RESUMEN
Claudins are a 27-member family of ~25 kDa membrane proteins that integrate into tight junctions to form molecular barriers at the paracellular spaces between endothelial and epithelial cells. As the backbone of tight junction structure and function, claudins are attractive targets for modulating tissue permeability to deliver drugs or treat disease. However, structures of claudins are limited due to their small sizes and physicochemical properties-these traits also make therapy development a challenge. Here we report the development of a synthetic antibody fragment (sFab) that binds human claudin-4 and the determination of a high-resolution structure of it bound to claudin-4/enterotoxin complexes using cryogenic electron microscopy. Structural and biophysical results reveal this sFabs mechanism of select binding to human claudin-4 over other homologous claudins and establish the ability of sFabs to bind hard-to-target claudins to probe tight junction structure and function. The findings provide a framework for tight junction modulation by sFabs for tissue-selective therapies.
Asunto(s)
Claudina-4 , Claudina-4/metabolismo , Humanos , Uniones Estrechas/metabolismo , Microscopía por Crioelectrón , Enterotoxinas/metabolismo , Enterotoxinas/química , Enterotoxinas/inmunología , Fragmentos de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/metabolismo , Unión Proteica , Modelos MolecularesRESUMEN
BACKGROUND: Chimeric antigen receptor-T (CAR-T) cells therapy is one of the novel immunotherapeutic approaches with significant clinical success. However, their applications are limited because of long preparation time, high cost, and interpersonal variations. Although the manufacture of universal CAR-T (U-CAR-T) cells have significantly improved, they are still not a stable and unified cell bank. METHODS: Here, we tried to further improve the convenience and flexibility of U-CAR-T cells by constructing novel modular universal CAR-T (MU-CAR-T) cells. For this purpose, we initially screened healthy donors and cultured their T cells to obtain a higher proportion of stem cell-like memory T (TSCM) cells, which exhibit robust self-renewal capacity, sustainability and cytotoxicity. To reduce the alloreactivity, the T cells were further edited by double knockout of the T cell receptor (TCR) and class I human leukocyte antigen (HLA-I) genes utilizing the CRISPR/Cas9 system. The well-growing and genetically stable universal cells carrying the CAR-moiety were then stored as a stable and unified cell bank. Subsequently, the SDcatcher/GVoptiTag system, which generate an isopeptide bond, was used to covalently connect the purified scFvs of antibody targeting different antigens to the recovered CAR-T cells. RESULTS: The resulting CAR-T cells can perform different functions by specifically targeting various cells, such as the eradication of human immunodeficiency virus type 1 (HIV-1)-latenly-infected cells or elimination of T lymphoma cells, with similar efficiency as the traditional CAR-T cells did. CONCLUSION: Taken together, our strategy allows the production of CAR-T cells more modularization, and makes the quality control and pharmaceutic manufacture of CAR-T cells more feasible.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Receptores Quiméricos de Antígenos , Humanos , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Fragmentos de Inmunoglobulinas/metabolismo , Linfocitos T , Receptores de Antígenos de Linfocitos T/metabolismo , Inmunoterapia Adoptiva/métodosRESUMEN
Single chain antibody fragment (scFv) is a small molecule composed of a variable region of heavy chain (VH) and a variable region of light chain (VL) of an antibody, and these two chains are connected by a flexible short peptide. scFv is the smallest functional fragment with complete antigen-binding activity, which contains both the antibody-recognizing site and the antigen-binding site. Compared with other antibodies, scFv has the advantages of small molecular weight, strong penetration, low immunogenicity, and easy expression. Currently, the most commonly used display systems for scFv mainly include the phage display system, ribosome display system, mRNA display system, yeast cell surface display system and mammalian cell display system. In recent years, with the development of scFv in the field of medicine, biology, and food safety, they have also attracted much attention in the sectors of biosynthesis and applied research. This review summarizes the advances of scFv display systems in recent years in order to facilitate scFv screening and application.
