Lentivirus transduction of bone marrow hemopoietic precursor cells with Lin-c-kit+ phenotype ex vivo using a genetic construct containing green fluorescent protein gene.

We developed a technology of labeling bone marrow precursor cells with the Lin-c-kit+ phenotype in culture by green fluorescent protein gene using a lentivirus vector. The proposed system provides effective transduction of bone marrow precursor cells and high transgene expression level in vitro (27%). The integration of the transgene into the transduced cell genome in vivo was verified by the method of splenic colonies.

[Triple interpolyelectrolyte complexes DNA-polycation-polyanion: a new approach to optimization of mixtures for transfection in eukaryotic cells].

A new approach to optimization of mixtures for the condensation and introduction of plasmid DNA into eukaryotic cells is proposed, which is based on the formation of ternary interpolyelectrolyte complexes (IPEC) DNA/polycation/polyanion. Polyethyleneimine (PEI) with M 30-40 kDa as polycation and polyacrylic acid (PA) with M 20 kDa or its grafted copolymer with polyethyleneglycol (PEG) as polyanion were used, and ternary complexes with various ratios of the components were prepared. The PA-PEG incorporation into a ternary complex (by itself or as a 1:1 mixture with PA) was shown to confer the solubility onto complexes in a wide range of DNA/PEI ratios.

Polylysine-Gd-DTPAn and polylysine-Gd-DOTAn coupled to anti-CEA F(ab')2 fragments as potential immunocontrast agents. Relaxometry, biodistribution, and magnetic resonance imaging in nude mice grafted with human colorectal carcinoma.

Rationale And Objectives: Immunocontrast agents used for magnetic resonance imaging require antibodies that preserve the immunoreactivity while containing a high number of chelated paramagnetic ions.

Methods: Anti-CEA F(ab')2 fragments were coupled to polylysine-Gd-DOTA and polylysine-Gd-DTPA. A paramagnetic load as high as n = 24 to 28 metal ions per antibody was reached.

Introduction of five potentially metabolizable linking groups between 111In-cyclohexyl EDTA derivatives and F(ab')2 fragments of anti-carcinoembryonic antigen antibody--II. Comparative pharmacokinetics and biodistribution in human colorectal carcinoma-bearing nude mice.

The five linker-containing immunoconjugates described in the preceding paper were labeled with 111In and tested for their biodistribution, pharmacokinetics and immunoscintigraphic imaging properties in tumor-xenografted nude mice. The results were compared with DTPADA and CDTAMA for reference. Results showed that, for immunoscintigraphy, the derivatives in decreasing order of effectiveness were: aliphatic (tumor/liver > 4.

Introduction of five potentially metabolizable linking groups between 111In-cyclohexyl EDTA derivatives and F(ab')2 fragments of anti-carcinoembryonic antigen antibody--I. A new reproducible synthetic method.

The purpose of this study was to synthesize new bifunctional linker-chelating agents for the modification of the in vivo distribution of 111In-labeled antibodies. A general simple synthetic method of preparing cyclohexyl EDTA (CDTA) derivatives containing a linker/spacer group is described. Linkers prepared included a diester, a six carbon aliphatic chain, two thioethers and a disulfide group.

Biodistribution of anti-CEA F(ab')2 fragments conjugated with chelating polymers: influence of conjugate electron charge on tumor uptake and blood clearance.

F(ab')2 fragments of anti-carcinoembryonic antigen (CEA) monoclonal antibody (mAb) were modified with three chain-terminal polylysine-based chelating polymers so as to carry different electron charges. Immunoreactive conjugates labeled with 111In up to a specific radioactivity of 120-140 microCi/micrograms were injected into nude mice bearing human colorectal carcinoma, and the biodistribution patterns were compared with each other and with that of an anti-CEA F(ab')2-DTPA control. Immunoconjugate modified with positively-charged polymer produced the highest tumor uptake [up to 20% injected dose per gram (ID/g)], with very significant non-specific radioactivity in normal organs (particularly kidneys).

Purification of radiolabeled monoclonal antibodies to angiotensin-converting enzyme significantly improves specificity and efficacy of its targeting into the lung.

The aim of this study was to improve the labeling/purification procedures for monoclonal antibody (MoAb) to angiotensin-converting enzyme (ACE). MoAb 9B9 was very stable upon iodination at a wide range of iodogen concentrations and incubation times, and was also very stable upon storage, indicating the high technological potential of this MoAb. Radiolabeled MoAb 9B9 was purified by (i) adsorption chromatography on cellulose, (ii) HPLC (gel filtration) and (iii) affinity chromatography on ACE-Sepharose.

[The conjugation of preliminarily immunosorbent-immobilized 5B4D6 monoclonal antibodies with a chelating polymer].

A study has been performed to investigate the change in antigen-binding capacity of antibodies as a result of their interaction with chelating agents, and polymers under various conditions. It has been demonstrated that antibody immobilization on the sorbent preceded by the antibody conjugation with chelating polymer allows better maintenance of specific activity of 5B4D6 monoclonal antibodies. Such modification yields a 10-fold increase in the antibody-antigen binding as compared with a standard conjugation technique in a mixture.

Site-specific conjugation of chain-terminal chelating polymers to Fab' fragments of anti-CEA mAb: effect of linkage type and polymer size on conjugate biodistribution in nude mice bearing human colorectal carcinoma.

