Otto A. Gansow

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.

The Multichain Interleukin-2 Receptor: A Target for Immunotherapy

Activation of resting T-lymphocytes induces synthesis of interleukin-2 (IL-2) and expression of cell surface receptors for this lymphokine. In contrast to resting normal T-cells that do not express high-affinity IL-2 receptors (IL-2R), abnormal T-cells of patients with leukemia-lymphoma, certain autoimmune disorders, and individuals rejecting allografts express this receptor. Exploiting this difference in receptor expression, antibodies to the IL-2 receptor have been used effectively to treat patients with leukemia and lymphoma. One approach is to use monoclonal antibodies produced in mice; the disadvantage is that they are highly immunogenic. In an effort to reduce the immunogenicity of the mouse monoclonal antibodies, monoclonal-antibody-mediated therapy has been revolutionized by generating humanized antibodies produced by genetic engineering in which the molecule is human except for the antigen-combining regions, which are retained from the mouse. Further, to increase its cytotoxic effectiveness, the monoclonal antibody has been armed with toxins or radionuclides. Alternatively, IL-2 itself has been linked to a toxin to kill IL-2 receptor-bearing cells. Thus, IL-2 receptor-directed therapy provides a new method for treating certain neoplastic diseases and autoimmune disorders and for preventing allograft rejection.

Newer approaches to the radiolabeling of monoclonal antibodies by use of metal chelates

Monoclonal antibodies (mAbs) radiolabeled by use of metal chelators are being investigated in the laboratory for use in clinical trials. 111In is presently employed for diagnostic scintigraphy, but its applications are limited by substantive and persistant uptake of radiometal in the liver. Much current research is focused on performing cancer therapy with 90Y and 212Bi chelate-linked mAbs. This report chronicles the development and evaluation of chelating agents for 111In-radioimmunoimaging and 90Y- and 212Bi-radioimmunotherapy.

Biodistribution and metabolism of targeted and nontargeted protein-chelate-gadolinium complexes: Evidence for gadolinium dissociation in vitro and in vivo

The intracellular metabolism of receptor-targeted 153Gd-DTPA-glycoproteins was studied in vitro and in vivo. These agents bound to cell surface receptors, underwent receptor mediated endocytosis, and were rapidly degraded to a metabolite which co-migrated with a 153Gd-DTPA-lysine standard on thin layer chromatography. The rates of dissociation of 153Gd and 111In from a glycoprotein-chelate conjugate were determined in vitro. Gadolinium readily dissociated, in a pH-sensitive manner, from glycoprotein-DTPA, and to a lesser degree glycoprotein-MX-DTPA. The biodistribution of targeted and blood pool 153Gd/111In labeled proteins also suggested that gadolinium dissociates from protein-DTPA and protein-MX-DTPA and their metabolites leading to an accumulation of gadolinium in bone. Metal-DTPA-glycoprotein agents targeted to cell surface receptors can still produce very high concentrations of radioactive or paramagnetic metals within the lysosome due to the high rate of accumulation afforded by receptor mediated endocytosis and the low release rate of metabolites such as metal-DTPA-lysine. However, the continued development of gadolinium based macromolecular agents will require improvements in bifunctional chelates.

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.

Comparative biological properties of a recombinant chimeric anti-carcinoma mAb and a recombinant aglycosylated variant

SummaryIt has been demonstrated previously that the degree of glycosylation of a molecule may alter its pharmacokinetic properties and, in the case of an antibody, its metabolism and other biological properties. Transfectomas producing aglycosylated chimeric B72.3(γ1) pancarcinoma monoclonal antibody (mAb) were developed by introduction of the eukaryotic expression construct pECMgpB72.3 HuG1-agly, into SP2/0 murine myeloma cells producing the chimeric κ chain of mAb B72.3. After cell cloning, one subclone with the highest binding to the TAG-72-positive human colon carcinoma was designated mAb aGcB72.3, and its biological and biochemical properties were compared with those of the chimeric B72.3(γ1), designated mAb cB72.3. Polyacrylamide gel electrophoresis showed that under non-reducing conditions, the molecular masses of the aGcB72.3 and cB72.3 mAbs were 162 kDa and 166 kDa respectively. The heavy chain of mAb aGcB72.3 had a slightly faster mobility than that of cB72.3, while the mobility of the light chains of the two chimeric mAbs was similar. No difference was observed in the isoelectric points of either chimeric mAb. Liquid competition radioimmunoassays demonstrated that the aGcB72.3 and cB72.3 mAbs have comparable binding properties to TAG-72. These studies demonstrate that aglycosylation of the chimeric IgG1 mAb B72.3 at theCh2 domain, as has been shown for other mAbs [Dorai H., Mueller B., Reisfeld R. A., Gillies S. D. (1991) Hybridoma 10:211; Morrison S. L., Oi V. T. (1989) Adv Immunol 44:65], eliminates antibody-dependent cell-mediated cytotoxicity activity, but does not substantially alter affinity or plasma clearance in mice. These studies also demonstrate for the first time (a) no difference in plasma clearance of an aglycosylated and a chimeric mAb in a primate after i.v. inoculation; (b) a difference (P ⩽0.05) in mice in the more rapid peritoneal clearance of a chimeric mAb versus an aglycosylated chimeric mAb; (c) higher (0.05 ⩽P ⩽0.1) tumor:liver ratios at 24, 72 and 168 h using111In-labeled aglycosylated chimeric mAb versus chimeric mAb. Since the liver is the major site of metastatic spread for most carcinomas, slight differences in tumor to normal liver ratios may be important in diagnostic applications. These studies thus indicate that comparative analyses of a novel recombinant construct (i.e., aglycosylated) and its standard chimeric counterpart require documentation in more than one system and are necessary if one is ultimately to define optimal recombinant/chimeric constructs for diagnosis and therapy in humans.