Asunto(s)
Región Variable de Inmunoglobulina , Anticuerpos de Cadena Única , Animales , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/metabolismo , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/metabolismo , Biblioteca de Péptidos , Mamíferos/genéticaRESUMEN
Genetically encoded site-specifically incorporated noncanonical amino acids (ncAAs) have been used to modulate properties of several proteins. Here, we describe a procedure for engineering photoactive antibody fragments that bind to their target antigen only after irradiation with 365 nm light. The procedure starts with identification of tyrosine residues in antibody fragments that are important for antibody-antigen binding and thus targets for replacement with photocaged tyrosine (pcY). This is followed by cloning of plasmids and expression of pcY-containing antibody fragments in E. coli. Finally, we describe a cost-effective and biologically-relevant method for measuring the binding affinity of photoactive antibody fragments to antigens expressed on the surface of live cancer cells.
Asunto(s)
Escherichia coli , Fragmentos de Inmunoglobulinas , Fragmentos de Inmunoglobulinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Aminoácidos/química , Tirosina/metabolismo , Proteínas/química , Antígenos/metabolismoRESUMEN
Mice are rarely used in pharmacokinetic (PK) studies of ocular therapeutics due to the small size of their eyes and challenges in drug administration, tissue collection, and analysis of drug concentrations. Therefore, ocular PK of protein therapeutics in mouse eye following intravitreal (IVT) administration is not known. Here, we have presented the first of its kind investigation, to study the PK of 4 different size non-binding protein therapeutics in mouse plasma, cornea/ICB, vitreous humor, retina, and posterior cup (including choroid) following IVT administration. Administered proteins include trastuzumab (150 kDa) and F(ab)2 (100 kDa), Fab, and scFv (27 kDa) fragments of trastuzumab. An imaging and injection apparatus suitable for performing small (50 nL) IVT injections in mice was developed, and techniques for enucleation of the eye and dissection of ocular tissues were developed. Furthermore, a sensitive enzyme-linked immunosorbent assay (ELISA) for detection of proteins in very small amounts of ocular tissues were developed. It was observed that elimination from the vitreous chamber was the primary driver of PK in the cornea/ICB, retina, posterior cup, and plasma. Trastuzumab displays first-order kinetics in the vitreous humor with a half-life of 18.8 h. F(ab)2, Fab, and ScFv show biphasic PK profiles with distribution phases becoming more rapid as molecular weight decreases, and terminal elimination becoming longer as molecular weight decreases, with terminal half-lives of 16.3, 20.6, and 48.9 h, respectively. The mean residence times of trastuzumab, F(ab)2, Fab, and scFv in the vitreous humor were 26.0, 12.2, 10.7, and 8.16 h, respectively. It was found that the mean residence time in vitreous humor doubles with an increase in molecular weight of â¼69 kDa. Interestingly, the PK of proteins measured in the un-injected eye suggest the presence of a pathway for drug transfer between the eyes, which needs to be further validated. Overall, the findings presented here pave the way for drug discovery and development studies of protein therapeutics for ophthalmic indications in mice.
Asunto(s)
Anticuerpos Monoclonales , Ojo , Ratones , Animales , Anticuerpos Monoclonales/metabolismo , Inyecciones Intravítreas , Ojo/metabolismo , Cuerpo Vítreo/metabolismo , Trastuzumab , Fragmentos de Inmunoglobulinas/metabolismoRESUMEN
The methylotrophic yeast Pichia pastoris has been one of the most widely used organisms in recent years as an expression system for a wide variety of recombinant proteins with therapeutic potential. Its popularity as an alternative system to Escherichia coli is mainly due to the easy genetic manipulation and the ability to produce high levels of heterologous proteins, either intracellularly or extracellularly. Being a eukaryotic organism, P. pastoris carries out post-translational modifications that allow it to produce soluble and correctly folded recombinant proteins. This work, evaluated the expression capacity in P. pastoris of two single-chain variable fragments (scFvs) of human origin, 10FG2 and LR. These scFvs were previously obtained by directed evolution against scorpion venom toxins and are able to neutralize different toxins and venoms of Mexican species. The yield obtained in P. pastoris was higher than that obtained in bacterial periplasm (E. coli), and most importantly, biochemical and functional properties were not modified. These results confirm that P. pastoris yeast can be a good expression system for the production of antibody fragments of a new recombinant antivenom.