Polylysine-based chelating polymers were used for site-specific modification of anti-CEA mAb Fab' fragments via their SH group distal to the antigen-binding site of the antibody molecule. Conjugation was performed using chain-terminal (pyridyldithio)propionate or 4-(p-maleimidophenyl)butyrate moieties to form reducible (S-S) or stable (S-C) bonds between a polymer and Fab' molecule, respectively. One S-S conjugate (S-S9) and two different S-C conjugates (S-C3 and S-C9) were prepared using 3- and 9-kDa molecular weight polymers.

Targeting of macromolecular carriers and liposomes by antibodies to myosin heavy chain.

Macromolecular carriers and liposomes were covalently coupled to monoclonal antibodies against cardiac myosin heavy chain. Deferoxamine-modified polymers bound tightly with 67Ga and 68Ga radioisotopes. Ternary deferoxamine-polylysine antibody conjugates specifically targeted the radioisotopes to a myosin-coated microplate.

Gamma imaging with negatively charge-modified monoclonal antibody: modification with synthetic polymers.

Antimyosin Fab has been modified to carry highly negatively charged synthetic polymers containing DTPAs (DTPA-PL) as chelating agents, of starting molecular weights 3.3 and 17 kD. The immunoreactivities of the modified antibodies were unaffected by the modification procedure.

Terminal-modified polylysine-based chelating polymers: highly efficient coupling to antibody with minimal loss in immunoreactivity.

A method is suggested for the preparation of chelating polymers containing a single terminal reactive group capable of interaction with proteins. These polymers were synthesized from N-CBZ-polylysine and DTPA and contain a terminal SH or pyridyldisulfide group. A polymer molecule with MW 13,500 is able to carry up to 40 DTPA residues.

Endotoxin reduces specific pulmonary uptake of radiolabeled monoclonal antibody to angiotensin-converting enzyme.

The biodistribution of radiolabeled monoclonal antibody (Mab) to angiotensin-converting enzyme (ACE) was examined in normal and endotoxin-treated rats. Endotoxin administration at a dose of 4 mg/kg induced mild or middle pulmonary edema. The ACE activity in lung homogenate remained virtually unchanged, while the activity of serum ACE increased 15 hr after endotoxin infusion.

Succinylated polylysine as a possible link between an antibody molecule and deferoxamine.

Modification of antibodies with chelating polymers may be helpful for radioimmunoimaging, radioimmunotherapy, and NMR tomography. Succinylated polylysine was activated with carbodiimide/N-hydroxysulfosuccinimide in dimethyl sulfoxide and isolated as a dry solid. Sulfosuccinimide-esterified polymer was used for the two-stage coupling of an amino-containing chelating agent (deferoxamine) to monoclonal R11D10 (IgG) or its Fab fragment.

[The chemical modification of immunoglobulins for accelerating their elimination from the blood flow during radioimmunodiagnosis].

Immunoglobulins were modified by diethylenetriaminepentaacetic acid anhydride and sulfosuccinimidy 16-(biotinamido) hexanoate and were conjugated with modified polylysine. Biodistribution of the samples was observed before and after avidin injection. Samples containing no polylysine accumulate in reticuloendothelial system, protein conjugate with polylysine concentrate in kidneys.

Radioimmunoimaging of lung vessels: an approach using indium-111-labeled monoclonal antibody to angiotensin-converting enzyme.

A murine monoclonal antibody against human angiotensin-converting enzyme was radiolabeled with 111In via diethylenetriaminepentaacetic acid without substantial loss of antigen-binding capacity. This monoclonal antibody designated 9B9 cross-reacted with rat and monkey angiotensin-converting enzyme. Indium-111-labeled 9B9 selectively accumulated 10-20 times greater in the lung than in blood or other organs following intravenous administration in rats.

Blood clearance of radiolabeled antibody: enhancement by lactosamination and treatment with biotin-avidin or anti-mouse IgG antibodies.

Methods of rapid blood clearance of 111In-labeled mouse monoclonal antibody 9B9 against angiotensin-converting enzyme were studied. Indium-111-9B9 is specifically accumulated in rat lung, but its blood clearance is relatively slow and target-to-blood radioactivity ratio/g tissue (localization ratio) increases from 11 to 30 only 48 hr postinjection. Injection of second (anti-mouse immunoglobulin) antibodies results in slight (1.

[A method of fluorescence immunoassay with temporal resolution and the use of europium label and polymeric complexon].

Recently developed solid-phase immunofluorescent assay with temporal distinction involved a newly produced procedure for binding of of the boron group label with antibodies via modified polymer, which is able to bind with antibodies across the avidin bridge. The assay developed is more simple than the widely used procedures, it exhibits high sensitivity being superior as compared with a corresponding immunoenzyme assay by a decimal order.

Monoclonal antibody modification with chelate-linked high-molecular-weight polymers: major increases in polyvalent cation binding without loss of antigen binding.

Polyethyleneimine or polylysines of differing molecular sizes were substituted with either EDTA or DTPA and then with succinic acid groups. These polymers were then reacted with the amino groups on myosin-specific monoclonal antibody or its Fab using a water soluble carbodiimide. The polymer-antibody complexes were capable of binding up to 150 di- or trivalent ions per mole (Mn++, Gd , or 111In ) without attendant loss of antigen binding.

[Modification of monoclonal antibodies by polymers possessing chelating properties].

Monoclonal antibodies to heavy chains of the heart myosin were chemically modified by chelate polymers containing EDTA or DTPA residues. The modification allows binding up to 90 atoms of heavy metals (e.g.