single-chain antibody fragment Dotaz Zobrazit nápovědu
Single-chain variable antibody fragments (scFvs) are molecules with immense therapeutic and diagnostic potential. Knowledge of their three-dimensional structure is important for understanding their antigen-binding mode as well as for protein-engineering approaches such as antibody humanization. A major obstacle to the crystallization of single-chain variable antibody fragments is their relatively poor homogeneity caused by spontaneous oligomerization. A new approach to optimization of the crystallizability of single-chain variable antibody fragments is demonstrated using a representative single-chain variable fragment derived from the anti-CD3 antibody MEM-57. A Thermofluor-based assay was utilized to screen for optimal conditions for antibody-fragment stability and homogeneity. Such an optimization of the protein storage buffer led to a significantly improved ability of the scFv MEM-57 to yield crystals.
Single-chain antibodies (scFv) exhibiting specific binding to Lawsonia intracellularis were isolated from a phagemid library expressing scFvs molecules on the surface of filamentous bacteriophages. For scFv selection whole bacterial cells were used and individual clones were tested in ELISA test. The total of seven unique clones with different fingerprint profiles was isolated. All clones were able to bind specifically in immunofluorescence assay. This is the first report of species specific recombinant antibodies against L. intracellularis.
- MeSH
- financování organizované MeSH
- Lawsonia (bakterie) imunologie MeSH
- lidé MeSH
- peptidová knihovna MeSH
- protilátky bakteriální genetika imunologie izolace a purifikace MeSH
- specificita protilátek MeSH
- variabilní oblast imunoglobulinu genetika imunologie izolace a purifikace MeSH
- Check Tag
- lidé MeSH
Single-chain antibodies (scFv) specific to Brachyspira hyodysenteriae were isolated from a phagemid library. Recombinant Bhlp 29.7 protein was used for scFv selection and individual clones were tested by ELISA and immunofluorescent test; four unique clones were isolated. One of selected clones was able to bind specifically B. hyodysenteriae in ELISA and immunofluorescence test. This is the first report of species-specific recombinant antibodies against B. hyodysenteriae.
- MeSH
- antigeny bakteriální imunologie MeSH
- bakteriální proteiny imunologie MeSH
- Brachyspira hyodysenteriae imunologie MeSH
- druhová specificita MeSH
- ELISA MeSH
- fluorescenční protilátková technika přímá MeSH
- gramnegativní bakteriální infekce diagnóza veterinární MeSH
- lipoproteiny imunologie MeSH
- monoklonální protilátky imunologie MeSH
- nemoci prasat diagnóza MeSH
- peptidová knihovna MeSH
- prasata MeSH
- protilátky bakteriální imunologie MeSH
- rekombinantní fúzní proteiny imunologie MeSH
- specificita protilátek MeSH
- variabilní oblast imunoglobulinu imunologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
A water-soluble polymer cancerostatic actively targeted against cancer cells expressing a disialoganglioside antigen GD2 was designed, synthesized and characterized. A polymer conjugate of an antitumor drug doxorubicin with a N-(2-hydroxypropyl)methacrylamide-based copolymer was specifically targeted against GD2 antigen-positive tumor cells using a recombinant single chain fragment (scFv) of an anti-GD2 monoclonal antibody. The targeting protein ligand was attached to the polymer-drug conjugate either via a covalent bond between the amino groups of the protein using a traditional nonspecific aminolytic reaction with a reactive polymer precursor or via a noncovalent but highly specific interaction between bungarotoxin covalently linked to the polymer and the recombinant scFv modified with a C-terminal bungarotoxin-binding peptide. The GD2 antigen binding activity and GD2-specific cytotoxicity of the targeted noncovalent polymer-scFv complex proved to be superior to the covalent polymer-scFv conjugate.