Asunto(s)
Escorpiones , Ponzoñas , Animales , Humanos , Escorpiones/química , Ponzoñas/metabolismo , Saccharomyces cerevisiae/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/química , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/metabolismoRESUMEN
Single chain antibody fragment (scFv) is a small molecule composed of a variable region of heavy chain (VH) and a variable region of light chain (VL) of an antibody, and these two chains are connected by a flexible short peptide. scFv is the smallest functional fragment with complete antigen-binding activity, which contains both the antibody-recognizing site and the antigen-binding site. Compared with other antibodies, scFv has the advantages of small molecular weight, strong penetration, low immunogenicity, and easy expression. Currently, the most commonly used display systems for scFv mainly include the phage display system, ribosome display system, mRNA display system, yeast cell surface display system and mammalian cell display system. In recent years, with the development of scFv in the field of medicine, biology, and food safety, they have also attracted much attention in the sectors of biosynthesis and applied research. This review summarizes the advances of scFv display systems in recent years in order to facilitate scFv screening and application.
Asunto(s)
Animales , Región Variable de Inmunoglobulina/genética , Fragmentos de Inmunoglobulinas/metabolismo , Anticuerpos de Cadena Única/metabolismo , Biblioteca de Péptidos , Mamíferos/genéticaRESUMEN
The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is the major target for antibody therapeutics. Shark-derived variable domains of new antigen receptors (VNARs) are the smallest antibody fragments with flexible paratopes that can recognize protein motifs inaccessible to classical antibodies. This study reported four VNARs binders (JM-2, JM-5, JM-17, and JM-18) isolated from Chiloscyllium plagiosum immunized with SARS-CoV-2 RBD. Biolayer interferometry showed that the VNARs bound to the RBD with an affinity KD ranging from 38.5 to 2720 nM, and their Fc fusions had over ten times improved affinity. Gel filtration chromatography revealed that JM-2-Fc, JM-5-Fc, and JM-18-Fc could form stable complexes with RBD in solution. In addition, five bi-paratopic VNARs, named JM-2-5, JM-2-17, JM-2-18, JM-5-18, and JM-17-18, were constructed by fusing two VNARs targeting distinct RBD epitopes based on epitope grouping results. All these bi-paratopic VNARs except for JM-5-18 showed higher RBD binding affinities than its component VNARs, and their Fc fusions exhibited further enhanced binding affinities, with JM-2-5-Fc, JM-2-17-Fc, JM-2-18-Fc, and JM-5-18-Fc having KD values lower than 1 pM. Among these Fc fusions of bi-paratopic VNARs, JM-2-5-Fc, JM-2-17-Fc, and JM-2-18-Fc could block the angiotensin-converting enzyme 2 (ACE2) binding to the RBD of SARS-CoV-2 wildtype, Delta, Omicron, and SARS-CoV, with inhibition rates of 48.9~84.3%. Therefore, these high-affinity VNAR binders showed promise as detectors and therapeutics of COVID-19.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Tiburones , Enzima Convertidora de Angiotensina 2 , Animales , Epítopos , Humanos , Fragmentos de Inmunoglobulinas/metabolismo , Peptidil-Dipeptidasa A/metabolismo , Unión Proteica , Receptores Virales/metabolismo , SARS-CoV-2 , Glicoproteína de la Espiga del CoronavirusRESUMEN
Molecular imaging with PET offers an alternative method to quantify programmed-death-ligand 1 (PD-L1) to accurately select patients for immunotherapies. More and more clinical and preclinical trials involve radiolabeling of antibody fragments for their desirably fast clearance and high tumor penetration. As the radiolabeling strategy can significantly impact pharmacokinetics and biodistribution, we explored in this work a site-specific radiofluorination strategy on an anti-PD-L1 fragment antigen-binding (Fab) and compared the pharmacokinetic and biodistribution properties with the same Fab labeled using stochastic radiolabeling chemistry. We applied an enzymatic bioconjugation mediated by a variant of the lipoic acid ligase (LplA) that promotes the formation of an amide bond between a short peptide cloned onto the C terminus of the Fab. A synthetic analogue of the enzyme natural substrate, lipoic acid, was radiolabeled with fluorine-18 for site-specific conjugation by LplA. We compared the biodistribution of the site-specifically labeled Fab with a stochastically labeled Fab on lysine side chains in tumor-bearing mice. The two methods of fluorination demonstrate a comparable whole-body biodistribution. The 89Zr-labeled Fab had different biodistribution compared to either 18F-labeled Fab. We attribute the difference to [89Zr] metabolism. Fab-LAP-[18F]FPyOctA therefore reflects better the true pharmacokinetic profile of the Fab.
