Ácidos Nucleicos de Péptidos , Trastuzumab , Animales , Proteínas Cromosómicas no Histona , Humanos , Ratones , Ácidos Nucleicos de Péptidos/farmacología , Radioisótopos , Receptor ErbB-2/metabolismo , Trastuzumab/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Radioimmunotherapy (RIT) is a cancer treatment that combines radiation therapy with tumor-directed monoclonal antibodies (Abs). Although RIT had been introduced for the treatment of CD20 positive non-Hodgkin lymphoma decades ago, it never found a broad clinical application. In recent years, researchers have developed theranostic agents based on Ab fragments or small Ab mimetics such as peptides, affibodies or single-chain Abs with improved tumor-targeting capacities. Theranostics combine diagnostic and therapeutic capabilities into a single pharmaceutical agent; this dual application can be easily achieved after conjugation to radionuclides. The past decade has seen a trend to increased specificity, fastened pharmacokinetics, and personalized medicine. In this review, we discuss the different strategies introduced for the noninvasive detection and treatment of hematological malignancies by radiopharmaceuticals. We also discuss the future applications of these radiotheranostic agents.
Asunto(s)
Neoplasias Hematológicas , Linfoma no Hodgkin , Neoplasias , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antineoplásicos , Neoplasias Hematológicas/tratamiento farmacológico , Humanos , Linfoma no Hodgkin/diagnóstico por imagen , Linfoma no Hodgkin/tratamiento farmacológico , Linfoma no Hodgkin/radioterapia , Neoplasias/tratamiento farmacológico , RadioinmunoterapiaRESUMEN
Small extracellular vesicles (sEVs) have in recent years evolved as a source of biomarkers for disease diagnosis and therapeutic follow up. sEV samples derived from multicellular organisms exhibit a high heterogeneous repertoire of vesicles which current methods based on ensemble measurements cannot capture. In this work we present droplet barcode sequencing for protein analysis (DBS-Pro) to profile surface proteins on individual sEVs, facilitating identification of sEV-subtypes within and between samples. The method allows for analysis of multiple proteins through use of DNA barcoded affinity reagents and sequencing as readout. High throughput single vesicle profiling is enabled through compartmentalization of individual sEVs in emulsion droplets followed by droplet barcoding through PCR. In this proof-of-concept study we demonstrate that DBS-Pro allows for analysis of single sEVs, with a mixing rate below 2%. A total of over 120,000 individual sEVs obtained from a NSCLC cell line and from malignant pleural effusion (MPE) fluid of NSCLC patients have been analyzed based on their surface proteins. We also show that the method enables single vesicle surface protein profiling and by extension characterization of sEV-subtypes, which is essential to identify the cellular origin of vesicles in heterogenous samples.
Asunto(s)
Vesículas Extracelulares , Humanos , Vesículas Extracelulares/genética , Biomarcadores/metabolismo , Línea Celular , Proteínas de la Membrana/metabolismoRESUMEN
Traditionally, labeling of antibodies has been performed by covalent conjugation to amine or carboxyl groups. These methods are efficient but suffer from nonspecificity, since all free and available amine/carboxyl groups have the possibility to react. This drawback may lead to uncontrolled levels and locations of the labeling. Hence, the labeled molecules might behave differently and, possibly, the binding site of the antibody will also be affected. In this project, we have developed a highly stringent method for labeling of antibodies by utilizing an immunoglobulin-binding domain from protein A, the Z domain. Domain Z has been synthesized with an amino acid analogue, benzoylphenylalanine, capable of forming covalent attachment to other amino acids upon UV-exposure. This feature has been used for directed labeling of immunoglobulins and subsequent use of these in different assays.
