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]. / Troinye interpoliélektrolitnye kompleksy DNK-polikation-polianion: novyi podkhod k optimizatsii smesei dlia transfektsii éukarioticheskikh kletok.

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. Incorporation of even minute amounts of PA-PEG (as a 1:9 mixture with PA), while not completely preventing the aggregation of ternary IPEC, drastically changed their sorption characteristics. Using a beta-galactosidase-encoding plasmid, efficiencies of transfection of the CHO-AA8 and 293 cells for different IPEC and DNA/lipofectin complex were compared. The maximum efficiency was exhibited by ternary complex DNA/PEI/polyanion where a 1:1 mixture of PA and PA-PEG was used as polyanion. Possible reasons for this effect and further ways of optimization of mixtures for expression of plasmid DNA in the context of the new approach are discussed.

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. RESULTS: The immunoreactivity of the gadolinium (Gd)-labeled anti-CEA F(ab')2 immunoconjugates was 80% to 85%. Compared with that of commercial chelates, the relaxivity (R1) increase is as follows: Gd-DTPA < Gd-DOTA < Gd-H2O < PL-Gd-DTPA24-28 < PL-Gd-DTPA24-28 F(ab')2 < PL-Gd-DOTA24-28 < PL-Gd-DOTA24-28 F(ab')2. 1H nuclear magnetic relaxation dispersion data of immunoconjugates showed that the high relaxivity enhancement was the result of a reduction of the molecular tumbling rate. Twenty-four hours after intravenous injection of 50 micrograms (1 mumol Gd/kg) of Gd-labeled immunoconjugates to nude mice grafted with human colorectal carcinoma LS 174T, the tumor uptake was 10% to 15%, resulting in an increase of R1 of up to 15% to 20% versus noninjected mice. No difference was found between PL-Gd-DTPA24-28 F(ab')2 and PL-Gd-DOTA24-28 F(ab')2 immunoconjugates for tumor, liver, and kidney uptake. A high signal intensity of tumor was observed in 50% of the tested mice.

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. The CDTA-linker compounds were coupled to F(Ab')2 fragments of anti-carcinoembryonic antigen monoclonal antibody and labeled with 111In with good retention of immunoreactivity.

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 > 4.5 and tumor/kidney > 6.5 at 96 h), thioether (tumor/liver > 3 and tumor/kidney > 1.2 at 24 h), ethylene glycol succinate (tumor/liver > 1.7 and tumor/kidney > 0.5 at 24 h) and disulfide (tumor/liver > 0.5 and tumor/kidney > 0.6 at 96 h). Pharmacokinetic results were complementary with those of the biodistribution studies and provide a basis for the study of in vivo metabolic mechanisms of linker-immunoconjugates. Indium-111-labeled linker-immunoconjugates appear promising for tumor imaging with better contrast than what is obtained with the use of the conventional 111In-DTPA dianhydride chelate.

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 best result was obtained with cellulose: specificity of MoAb 9B9 accumulation in the lung increased 2-fold. We conclude that the phenomenon of specific lung accumulation of MoAb 9B9 may serve as an ideal (convenient, cheap and technological) assay system for evaluation of monoclonal antibody modification and labeling.

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). When modified with a polymer carrying only a slight negative charge, the immunoconjugate also produced fairly high tumor uptake (up to 18% ID/g), with much lower non-specific radioactivity in normal organs. Highly negatively-charged conjugate produced the lowest tumor uptake (up to 8% ID/g), whereas blood and whole-body clearances were the fastest but slower than those of conventionally labeled F(ab')2 mAb. The possible mechanisms for the effects described are discussed.

[The conjugation of preliminarily immunosorbent-immobilized 5B4D6 monoclonal antibodies with a chelating polymer]. / Kon''iugirovanie predvaritel'no immobilizovannykh na immunosorbente monoklonal'nykh antitel 5B4D6 s khelatiruiushchim polimerom.

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. No significant loss of immunoreactivity was observed in solid-phase immunoassay, 90-95% of 111In-labeled conjugates being bound to CEA-coated Sepharose beads. After labeling with 111In, the conjugates had a specific radioactivity of 90-120 microCi/micrograms. Injected in nude mice bearing LS 174T carcinoma, the conjugates produced different biodistribution patterns. S-S9 was practically unable to accumulate in tumor and produced very rapid blood clearance of radioactivity and high uptake of radioactivity in liver, spleen, and especially kidneys (225% ID/g 24 h postinjection). S-C3 and S-C9 produced practically the same blood clearances (much slower than that of S-S9) and significant tumor uptake (9-10% ID/g at 24 h). S-C3 gave significantly lower radioactivity in spleen, skin, and bones, and cleared more rapidly from liver and kidneys. Renal uptake for S-C3 and S-C9 was rather high (45% ID/g at 24 h), but much lower than for S-S9.

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. Scatchard analysis revealed a high affinity of the conjugate for the target with a Kas of approximately 10(8) M-1. Liposomes that contained immobilized antimyosin antibodies were targeted specifically to the myosin-coated plate. Additional coating of these liposomes with polyethylene glycol reduced specific binding to the target in vitro. However, because of the presence of polyethylene glycol on the surface of liposomes, these liposomes had a long half-life and slowly cleared from the blood-stream after intravenous injection. These immunoliposomes showed up to 16- to 18-fold specific localization to the necrotic areas of the myocardium in rabbits with experimental infarction.