Asunto(s)
Neoplasias , Ácido Tióctico , Amidas , Animales , Antígeno B7-H1 , Línea Celular Tumoral , Radioisótopos de Flúor , Fragmentos de Inmunoglobulinas/metabolismo , Ligandos , Ligasas/metabolismo , Lisina/metabolismo , Ratones , Péptidos/metabolismo , Tomografía de Emisión de Positrones/métodos , Radiofármacos/farmacocinética , Distribución TisularRESUMEN
Yeast surface display (YSD) has been shown to represent a powerful tool in the field of antibody discovery and engineering as well as for selection of high producer clones. However, YSD is predominantly applied in Saccharomyces cerevisiae, whereas expression of heterologous proteins is generally favored in the non-canonical yeast Pichia pastoris (Komagataella phaffii). Establishment of surface display in P. pastoris would therefore enable antibody selection and expression in a single host. Here we describe the generation of a Pichia surface display (PSD) system based on antibody expression from episomal plasmids. By screening a diverse set of expression vectors using Design of Experiments (DoE), the effect of different genetic elements on the surface expression of antibody fragments was analyzed. Among the tested genetic elements, we found that the combination of P. pastoris formaldehyde dehydrogenase (FLD1) promoter, S. cerevisiae invertase 2 signal peptide (SUC2), and α-agglutinin cell wall protein (SAG1) including an autonomously replicating sequence of Kluyveromyces lactis (panARS) were contributing most strongly to higher display levels of three tested antibody fragments. Employing this combination resulted in the display of antibody fragments for up to 25% of cells. Despite significantly reduced expression levels in PSD compared to well-established YSD in S. cerevisiae, similar fractions of antigen binding single-chain variable fragments (scFvs) were observed (80% vs. 84%). In addition, plasmid stability assays and flow cytometric analysis demonstrated the efficient plasmid clearance of cells and associated loss of antibody fragment display after removal of selective pressure. KEY POINTS: ⢠First report of antibody display in P. pastoris using episomal plasmids. ⢠Identification of genetic elements conferring highest levels of antibody display. ⢠Comparable antigen binding capacity of displayed scFvs for PSD compared to YSD.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/metabolismo , Proteínas de la Membrana/genética , Pichia/genética , Pichia/metabolismo , Plásmidos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , SaccharomycetalesRESUMEN
New insecticidal genes and approaches for pest control are a hot research area. In the present study, we explored a novel strategy for the generation of insecticidal proteins. The midgut cadherin of Helicoverpa armigera (H. armigera) was used as a target to screen materials that have insecticidal activity. After three rounds of panning, the phage-displayed human domain antibody B1F6, which not only binds to the H. armigera cadherin CR9-CR11 but also significantly inhibits Cry1Ac toxins from binding to CR9-CR11, was obtained from a phage-displayed human domain antibody (DAb) library. To better analyze the relevant activity of B1F6, soluble B1F6 protein was expressed by Escherichia coli BL21 (DE3). The cytotoxicity assays demonstrated that soluble B1F6 induced Sf9 cell death when expressing H. armigera cadherin on the cell membrane. The insect bioassay results showed that soluble B1F6 protein (90 µg/cm2) caused 49.5 ± 3.3% H. armigera larvae mortality. The midgut histological results showed that soluble B1F6 caused damage to the midgut epithelium of H. armigera larvae. The present study explored a new strategy and provided a basic material for the generation of new insecticidal materials.