Asunto(s)
Anticuerpos/química , Inmunoglobulinas/química , Proteína Estafilocócica A/química , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Sitios de Unión , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Inmunoglobulinas/inmunología , Ratones , Datos de Secuencia Molecular , Péptidos/química , Péptidos/inmunología , Estructura Terciaria de Proteína , Proteína Estafilocócica A/inmunología , Rayos UltravioletaRESUMEN
Radionuclide molecular imaging has the potential to improve cancer treatment by selection of patients for targeted therapy. Affibody molecules are a class of small (7 kDa) high-affinity targeting proteins with appreciable potential as molecular imaging probes. The NOTA chelator forms stable complexes with a number of radionuclides suitable for SPECT or PET imaging. A maleimidoethylmonoamide NOTA (MMA-NOTA) has been prepared for site-specific labeling of Affibody molecules having a unique C-terminal cysteine. Coupling of the MMA-NOTA to the anti-HER2 Affibody molecule Z(HER2:2395) resulted in a conjugate with an affinity (dissociation constant) to HER2 of 72 pM. Labeling of [MMA-NOTA-Cys(61)]-Z(HER2:2395) with (111)In gave a yield of >95% after 20 min at 60 °C. In vitro cell tests demonstrated specific binding of [(111)In-MMA-NOTA-Cys(61)]-Z(HER2:2395) to HER2-expressing cell lines. In mice bearing prostate cancer DU-145 xenografts, the tumor uptake of [(111)In-MMA-NOTA-Cys(61)]-Z(HER2:2395) was 8.2 ± 0.9% IA/g and the tumor-to-blood ratio was 31 ± 1 (4 h postinjection). DU-145 xenografts were clearly visualized by a gamma camera. Direct in vivo comparison of [(111)In-MMA-NOTA-Cys(61)]-Z(HER2:2395) and [(111)In-MMA-DOTA-Cys(61)]-Z(HER2:2395) demonstrated that both conjugates provided equal radioactivity uptake in tumors, but the tumor-to-organ ratios were better for [(111)In-MMA-NOTA-Cys(61)]-Z(HER2:2395) due to more efficient clearance from normal tissues. In conclusion, coupling of MMA-NOTA to a cysteine-containing Affibody molecule resulted in a site-specifically labeled conjugate, which retains high affinity, can be efficiently labeled, and allows for high-contrast imaging.
Asunto(s)
Compuestos Heterocíclicos , Maleimidas , Imagen Molecular , Radiofármacos , Proteínas Recombinantes de Fusión , Coloración y Etiquetado , Animales , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/farmacocinética , Compuestos Heterocíclicos con 1 Anillo , Masculino , Maleimidas/química , Maleimidas/farmacocinética , Ratones , Ratones Desnudos , Estructura Molecular , Radiofármacos/química , Radiofármacos/farmacocinética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacocinética , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The Z-molecule is a small, engineered IgG-binding affinity protein derived from the immunoglobulin-binding domain B of Staphylococcus aureus protein A. The Z-domain consists of 58 amino acids forming a well-defined antiparallel three-helix structure. Two of the three helices are involved in ligand binding, whereas the third helix provides structural support to the three-helix bundle. The small size and the stable three-helix structure are two attractive properties comprised in the Z-domain, but a further reduction in size of the protein is valuable for several reasons. Reduction in size facilitates synthetic production of any protein-based molecule, which is beneficial from an economical viewpoint. In addition, a smaller protein is easier to manipulate through chemical modifications. By omitting the third stabilizing helix from the Z-domain and joining the N- and C-termini by a native peptide bond, the affinity protein obtains the advantageous properties of a smaller scaffold and in addition becomes resistant to exoproteases. We here demonstrate the synthesis and evaluation of a novel cyclic two-helix Z-domain. The molecule has retained affinity for its target protein, is resistant to heat treatment, and lacks both N- and C-termini.
