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.

• Klíčová slova
• Thermofluor assay, crystallizability optimization, crystallization, differential scanning fluorimetry, oligomerization, single-chain antibody fragment,
• MeSH
• gelová chromatografie MeSH
• jednořetězcové protilátky chemie   MeSH
• krystalizace * MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• sekvence aminokyselin MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• jednořetězcové protilátky MeSH

This review discusses methods for the single-chain antibody fragment ($cFv) generation and scFv expression systems, and describes potential applications of scFv in the therapy of viral diseases and cancer, with emphasis on intracellularly expressed scFvs (intrabodies), application of scFvs in detection and diagnostics, and their use in proteomics.

• MeSH
• genetická terapie * MeSH
• imunoglobuliny - fragmenty genetika   terapeutické užití   MeSH
• klonování DNA metody   MeSH
• lidé MeSH
• monoklonální protilátky genetika   terapeutické užití   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• imunoglobuliny - fragmenty MeSH
• monoklonální protilátky MeSH

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
• 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
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• peptidová knihovna * MeSH
• protilátky bakteriální MeSH
• variabilní oblast imunoglobulinu 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.

• Klíčová slova
• NAALADase, antibody fragment, glutamate carboxypeptidase II, in vivo imaging, monoclonal antibody, prostate cancer, prostate-specific membrane antigen,
• 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
• Názvy látek
• antigeny povrchové MeSH
• FOLH1 protein, human MeSH Prohlížeč
• glutamátkarboxypeptidasa II MeSH
• jednořetězcové protilátky MeSH
• monoklonální protilátky MeSH
• rekombinantní proteiny 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
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny bakteriální MeSH
• bakteriální proteiny MeSH
• lipoproteiny MeSH
• monoklonální protilátky MeSH
• peptidová knihovna MeSH
• protilátky bakteriální MeSH
• rekombinantní fúzní proteiny MeSH
• variabilní oblast imunoglobulinu MeSH

A single-chain antibody fragments (scFv) was developed directed against transmembrane envelope glycoprotein gp46 of the virus maedi-visna, by the application of the antibody phage display library. To get specific scFv binders, the library was panned against the biotinylated peptide of 20 amino acids corresponding to the principal immunodominant domain of gp46 protein. The number of positively binding scFvs was evaluated by scFv-phage ELISA, BstN1 fingerprinting and DNA sequencing. The scFvs were expressed in soluble form and purified by immobilized metal affinity chromatography (IMAC) with a yield of 2-2.5 mg/l. Two scFvs have shown to recognize gp46 and gp150 proteins in Western blot analysis. The scFvs also recognized the virus in infected cells as shown by immunofluorescence assay. The affinity of the obtained antibody fragments to gp46 peptide was measured by surface plasmon resonance, and the resulting K(A) was in the 10(6)-10(7)lmol(-1) range. The application of characterized scFvs for expression as intrabodies in intracellular immunization against virus maedi-visna infection and for the diagnosis of this virus is discussed.

• MeSH
• buněčné linie MeSH
• DNA fingerprinting metody   MeSH
• ELISA MeSH
• imunoglobuliny - fragmenty chemie   imunologie   MeSH
• lehké řetězce imunoglobulinů MeSH
• molekulární sekvence - údaje MeSH
• ovce MeSH
• peptidová knihovna * MeSH
• povrchová plasmonová rezonance MeSH
• proteiny virového obalu imunologie   MeSH
• protilátky virové imunologie   MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza DNA MeSH
• specificita protilátek MeSH
• variabilní oblast imunoglobulinu MeSH
• virus visna-maedi imunologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• imunoglobuliny - fragmenty MeSH
• lehké řetězce imunoglobulinů MeSH
• peptidová knihovna * MeSH
• proteiny virového obalu MeSH
• protilátky virové MeSH
• variabilní oblast imunoglobulinu 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
• Názvy látek
• antitumorózní látky MeSH
• bungarotoxiny MeSH
• doxorubicin MeSH
• Duxon MeSH Prohlížeč
• ganglioside, GD2 MeSH Prohlížeč
• gangliosidy MeSH
• jednořetězcové protilátky MeSH
• kyseliny polymethakrylové MeSH
• nanokonjugáty MeSH

Single chain Fv (scFv) antibodies (generated by phage display technology, molecules representing new and efficient tools in the research and diagnostics of infectious diseases) against the capsid protein (p25) of Maedi-Visna virus were selected. Several clones of p25 specific scFv antibodies were identified; one of them was expressed as a soluble scFv molecule, purified by immobilized metal-affinity chromatography and further characterized by sequencing and determination of the kinetic equilibrium association constant. Sequence analysis showed that the rearranged VL and VH domains of the analyzed scFv clone used sequences from the VL3 family (germline DPL16/VL3.1) and VH1 family (germline VH20), respectively. The kinetic equilibrium association constant was determined as KA = 1.12 +/- 0.52 L/mumol.

• MeSH
• imunoglobuliny - fragmenty genetika   imunologie   izolace a purifikace   MeSH
• kinetika MeSH
• lehké řetězce imunoglobulinů genetika   imunologie   MeSH
• molekulární sekvence - údaje MeSH
• peptidová knihovna MeSH
• rekombinantní proteiny genetika   imunologie   izolace a purifikace   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• těžké řetězce imunoglobulinů genetika   imunologie   MeSH
• variabilní oblast imunoglobulinu genetika   imunologie   izolace a purifikace   MeSH
• virové plášťové proteiny genetika   imunologie   MeSH
• virus visna-maedi genetika   imunologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• imunoglobuliny - fragmenty MeSH
• lehké řetězce imunoglobulinů MeSH
• peptidová knihovna MeSH
• rekombinantní proteiny MeSH
• těžké řetězce imunoglobulinů MeSH
• variabilní oblast imunoglobulinu MeSH
• virové plášťové proteiny 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
• Názvy látek
• antigeny CD20 * MeSH
• epitopy MeSH
• jednořetězcové protilátky MeSH
• lehké řetězce imunoglobulinů MeSH
• peptidová knihovna MeSH
• peptidy MeSH
• rekombinantní fúzní proteiny MeSH
• těžké řetězce imunoglobulinů 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.

• Klíčová slova
• cancer therapy, coiled coil, drug targeting, hydrophilic polymer, scFv,
• 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
• Názvy látek
• akrylamidy MeSH
• cyklin D1 MeSH
• imunoglobuliny - fragmenty MeSH
• imunokonjugáty MeSH
• monoklonální protilátky MeSH
• N-(2-hydroxypropyl)methacrylamide MeSH Prohlížeč
• nosiče léků MeSH
• peptidy MeSH
• polymery MeSH