Asunto(s)
Bacillus thuringiensis , Insecticidas , Mariposas Nocturnas , Animales , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Endotoxinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Humanos , Fragmentos de Inmunoglobulinas/metabolismo , Insecticidas/química , Larva/genética , Larva/metabolismo , Mariposas Nocturnas/metabolismoRESUMEN
Engineered antibody fragments (Fabs) have made major impacts on structural biology research, particularly to aid structural determination of membrane proteins. Nonetheless, Fabs generated by traditional monoclonal technology suffer from challenges of routine production and storage. Starting from the known IgG paratopes of an antibody that binds to the "turret loop" of the KcsA K+ channel, we engineered a synthetic Fab (sFab) based upon the highly stable Herceptin Fab scaffold, which can be recombinantly expressed in Escherichia coli and purified with single-step affinity chromatography. This synthetic Fab was used as a crystallization chaperone to obtain crystals of the KcsA channel that diffracted to a resolution comparable to that from the parent Fab. Furthermore, we show that the turret loop can be grafted into the unrelated voltage-gated Kv1.2-Kv2.1 channel and still strongly bind the engineered sFab, in support of the loop grafting strategy. Macroscopic electrophysiology recordings show that the sFab affects the activation and conductance of the chimeric voltage-gated channel. These results suggest that straightforward engineering of antibodies using recombinant formats can facilitate the rapid and scalable production of Fabs as structural biology tools and functional probes. The impact of this approach is expanded significantly based on the potential portability of the turret loop to a myriad of other K+ channels.
Asunto(s)
Canales de Potasio con Entrada de Voltaje , Canales de Potasio , Secuencia de Aminoácidos , Fragmentos de Inmunoglobulinas/metabolismo , Canales de Potasio/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismoRESUMEN
The ocular pharmacokinetics (PK) of antibody-based therapies are infrequently studied in mice due to the technical difficulties in working with the small murine eye. This study is the first of its kind to quantitatively measure the PK of variously sized proteins in the plasma, cornea/ICB, vitreous humor, retina, and posterior cup (including choroid) of the mouse and to evaluate the relationship between molecular weight (MW) and antibody biodistribution coefficient (BC) to the eye. Proteins analyzed include trastuzumab (150 kDa), trastuzumab-vc-MMAE (T-vc-MMAE, 155 kDa), F(ab)2 (100 kDa), Fab (50 kDa), and scFv (27 kDa). As expected, ocular PK mirrored the systemic PK as plasma was the driving force for ocular exposure. For trastuzumab, T-vc-MMAE, F(ab)2, Fab, and scFv, respectively, the BCs in the cornea/ICB were 0.610%, 0.475%, 1.74%, 3.39%, and 13.7%; the BCs in the vitreous humor were 0.0198%, 0.0427%, 0.0934%, 0.234%, and 5.56%; the BCs for the retina were 0.539%, 0.230%, 0.704%, 2.44%, and 20.4%; the BCs for the posterior cup were 0.557%, 0.650%, 1.47%, 4.06%, and 13.9%. The relationship between BC and MW was best characterized by a log-log regression in which BC decreased as MW increased, with every doubling in MW leading to a decrease in BC by a factor of 3.44 × , 6.76 × , 4.74 × , and 3.43 × in cornea/ICB, vitreous humor, retina, and posterior cup, respectively. In analyzing the disposition of protein therapeutics to the eye, these findings enhance our understanding of the potential for ocular toxicity of systemically administered protein therapeutics and may aid in the discovery of systemically administered protein therapeutics for ocular disorders.
Asunto(s)
Ojo/metabolismo , Inmunoconjugados/farmacocinética , Fragmentos Fab de Inmunoglobulinas/metabolismo , Oligopéptidos/farmacocinética , Trastuzumab/farmacocinética , Animales , Inmunoconjugados/administración & dosificación , Inmunoconjugados/química , Fragmentos Fab de Inmunoglobulinas/administración & dosificación , Fragmentos de Inmunoglobulinas/administración & dosificación , Fragmentos de Inmunoglobulinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Peso Molecular , Oligopéptidos/administración & dosificación , Oligopéptidos/química , Distribución Tisular , Trastuzumab/administración & dosificación , Trastuzumab/químicaRESUMEN
Superantigens are unconventional antigens which recognise immune receptors outside their usual recognition sites e.g. complementary determining regions (CDRs), to elicit a response within the target cell. T-cell superantigens crosslink T-cell receptors and MHC Class II molecules on antigen-presenting cells, leading to lymphocyte recruitment, induction of cytokine storms and T-cell anergy or apoptosis among many other effects. B-cell superantigens, on the other hand, bind immunoglobulins on B-cells, affecting opsonisation, IgG-mediated phagocytosis, and driving apoptosis. Here, through a review of the structural basis for recognition of immune receptors by superantigens, we show that their binding interfaces share specific physicochemical characteristics when compared with other protein-protein interaction complexes. Given that antibody-binding superantigens have been exploited extensively in industrial antibody purification, these observations could facilitate further protein engineering to optimize the use of superantigens in this and other areas of biotechnology.