Asunto(s)
Fragmentos de Péptidos/síntesis química , Péptidos Cíclicos/síntesis química , Proteína Estafilocócica A/química , Secuencia de Aminoácidos , Dicroismo Circular , Humanos , Proteínas Inmovilizadas/química , Inmunoglobulina G/química , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Péptidos Cíclicos/química , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Resonancia por Plasmón de SuperficieRESUMEN
We present an approach to improve the detection sensitivity of a streaming current-based biosensor for membrane protein profiling of small extracellular vesicles (sEVs). The experimental approach, supported by theoretical investigation, exploits electrostatic charge contrast between the sensor surface and target analytes to enhance the detection sensitivity. We first demonstrate the feasibility of the approach using different chemical functionalization schemes to modulate the zeta potential of the sensor surface in a range -16.0 to -32.8 mV. Thereafter, we examine the sensitivity of the sensor surface across this range of zeta potential to determine the optimal functionalization scheme. The limit of detection (LOD) varied by 2 orders of magnitude across this range, reaching a value of 4.9 × 106 particles/mL for the best performing surface for CD9. We then used the optimized surface to profile CD9, EGFR, and PD-L1 surface proteins of sEVs derived from non-small cell lung cancer (NSCLC) cell-line H1975, before and after treatment with EGFR tyrosine kinase inhibitors, as well as sEVs derived from pleural effusion fluid of NSCLC adenocarcinoma patients. Our results show the feasibility to monitor CD9, EGFR, and PD-L1 expression on the sEV surface, illustrating a good prospect of the method for clinical application.
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Técnicas Biosensibles/métodos , Vesículas Extracelulares/química , Electricidad Estática , Anticuerpos Inmovilizados/inmunología , Antígeno B7-H1/análisis , Antígeno B7-H1/metabolismo , Línea Celular Tumoral , Técnicas Electroquímicas , Receptores ErbB/análisis , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Vesículas Extracelulares/efectos de los fármacos , Vesículas Extracelulares/inmunología , Humanos , Límite de Detección , Inhibidores de Proteínas Quinasas/farmacología , Tetraspanina 29/análisis , Tetraspanina 29/metabolismoRESUMEN
An electrical immuno-sandwich assay utilizing an electrokinetic-based streaming current method for signal transduction is proposed. The method records the changes in streaming current, first when a target molecule binds to the capture probes immobilized on the inner surface of a silica micro-capillary, and then when the detection probes interact with the bound target molecules on the surface. The difference in signals in these two steps constitute the response of the assay, which offers better target selectivity and a linear concentration dependent response for a target concentration within the range 0.2-100 nM. The proof of concept is demonstrated by detecting different concentrations of Immunoglobulin G (IgG) in both phosphate buffered saline (PBS) and spiked in E. coli cell lysate. A superior target specificity for the sandwich assay compared to the corresponding direct assay is demonstrated along with a limit of detection of 90 pM in PBS. The prospect of improving the detection sensitivity was theoretically analysed, which indicated that the charge contrast between the target and the detection probe plays a crucial role in determining the signal. This aspect was then experimentally validated by modulating the zeta potential of the detection probe by conjugating negatively charged DNA oligonucleotides. The length of the conjugated DNA was varied from 5 to 30 nucleotides, altering the zeta potential of the detection probe from -9.3 ± 0.8 mV to -20.1 ± 0.9 mV. The measurements showed a clear and consistent enhancement of detection signal as a function of DNA lengths. The results presented here conclusively demonstrate the role of electric charge in detection sensitivity as well as the prospect for further improvement. The study therefore is a step forward in developing highly selective and sensitive electrokinetic assays for possible application in clinical investigations.
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Técnicas Biosensibles , ADN , Sondas de ADN/genética , Escherichia coli/genética , Sensibilidad y EspecificidadRESUMEN
Affibody-mediated PNA-based pretargeting is a promising approach to radionuclide therapy of HER2-expressing tumors. In this study, we test the hypothesis that shortening the PNA pretargeting probes would increase the tumor-to-kidney dose ratio. The primary probe ZHER2:342-SR-HP15 and the complementary secondary probes HP16, HP17, and HP18, containing 9, 12, and 15 nucleobases, respectively, and carrying a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator were designed, synthesized, characterized in vitro, and labeled with 177Lu. In vitro pretargeting was studied in HER2-expressing SKOV3 and BT474 cell lines. The biodistribution of these novel probes was evaluated in immunodeficient mice bearing SKOV3 xenografts and compared to the previously studied [177Lu]Lu-HP2. Characterization confirmed the formation of high-affinity duplexes between HP15 and the secondary probes, with the affinity correlating with the length of the complementary PNA sequences. All the PNA-based probes were bound specifically to HER2-expressing cells in vitro. In vivo studies demonstrated HER2-specific uptake of all 177Lu-labeled probes in xenografts in a pretargeting setting. The ratio of cumulated radioactivity in the tumor to the radioactivity in kidneys was dependent on the secondary probe's size and decreased with an increased number of nucleobases. The shortest PNA probe, [177Lu]Lu-HP16, showed the highest tumor-to-kidney ratio. [177Lu]Lu-HP16 is the most promising secondary probe for affibody-mediated tumor pretargeting.
