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

Liposomes functionalized with monoclonal antibodies or their antigen-binding fragments have attracted much attention as specific drug delivery devices for treatment of various diseases including cancer. The conjugation of antibodies to liposomes is usually achieved by covalent coupling using cross-linkers in a reaction that might adversely affect the characteristics of the final product. Here we present an alternative strategy for liposome functionalization: we created a recombinant Fab antibody fragment genetically fused on its C-terminus to the hydrophobic peptide derived from pulmonary surfactant protein D, which became inserted into the liposomal bilayer during liposomal preparation and anchored the Fab onto the liposome surface. The Fab-conjugated liposomes specifically recognized antigen-positive cells and efficiently delivered their cargo, the Alexa Fluor 647 dye, into target cells in vitro and in vivo. In conclusion, our approach offers the potential for straightforward development of nanomedicines functionalized with an antibody of choice without the need of harmful cross-linkers.

• Klíčová slova
• Active targeting, Antibody engineering, Immunoliposome, Liposome functionalization, Recombinant Fab antibody fragment,
• MeSH
• antigen CD48 metabolismus   MeSH
• antigeny CD59 metabolismus   MeSH
• imunoglobuliny - Fab fragmenty chemie   imunologie   metabolismus   MeSH
• Jurkat buňky MeSH
• lidé MeSH
• liposomy chemie   MeSH
• lymfom imunologie   metabolismus   patologie   MeSH
• monoklonální protilátky chemie   imunologie   metabolismus   MeSH
• myši MeSH
• nádorové buňky kultivované MeSH
• peptidové fragmenty imunologie   metabolismus   MeSH
• protein D asociovaný s plicním surfaktantem imunologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigen CD48 MeSH
• antigeny CD59 MeSH
• CD48 protein, human MeSH Prohlížeč
• CD59 protein, human MeSH Prohlížeč
• imunoglobuliny - Fab fragmenty MeSH
• liposomy MeSH
• monoklonální protilátky MeSH
• peptidové fragmenty MeSH
• protein D asociovaný s plicním surfaktantem 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

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

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

Precipitating and non-precipitating anti-Dnp antibodies and S-sulpho non-specific IgG in gram quantities were subjected to limited cleavage by trypsin. Upon gel chromatography on Sephadex G-100 the fraction of Fab and Fc fragments was separated from incompletely split molecules and from tFc' fragments. The Fab and Fc fragments were separated from each other either by ion-exchange chromatography on QAE-Sephadex or by preparative electrophoresis in starch block. Both Fab and Fc fragments appeared to be heterogeneous as to electric charge. The Fc fragments were characterized by amino acid composition and N-terminal amino acids. The Fc fragment of non-specific IgG was cleaved by cyanogen bromide, and a C-terminal peptide containing 18 residues was isolated. Partial amino acid sequence of this peptide pointed to a high degree of homology with immunoglobulins of other animal species.

• MeSH
• aminokyseliny analýza   MeSH
• bromkyan MeSH
• dinitrofenoly imunologie   MeSH
• imunoglobulin G analýza   MeSH
• imunoglobuliny - Fab fragmenty izolace a purifikace   MeSH
• imunoglobuliny - Fc fragmenty izolace a purifikace   MeSH
• imunoglobuliny - fragmenty izolace a purifikace   MeSH
• peptidy analýza   MeSH
• prasata imunologie   MeSH
• precipitiny analýza   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aminokyseliny MeSH
• bromkyan MeSH
• dinitrofenoly MeSH
• imunoglobulin G MeSH
• imunoglobuliny - Fab fragmenty MeSH
• imunoglobuliny - Fc fragmenty MeSH
• imunoglobuliny - fragmenty MeSH
• peptidy MeSH
• precipitiny MeSH

Preimmune antibody repertoire is not a statistical representation of all germ-line VH, D, JH and VL, JL segments encoding heavy and light chains, respectively. The antibody repertoire is biased towards a fraction of VH and VL gene segments in fetal/neonatal as well as adult life. The repertoire bias starts at the surface Ig-negative pre-B cells and persists throughout B-cell ontogeny. Antigen-independent processes like preferential rearrangements of VH, D and JH segments and pairing of the heavy and surrogate light chain operate at the surface Ig-negative pre-B cell stage. Subsequently B cells may be subject to self-antigen selection based on the specificity of their surface receptors.

• MeSH
• B-lymfocyty imunologie   MeSH
• dospělí MeSH
• geny pro imunoglobuliny * MeSH
• imunoglobuliny - fragmenty genetika   MeSH
• imunoglobuliny genetika   MeSH
• lidé MeSH
• modely imunologické MeSH
• novorozenec MeSH
• plod MeSH
• stárnutí imunologie   MeSH
• tvorba protilátek MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• novorozenec MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• imunoglobuliny - fragmenty MeSH
• imunoglobuliny MeSH

BACKGROUND: Since the demonstration that the protease of the human immunodeficiency virus (HIV Pr) is essential in the viral life cycle, this enzyme has become one of the primary targets for antiviral drug design. The murine monoclonal antibody 1696 (mAb1696), produced by immunization with the HIV-1 protease, inhibits the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates with inhibition constants in the low nanomolar range. The antibody cross-reacts with peptides that include the N terminus of the enzyme, a region that is highly conserved in sequence among different viral strains and that, furthermore, is crucial for homodimerization to the active enzymatic form. RESULTS: We report here the crystal structure at 2.7 A resolution of a recombinant single-chain Fv fragment of mAb1696 as a complex with a cross-reactive peptide of the HIV-1 protease. The antibody-antigen interactions observed in this complex provide a structural basis for understanding the origin of the broad reactivity of mAb-1696 for the HIV-1 and HIV-2 proteases and their respective N-terminal peptides. CONCLUSION: A possible mechanism of HIV-protease inhibition by mAb1696 is proposed that could help the design of inhibitors aimed at binding inactive monomeric species.

• MeSH
• aspartátové endopeptidasy chemie   imunologie   metabolismus   MeSH
• chemické modely MeSH
• HIV-proteasa chemie   imunologie   metabolismus   MeSH
• imunoglobuliny - Fab fragmenty chemie   metabolismus   MeSH
• inhibitory HIV-proteasy chemie   farmakologie   MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• monoklonální protilátky chemie   farmakologie   MeSH
• peptidové fragmenty imunologie   metabolismus   MeSH
• protilátky virové chemie   metabolismus   MeSH
• vazebná místa protilátek MeSH
• zkřížené reakce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• aspartátové endopeptidasy MeSH
• HIV-proteasa MeSH
• imunoglobuliny - Fab fragmenty MeSH
• inhibitory HIV-proteasy MeSH
• monoklonální protilátky MeSH
• p16 protease, Human immunodeficiency virus 2 MeSH Prohlížeč
• peptidové fragmenty MeSH
• protilátky virové 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

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