Asunto(s)
Células Presentadoras de Antígenos/metabolismo , Linfocitos B/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Fragmentos de Inmunoglobulinas/metabolismo , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/metabolismo , Superantígenos/metabolismo , Linfocitos T/metabolismo , Animales , Anticuerpos/aislamiento & purificación , Células Presentadoras de Antígenos/inmunología , Apoptosis , Linfocitos B/inmunología , Linfocitos B/patología , Anergia Clonal , Síndrome de Liberación de Citoquinas/inmunología , Síndrome de Liberación de Citoquinas/metabolismo , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Fragmentos de Inmunoglobulinas/inmunología , Ingeniería de Proteínas , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal , Superantígenos/inmunología , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
Antibody fragments are promising building blocks for developing targeted therapeutics, thus improving treatment efficacy while minimising off-target toxicity. Despite recent advances in targeted therapeutics, patients with Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL), a high-risk malignancy, lack specific and effective targeted treatments. Cytokine receptor-like factor 2 (CRLF2) is overexpressed in 50% of Ph-like ALL cases, conferring the survival of leukemia blasts through activation of the JAK/STAT signalling pathway. Targeting such a vital cell-surface protein could result in potent anti-leukaemic efficacy and reduce the likelihood of relapse associated with antigen loss. Herein, we developed a novel single-chain variable fragment (scFv) against CRLF2 based on a monoclonal antibody raised against the recombinant extracellular domain of human TSLPRα chain. The scFv fragment demonstrated excellent binding affinity with CRLF2 protein in the nanomolar range. Cellular association studies in vitro using an inducible CRLF2 knockdown cell line and ex vivo using patient-derived xenografts revealed the selective association of the scFv with CRLF2. The fragment exhibited significant receptor antagonistic effects on STAT5 signalling, suggesting possible therapeutic implications in vivo. This study is the first to describe the potential use of a novel scFv for targeting Ph-like ALL.
Asunto(s)
Fragmentos de Inmunoglobulinas/metabolismo , Cromosoma Filadelfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Receptores de Citocinas/metabolismo , Proteínas Recombinantes/metabolismo , Animales , Línea Celular Tumoral , Niño , Endocitosis , Células HEK293 , Humanos , Ratones , Fosforilación , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Anticuerpos de Cadena Única/aislamiento & purificaciónRESUMEN
The research described in this report seeks to present proof-of-concept for a novel and robust platform for purification of antibody fragments and to define and optimize the controlling parameters. Purification of antigen-binding F(ab')2 fragments is achieved in the absence of chromatographic media or specific ligands, rather by using clusters of non-ionic detergent (e.g. Tween-60, Brij-O20) micelles chelated via Fe2+ ions and the hydrophobic chelator, bathophenanthroline (batho). These aggregates, quantitatively capture the F(ab')2 fragment in the absence or presence of E. coli lysate and allow extraction of only the F(ab')2 domain at pH 3.8 without concomitant aggregate dissolution or coextraction of bacterial impurities. Process yields range from 70 to 87% by densitometry. Recovered F(ab')2 fragments are monomeric (by dynamic light scattering), preserve their secondary structure (by circular dichroism) and are as pure as those obtained via Protein A chromatography (from a mixture of F(ab')2 and Fc fragments). The effect of process parameters on Ab binding and Ab extraction (e.g. temperature, pH, ionic strength, incubation time, composition of extraction buffer) are reported, using a monoclonal antibody (mAb) and polyclonal human IgG's as test samples.
Asunto(s)
Fragmentos Fab de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/metabolismo , Proteína Estafilocócica A/química , Anticuerpos Monoclonales/química , Cromatografía de Afinidad , Escherichia coli/metabolismo , Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/química , MicelasRESUMEN
A computational method was developed to recover the three-dimensional coordinates of gold nanoparticles specifically attached to a protein complex from tilt-pair images collected by electron microscopy. The program was tested on a simulated dataset and applied to a real dataset comprising tilt-pair images recorded by cryo electron microscopy of RNA polymerase II in a complex with four gold-labeled single-chain antibody fragments. The positions of the gold nanoparticles were determined, and comparison of the coordinates among the tetrameric particles revealed the range of motion within the protein complexes.
