Chuanchu Wu

Metal-chelate-dendrimer-antibody constructs for use in radioimmunotherapy and imaging

Polyamidoamine dendrimers were modified chemically by reaction with DOTA and DTPA type bifunctional metal chelators and were coupled to monoclonal antibody 2E4 without loss of protein immunoreactivity. Both the DTPA- and DOTA-dendrimer-antibody constructs were easily labeled with 90Y, 111In, 212Bi or cold Gd(III) suggesting use of this dendrimer-macrocycle for mAb guided radiotherapy or imaging.

Synthesis, characterization, and evaluation of a novel bifunctional chelating agent for the lead isotopes 203Pb and 212Pb

Radioisotopes of Pb(II) have been of some interest in radioimmunotherapy and radioimmunoimaging (RII). However, the absence of a kinetically stable bifunctional chelating agent for Pb(II) has hampered its use for these applications. 203Pb (T(1/2) = 52.02 h) has application potential in RII, with a gamma-emission that is ideal for single photon emission computerized tomography, whereas 212Pb (T(1/2) = 10 h) is a source of highly cytotoxic alpha-particles via its decay to its 212Bi (T(1/2) = 60 min) daughter. The synthesis of the novel bifunctional chelating agent 2-(4-isothiocyanotobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra- (2-carbamoyl methyl)-cyclododecane (4-NCS-Bz-TCMC) is reported herein. The Pb[TCMC]2+ complex was less labile to metal ion release than Pb[DOTA]2- at pH 3.5 and below in isotopic exchange experiments. In addition to increased stability to Pb2+ ion release at low pH, the bifunctional TCMC ligand was found to have many other advantages over the bifunctional 1,4,7,10-tetraazacyclodocane-1,4,7,10-tetraacetic acid (DOTA) ligand. These include a shorter and more straightforward synthetic route, a more efficient conjugation reaction to a monoclonal antibody (mAb), with a higher chelate to protein ratio, a higher percent immuroreactivity, and a more efficient radiolabeling reaction of the mAb-ligand conjugate with 203Pb.

Comparative biodistribution of indium- and yttrium-labeled B3 monoclonal antibody conjugated to either 2-(p-SCN-Bz)-6-methyl-DTPA (1 B4M-DTPA) or 2-(p-SCN-Bz)-1,4,7,10-tetraazacyclododecane tetraacetic acid (2B-DOTA)

The biodistribution of indium-111/yttrium-88-labeled B3 monoclonal antibody, a murine IgG1k, was evaluated in non-tumor-bearing mice. B3 was conjugated to either 2-(p-SCN-Bz)-6-methyl-DTPA (1B4M) or 2-(p-SCN-Bz)-1,4,7,10 tetraazacyclododecane tetra-acetic acid (2B-DOTA) and labeled with 111In at 1.4–2.4 mCi/mg and 88Y at 0.1–0.3 mCi/mg. Non-tumor-bearing nude mice were co-injected i.v. with 5–10 μCi/4–10 μg of 111In/88Y-labeled B3 conjugates and sacrificed at 6 h and daily up to 168 h post-injection. Mice injected with 111In/88Y (IB4M)-B3 showed a similar biodistribution of the two radiolabels in all tissues except the bones, where significantly higher accretion of 88Y than 111In was observed, with 2.8% ± 0.2% vs 1.3% ± 0.16% ID/g in the femur at 168 h, respectively (P<0.0001). In contrast, mice receiving the 111In/88Y-(DOTA)-B3 conjugate showed significantly higher accumulation of 111In than 88Y in most tissues, including the bones, with 2.0% ± 0.1% vs 1.2% ± 0.09% ID/g in the femur at 168 h, respectively (P<0.0001). Whereas the ratios of the areas underneath the curve (%ID × h/g) in the blood, liver, kidney and bone were 0.96, 1.12, 1.13, and 0.74 for 111In/88Y-(IB4M)-B3 and 0.84, 1.23, 1.56, and 1.31 for 111In/88Y (DOTA)-B3, respectively, ratios ≈ 1 were observed between 111In-(IB4M)-B3 and 88Y-(DOTA)-B3. In summary, while neither IB4M nor DOTA was equally stable for 111In and 88Y, the fate of 88Y- (DOTA)-B3 could be closely traced by that of 111 In-(IB4M)-B3.