RESUMEN
Precision cancer medicine for non-small-cell lung cancer (NSCLC) has increased patient survival. Nevertheless, targeted agents towards tumor-associated membrane receptors only result in partial remission for a limited time, calling for approaches which allow longitudinal treatment monitoring. Rebiopsy of tumors in the lung is challenging, and metastatic lesions may have heterogeneous signaling. One way ahead is to use liquid biopsies such as circulating tumor DNA or small extracellular vesicles (sEVs) secreted by the tumor into blood or other body fluids. Herein, an immuno-PCR-based detection of the tumor-associated membrane receptors EGFR, HER2, and IGF-1R on CD9-positive sEVs from NSCLC cells and pleural effusion fluid (PE) of NSCLC patients is developed utilizing DNA conjugates of antibody mimetics and affibodies, as detection agents. Results on sEVs purified from culture media of NSCLC cells treated with anti-EGFR siRNA, showed that the reduction of EGFR expression can be detected via immuno-PCR. Protein profiling of sEVs from NSCLC patient PE samples revealed the capacity to monitor EGFR, HER2, and IGF-1R with the immuno-PCR method. We detected a significantly higher EGFR level in sEVs derived from a PE sample of a patient with an EGFR-driven NSCLC adenocarcinoma than in sEVs from PE samples of non-EGFR driven adenocarcinoma patients or in samples from patients with benign lung disease. In summary, we have developed a diagnostic method for sEVs in liquid biopsies of cancer patients which may be used for longitudinal treatment monitoring to detect emerging bypassing resistance mechanisms in a noninvasive way.
RESUMEN
PURPOSE: Affibody molecules are a novel class of tumour-targeting proteins, which combine small size (7 kDa) and picomolar affinities. The Affibody molecule Z(HER2:342) has been suggested for imaging of HER2 expression in order to select patients for trastuzumab therapy. When optimizing chelators for (99m)Tc-labelling, we have found that synthetic Z(HER2:342) conjugated with mercaptoacetyl-glycyl-glycyl-glycyl (maGGG) and mercaptoacetyl-glycyl-seryl-glycyl (maGSG) chelators provides relatively low renal uptake of radioactivity and could be suitable for therapy. METHODS: maGGG-Z(HER2:342) and maGSG-Z(HER2:342) were labelled with (186)Re and their biodistribution was studied in normal mice. Dosimetric evaluation and tumour targeting to HER2-overexpressed xenografts (SKOV-3) by (186)Re-maGSG-Z(HER2:342) were studied. RESULTS: Gluconate-mediated labelling of maGGG-Z(HER2:342) and maGSG-Z(HER2:342) with (186)Re provided a yield of more than 95% within 60 min. The conjugates were stable and demonstrated specific binding to HER2-expressing SKOV-3 cells. Biodistribution in normal mice demonstrated rapid blood clearance, low accumulation of radioactivity in the kidney and other organs, accumulating free perrhenate. Both (186)Re-maGGG-Z(HER2:342) and (186)Re-maGSG-Z(HER2:342) demonstrated lower renal uptake than their (99m)Tc-labelled counterparts. (186)Re-maGSG-Z(HER2:342) provided the lowest uptake in healthy tissues. Biodistribution of (186)Re-maGSG-Z(HER2:342) in nude mice bearing SKOV-3 xenografts showed specific targeting of tumours. Tumour uptake 24 h after injection (5.84+/-0.54%ID/g) exceeded the concentration in blood by more than 500-fold, and uptake in kidneys by about 8-fold. Preliminary dosimetric evaluation showed that dose-to-tumour should exceed dose-to-kidney by approximately 5-fold. CONCLUSION: Optimization of chelators improves biodistribution properties of rhenium-labelled small scaffold proteins and enables selection of promising radiotherapeutic agents based on the Affibody molecule.