- MeSH
- antitumorózní látky aplikace a dávkování chemie farmakologie MeSH
- bungarotoxiny chemie MeSH
- buňky 3T3 MeSH
- doxorubicin aplikace a dávkování chemie farmakologie MeSH
- gangliosidy imunologie MeSH
- jednořetězcové protilátky chemie imunologie MeSH
- kyseliny polymethakrylové chemie MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nanokonjugáty chemie MeSH
- proliferace buněk účinky léků MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Prostate-Specific Membrane Antigen (PSMA) is an established biomarker for the imaging and experimental therapy of prostate cancer (PCa), as it is strongly upregulated in high-grade primary, androgen-independent, and metastatic lesions. Here, we report on the development and functional characterization of recombinant single-chain Fv (scFv) and Fab fragments derived from the 5D3 PSMA-specific monoclonal antibody (mAb). These fragments were engineered, heterologously expressed in insect S2 cells, and purified to homogeneity with yields up to 20 mg/L. In vitro assays including ELISA, immunofluorescence and flow cytometry, revealed that the fragments retain the nanomolar affinity and single target specificity of the parent 5D3 antibody. Importantly, using a murine xenograft model of PCa, we verified the suitability of fluorescently labeled fragments for in vivo imaging of PSMA-positive tumors and compared their pharmacokinetics and tissue distribution to the parent mAb. Collectively, our data provide an experimental basis for the further development of 5D3 recombinant fragments for future clinical use.
- MeSH
- antigeny povrchové imunologie MeSH
- buněčné linie MeSH
- buňky PC-3 MeSH
- fluorescence MeSH
- glutamátkarboxypeptidasa II imunologie MeSH
- hmyz MeSH
- jednořetězcové protilátky imunologie MeSH
- lidé MeSH
- monoklonální protilátky imunologie MeSH
- myši nahé MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádory prostaty imunologie MeSH
- rekombinantní proteiny imunologie MeSH
- xenogenní modely - testy antitumorózní aktivity metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
A BCL1 leukemia-cell-targeted polymer-drug conjugate with a narrow molecular weight distribution consisting of an N-(2-hydroxypropyl)methacrylamide copolymer carrier and the anticancer drug pirarubicin is prepared by controlled radical copolymerization followed by metal-free click chemistry. A targeting recombinant single chain antibody fragment (scFv) derived from a B1 monoclonal antibody is attached noncovalently to the polymer carrier via a coiled coil interaction between two complementary peptides. Two pairs of coiled coil forming peptides (abbreviated KEK/EKE and KSK/ESE) are used as linkers between the polymer-pirarubicin conjugate and the targeting protein. The targeted polymer conjugate with the coiled coil linker KSK/ESE exhibits 4× better cell binding activity and 2× higher cytotoxicity in vitro compared with the other conjugate. Treatment of mice with established BCL1 leukemia using the scFv-targeted polymer conjugate leads to a markedly prolonged survival time of the experimental animals compared with the treatment using the free drug and the nontargeted polymer-pirarubicin conjugate.