AN IMPROVED GENERATOR FOR THE PRODUCTION OF 213BI FROM 225AC

An improved generator was developed using a silica-based extraction chromatographic resin, Eichrom Silica Actinide Resin (Eichrom, Danen, IL), for the production of the α-emitting radionuclide Bi and to minimize radiolysis of the Ac/Bi generator. Ac-225 was adsorbed and evenly distributed on the top two-thirds of the generator resin. Bi-213 was eluted quantitatively with 1.0 Μ HCl. Simultaneous elution of the generator, subsequent dilution and re-adsorption of Bi onto an MP-50 column to concentrate the activity was performed by assembling the columns in series. Breakthrough of Ac from the generator was <0.05%, and no Ac was found when Bi was eluted from the second column. Bi-213 obtained can be easily used to radiolabel appropriate protein chelating agent conjugates. Hypothetically, resin damage by α-radiolysis should be obviated by employing such a silica-based resin and by broad distribution of the Ac on the column.

In vitro and in vivo evaluation of structure-stability relationship of 111In- and 67Ga-labeled antibody via 1B4M or C-NOTA chelates

2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (C-NOTA) or 2-(p-isothiocyanatobenzyl)-6-methyl-diethylenetriamine pentaacetic acid (1B4M) was conjugated to monoclonal antibody T101 (IgG2a), radiolabeled with 111In or 67Ga and then purified through size-exclusion HPLC. 111In 1B4M-T101 and 67Ga C-NOTA-T101 were stable in in vitro serum at 37 degrees C. In contrast, 111In C-NOTA-T101 and 67Ga 1B4M-T101 were unstable. The biodistribution in normal mice reflected the instability of the metal complex; the less-stable 111In C-NOTA conjugate left less tracer in blood, but more in liver and kidney whereas the less-stable 67Ga 1B4M conjugate left less tracer in blood, but more in bone. The biodistribution data suggest that the difference shown between the 111In and 67Ga conjugates might be mediated by differences in the in vivo chemistry of the metallic ions.

111Indium-labeled monoclonal antibody K1: Biodistribution study in nude mice bearing a human carcinoma xenograft expressing mesothelin

The monoclonal antibody (MAb) K1 is a murine IgG1 that recognizes mesothelin, a differentiation antigen present on mesothelium which is highly expressed on cancers derived from mesothelium, including most ovarian cancers and epithelioid mesotheliomas. MAb K1 was conjugated to 2‐(p‐isothiocyanatobenzyl)‐cyclohexyl‐ diethylenetriaminepentaacetic acid and labeled with 111In. The biodistribution of 111In‐K1 was studied in athymic nude mice bearing 2 s.c. tumors, one expressing a stably transfected plasmid encoding mesothelin and one composed of the parental untransfected A431 epidermoid carcinoma cells which do not express mesothelin. Tumor‐bearing mice were given an i.v. injection of 111In‐K1 and killed at different time points to determine the uptake of radiolabeled antibody. Significantly higher uptake was seen in antigen‐positive tumors at all time points, with peak values at 72 hr (52.9% vs. 8% of the injected dose/g tissue for antigen‐positive and antigen‐negative tumors, respectively). Uptake in antigen‐positive tumors was higher than the blood level at all time points, and the tumors contained a high level of the radiolabeled MAb even at 7 days (28.6% of the injected dose/g tumor). Int. J. Cancer80:559–563, 1999. Published 1999 Wiley‐Liss, Inc.

Evaluation of methods for large scale preparation of antibody ligand conjugates.

A rapid, single vessel method for preparation of clinical grade monoclonal antibody-chelating agent conjugates has been evaluated. By use of diafiltration methodology, currently employed dialysis step(s) that are normally used for the production of immunoconjugates may be eliminated. This technique has the advantage of eliminating the use of large amounts of buffer and the several days of time associated with purification of the product conjugate from unreacted ligand by dialysis, reduced risk of metal, pyrogen, and bacterial contamination, all coupled with the ability to produce multi-hundred milligram amounts of product suitable for vialing under GMP conditions for clinical applications. Evaluation of the product by this method indicates a superior quality of purity as compared with immunoconjugates prepared by the established methods.