Comparative biodistribution studies of DTPA-derivative bifunctional chelates for radiometal labeled monoclonal antibodies

Biodistribution of five different backbone-substituted derivatives of SCN-Bz-DTPA (1B4M-DTPA, 1M3B-DTPA, 1B3M-DTPA, GEM-DTPA and 2B-DTPA) linked to MAb B72.3 were compared to that of the parent molecule after labeling with 111indium. Athymic mice, bearing human colon carcinoma xenografts (LS-174T) were injected i.v. to determine the biodistribution of the MAb chelate conjugates. Three of the MAb metal chelate conjugates (1B4M-DTPA, 1M3B-DTPA, and 1B3M-DTPA), labeled with 111In showed efficient and stable tumor localization as well as a slower blood clearance rate than SCN-Bz-DTPA, GEM-DTPA or 2B-DTPA MAb chelate conjugates. Major differences were also seen in normal organ uptake, especially liver and spleen. Tumor-to-liver ratios rose as a function of time for 1B4M-DTPA, 1M3B-DTPA and 1B3M-DTPA MAb chelate conjugates with virtually no accumulation of the radiometal into this organ, as revealed by no increase in the liver-to-blood values. Small accretion in normal liver was noted for SCN-Bz-DTPA, GEM-DTPA or 2B-DTPA MAb chelate conjugates. The results reviewed here, and described previously (Roselli et al., 1991) demonstrate that the use in vivo of backbone-substituted forms of the SCN-Bz-DTPA, such as 1B4M-DTPA, 1M3B-DTPA, and 1B3M-DTPA bound to MAbs, can reduce uptake of indium to normal organs while maximizing the dose to tumor.

Preparation of 211At-Labeled Humanized Anti-Tac Using 211At Produced in Disposable Internal and External Bismuth Targets

These studies describe the production and purification of 211At as well as the procedure for labeling humanized anti-Tac, the antibody to the alpha-chain of the IL-2 receptor (IL-2R alpha), which has been shown to be a useful target for immunotherapy. The optimized protocol combines the advantages of the two-stage dry distillation procedure with the astatination of trialkylstannyl substances as labeling compounds for proteins. The 211At was produced by bombarding either an external or a recently developed disposable internal bismuth target with alpha-particles from a Cyclotron Corporation CS-30 cyclotron. The 211At was found to contain less than 0.01% 210At. The production rate for the external target was 0.15 mCi +/- 0.056 microA(-1) h(-1) (n = 9) (5.55 MBq mcroA[-1] h[-1]). The production rate for the internal target was 0.44 +/- 0.14 mCi microA(-1) h(-1) (n = 16) (16.28 MBq mcroA[-1] h[-1]).

Preparation and in vivo evaluation of linkers for 211At labeling of humanized anti-Tac

The syntheses, radiolabeling, antibody conjugation, and in vivo evaluation of new linkers for 211At labeling of humanized anti-Tac (Hu-anti-Tac), an antibody to the alpha-chain of the IL-2 receptor (IL-2Ralpha) shown to be a useful target for radioimmunotherapy are described. Synthesis of the organometallic linker precursors is accomplished by reaction of the corresponding bromo- or iodoaryl esters with bis(tributyltin) in the presence of a palladium catalyst. Subsequent conversion to the corresponding N-succinimidyl ester and labeling with 211At of two new linkers, N-succinimidyl 4-[211At]astato-3-methylbenzoate and N-succinimidyl N-(4-[211At]astatophenethyl)succinamate (SAPS), together with the previously reported N-succinimidyl 4-[211At]astatobenzoate and N-succinimidyl 3-[211At]astato-4-methylbenzoate, are each conjugated to Hu-anti-Tac. The plasma survival times of these conjugates are compared to those of directly iodinated (125I) Hu-anti-Tac. The N-succinimidyl N-(4-[211At]astatophenethyl)succinamate compound (SAPS) emerged from this assay as the most viable candidate for 211At-labeling of Hu-anti-Tac. SAPS, along with the directly analogous radio-iodinated reagent, N-succinimidyl N-(4-[125I]astatophenethyl)succinamate (SIPS), are evaluated in a biodistribution study along with directly iodinated (125I) Hu-anti-Tac. Blood clearance and biological accretion results indicate that SAPS is a viable candidate for further evaluation for radioimmunotherapy of cancer.

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.