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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.

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.

The purpose of this study was to synthesize new bifunctional linker-chelating agents for the modification of the in vivo distribution of 111 In-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 111 In with good retention of immunoreactivity.

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.

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 111 In up to a specific radioactivity of 120–140 μCi/μg 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 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.

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.

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.

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.