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. The isoelectric points (PI) of unmodified antimyosin (AM) Fab (PI range 7-9, Mr = 52kD) were changed to PIs predominantly between 4 and 5 (Mr = 59 kD for DTPA-PL3.3kD-AM-Fab and 67kD for DTPA-PL17kD-AM-Fab). These AM-Fab preparations were tested for specific target localization and visualization in vivo in an experimental canine model of acute myocardial infarction. The charge-modified 111In-labeled AM-Fab preparations showed enhanced target (necrotic myocardium) visualization within 30 min of intravenous infusion and decreased background activity in normal myocardium (mean %ID/g +/- s.e.m., 0.0076 +/- 0.0006, n = 164, and 0.0056 +/- 0.0004, n = 92, for 111In-DTPA-PL3.3kD- and DTPA-PL17kD-AM-Fab respectively) relative to conventional 111In-DTPA-AM-Fab (0.0263 +/- 0.0037, n = 135) (p less than 0.001) or radioiodinated AM-Fab (0.0098 +/- 0.0006, n = 256) (p less than or equal to 0.001). Furthermore, the concentration of negatively charged 111In-labeled antimyosin Fab decreased in non-target organs such as the liver and kidneys. In diagnostic and therapeutic applications, charge-modified macromolecules may improve target localization and reduce non-target organ activity.

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. Polymers easily and quantitatively bind with antibodies (Fab fragments of antimyosin antibodies R11D10) with minimal effect on antibody immunoreactivity as revealed in ELISA assay and in direct immunoanalysis. Conjugates prepared can chelate radioactive metal ions reaching very high specific radioactivity (greater than 1 mCi 111In/10 micrograms of protein). Perspectives for their application are discussed.

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. In normal rats, anti-ACE Mab accumulates specifically in the lung after i.v. injection. Endotoxin injection induces reduction of specific pulmonary uptake of this antibody. Even in non-edematous endotoxemia, the accumulation of anti-ACE Mab antibody (Mab 9B9) decreased from 19.02 to 11.91% of ID/g of tissue without any change in accumulation of control nonspecific IgG. The antibody distribution in other organs and its blood level were almost the same as in the control. In a case of endotoxemia accompanied by increased microvascular permeability, the lung accumulation of Mab 9B9 was reduced to 9.17% of ID/g of tissue, while the accumulation of nonspecific IgG increased to 1.44% versus 0.89% in the control.

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. Conjugates were separated from free components by using gel-chromatography and anion-exchange chromatography. Antibody-coupling efficiency and the loss of its immunoreactivity upon modification have been studied for polymers with different deferoxamine content. Specific binding of 67Ga to the corresponding antigen via the conjugate has been demonstrated.

[The chemical modification of immunoglobulins for accelerating their elimination from the blood flow during radioimmunodiagnosis]. / Khimicheskaia modifikatsiia immunoglobulinov dlia uskoreniia ikh vyvedeniia iz krovotoka v protsesse radioimmunodiagnostiki.

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. The results demonstrate that drug distribution depends on the type of modification.

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. Kinetics of lung accumulation and blood clearance were studied for 111In-9B9-antibody and compared to that of 125I-labeled 9B9 in rat. Highly specific accumulation of 111In-9B9-antibody in the lung of Macaca Rhesus monkeys after intravenous injection was monitored by gamma-imaging. Images of 111In-labeled antibody 9B9 biodistribution in monkey lung noticeably differ from the images of biodistribution of 99mTc-labeled albumin microspheres. This difference may provide information concerning the state of the endothelium of lung capillaries, which is different from the blood flow characteristics determined with routine microsphere technique.

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 (1.8-fold) increase of 9B9 localization ratio. Chemical modification of 9B9 aminogroups with lactose results in enhanced liver uptake and rapid blood clearance of antibody. Blood radioactivity level decreases tenfold, and as a result localization ratio increases threefold (up to 38 in 30 min). Injection of avidin following the injection of biotinylated 9B9 results in rapid clearance of blood radioactivity with increased uptake in liver and spleen. Lung uptake is not changed. Localization ratio increases fivefold over the avidin-untreated animal value. Implications of these approaches for various applications in immunoimaging are discussed.

[A method of fluorescence immunoassay with temporal resolution and the use of europium label and polymeric complexon]. / Metod fliuorestsentnogo immunoanaliza s vremennym razresheniem s ispol'zovaniem evropievoi metki i polimernogo kompleksona.

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. The polylysine derivatives of the intact antibody were rapidly cleared and sequestered in the liver, whereas the polylysine 14-kilodalton (kd) derivative of Fab was cleared from the circulation with minimal hepatic and kidney sequestration. This differed from the biodistribution of intact antimyosin or its Fab labeled with 111In via direct attachment of DTPA to the epsilon amino group of the lysyl residues. Applications in magnetic resonance and nuclear imaging are envisioned.

[Modification of monoclonal antibodies by polymers possessing chelating properties]. / Modifikatsiia monoklonal'nykh antitel polimerami, obladaiushchimi khelatiruiushchimi svoistvami.

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. 111In, Mn2+, Cd3+) per protein globule, i.e. an increase in the specific activity of labeling 20 to 50-fold in comparison with previous methods. Specific affinity of the modified protein is preserved. Conjugates obtained may be useful for in vivo immuno-imaging.