- MeSH
- akrylamidy chemie MeSH
- cílená molekulární terapie MeSH
- cyklin D1 antagonisté a inhibitory imunologie MeSH
- imunoglobuliny - fragmenty aplikace a dávkování imunologie MeSH
- imunokonjugáty aplikace a dávkování chemie MeSH
- leukemie imunologie patologie terapie MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- monoklonální protilátky chemie imunologie MeSH
- myši MeSH
- nosiče léků aplikace a dávkování chemie MeSH
- peptidy chemie imunologie MeSH
- polymery aplikace a dávkování chemie MeSH
- syntetická chemie okamžité shody MeSH
- systémy cílené aplikace léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Monoclonal antibodies are leading agents for therapeutic treatment of human diseases, but are limited in use by the paucity of clinically relevant models for validation. Sporadic canine tumours mimic the features of some human equivalents. Developing canine immunotherapeutics can be an approach for modeling human disease responses. Rituximab is a pioneering agent used to treat human hematological malignancies. Biologic mimics that target canine CD20 are just being developed by the biotechnology industry. Towards a comparative canine-human model system, we have developed a novel anti-CD20 monoclonal antibody (NCD1.2) that binds both human and canine CD20. NCD1.2 has a sub-nanomolar Kd as defined by an octet red binding assay. Using FACS, NCD1.2 binds to clinically derived canine cells including B-cells in peripheral blood and in different histotypes of B-cell lymphoma. Immunohistochemical staining of canine tissues indicates that the NCD1.2 binds to membrane localized cells in Diffuse Large B-cell lymphoma, Marginal Zone Lymphoma, and other canine B-cell lymphomas. We cloned the heavy and light chains of NCD1.2 from hybridomas to determine whether active scaffolds can be acquired as future biologics tools. The VH and VL genes from the hybridomas were cloned using degenerate primers and packaged as single chains (scFv) into a phage-display library. Surprisingly, we identified two scFv (scFv-3 and scFv-7) isolated from the hybridoma with bioactivity towards CD20. The two scFv had identical VH genes but different VL genes and identical CDR3s, indicating that at least two light chain mRNAs are encoded by NCD1.2 hybridoma cells. Both scFv-3 and scFv-7 were cloned into mammalian vectors for secretion in CHO cells and the antibodies were bioactive towards recombinant CD20 protein or peptide. The scFv-3 and scFv-7 were cloned into an ADEPT-CPG2 bioconjugate vector where bioactivity was retained when expressed in bacterial systems. These data identify a recombinant anti-CD20 scFv that might form a useful tool for evaluation in bioconjugate-directed anti-CD20 immunotherapies in comparative medicine.
- MeSH
- antigeny CD20 * chemie genetika imunologie metabolismus MeSH
- buněčné linie MeSH
- epitopy imunologie MeSH
- exprese genu MeSH
- hybridomy imunologie metabolismus MeSH
- jednořetězcové protilátky imunologie farmakologie MeSH
- klonování DNA MeSH
- lehké řetězce imunoglobulinů genetika MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- myši MeSH
- peptidová knihovna MeSH
- peptidy chemie metabolismus MeSH
- psi MeSH
- rekombinantní fúzní proteiny farmakologie MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- specificita protilátek imunologie MeSH
- těžké řetězce imunoglobulinů genetika MeSH
- tvorba protilátek imunologie MeSH
- vazba proteinů imunologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Hybridoma technology for synthesis of monoclonal antibodies was one of the major breakthroughs in biotechnology of the 20th century. Since then, it has been possible to produce the specific molecules with typical immunoglobulin domain structure in sufficient quantities and qua¬lity. So far the synthesis of these complex molecules is labour-intensive and highly expensive. Also the non-human origin of immunoglobulins limits their application in clinical practice. These circumstances led to the deve¬lopment of new formats of antigen-binding molecules. The main goal was to prepare simple and stable low-molecular-weight substances, which can be utilized not only in analytical chemistry, in separation science but also as diagnostics or therapeutics. Molecules with compact protein core (scaffold) with exposed hypervariable loops on the surface show a possible direction of efforts. Recently, very pro¬mising antigen-binding formats such as Affibody, DARPins, Affilins or Anticalins were tested not only for their diagnostic or therapeutic potentials, but also in analytical chemistry as highly specific ligands for isolation and purification of bioactive molecules, e.g. for affinity chromatography. The classification, origin and structure of new antigen-binding molecules with some examples of their applications are described.
- MeSH
- afinita protilátek MeSH
- ankyrinová repetice MeSH
- hybridomy * chemie MeSH
- imunoglobuliny - fragmenty * chemie MeSH
- jednořetězcové protilátky MeSH
- jednořetězcové zlomy DNA MeSH
- krevní proteiny chemie klasifikace MeSH
- lipokaliny chemie MeSH
- monoklonální protilátky * chemie MeSH
- repetitivní sekvence aminokyselin MeSH
- techniky in vitro MeSH
- Publikační typ
- práce podpořená grantem MeSH