Asunto(s)
Oro/química , Procesamiento de Imagen Asistido por Computador/métodos , Fragmentos de Inmunoglobulinas , Nanopartículas del Metal/química , ARN Polimerasa II , Microscopía por Crioelectrón/métodos , Fragmentos de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/metabolismo , Modelos Moleculares , Unión Proteica , ARN Polimerasa II/química , ARN Polimerasa II/metabolismoRESUMEN
The use of intracellular antibodies as templates to derive surrogate compounds is an important objective because intracellular antibodies can be employed initially for target validation in pre-clinical assays and subsequently employed in compound library screens. LMO2 is a T cell oncogenic protein activated in the majority of T cell acute leukaemias. We have used an inhibitory intracellular antibody fragment as a competitor in a small molecule library screen using competitive surface plasmon resonance (cSPR) to identify compounds that bind to LMO2. We selected four compounds that bind to LMO2 but not when the anti-LMO2 intracellular antibody fragment is bound to it. These findings further illustrate the value of intracellular antibodies in the initial stages of drug discovery campaigns and more generally antibodies, or antibody fragments, can be the starting point for chemical compound development as surrogates of the antibody combining site.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antígenos de Neoplasias/metabolismo , Fragmentos de Inmunoglobulinas/metabolismo , Proteínas con Dominio LIM/metabolismo , Leucemia de Células T/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Linfocitos T/metabolismo , Anticuerpos/metabolismo , Unión Competitiva , Células Cultivadas , Descubrimiento de Drogas , Humanos , Fragmentos de Inmunoglobulinas/genética , Espacio Intracelular , Conformación Proteica , Bibliotecas de Moléculas Pequeñas , Resonancia por Plasmón de Superficie , Proteína 1 de la Leucemia Linfocítica T Aguda/metabolismo , Linfocitos T/inmunologíaRESUMEN
Antibodies are essential tools in research and diagnostics. Although antibody fragments typically obtained from in vitro selection can be rapidly produced in bacteria, the generation of full-length antibodies or the modification of antibodies with probes is time and labor intensive. Protein ligation such as SpyTag technology could covalently attach domains and labels to antibody fragments equipped with a SpyTag. However, we found that the established periplasmic expression of antibody fragments in E. coli led to quantitative cleavage of the SpyTag by the proteases Tsp and OmpT. Here we report successful periplasmic expression of SpyTagged Fab fragments and demonstrate the coupling to separately prepared SpyCatcher modules. We used this modular toolbox of SpyCatcher proteins to generate reagents for a variety of immunoassays and measured their performance in comparison with traditional reagents. Furthermore, we demonstrate surface immobilization, high-throughput screening of antibody libraries, and rapid prototyping of antibodies based on modular antibody assembly.
Asunto(s)
Anticuerpos/metabolismo , Fragmentos de Inmunoglobulinas/genética , Péptido Hidrolasas/genética , Proteínas Periplasmáticas/genética , Anticuerpos/química , Línea Celular Tumoral , Femenino , Humanos , Fragmentos de Inmunoglobulinas/aislamiento & purificación , Fragmentos de Inmunoglobulinas/metabolismo , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Proteínas Periplasmáticas/química , Proteínas Periplasmáticas/metabolismoRESUMEN
This work presents a method for introducing synthetic symmetry into protein crystallization samples using an antibody fragment termed a diabody (Dab). These Dabs contain two target binding sites, and engineered disulfide bonds have been included to modulate Dab flexibility. The impacts of Dab engineering have been observed through assessment of thermal stability, small-angle X-ray scattering, and high-resolution crystal structures. Complexes between the engineered Dabs and HIV-1 reverse transcriptase (RT) bound to a high-affinity DNA aptamer were also generated to explore the capacity of engineered Dabs to enable the crystallization of bound target proteins. This strategy increased the crystallization hit frequency obtained for RT-aptamer, and the structure of a Dab-RT-aptamer complex was determined to 3.0-Å resolution. Introduction of synthetic symmetry using a Dab could be a broadly applicable strategy, especially when monoclonal antibodies for a target have previously been identified.