Irwin Fand

The effect of antibody protein dose on the uniformity of tumor distribution of radioantibodies : an autoradiographic study

SummaryThe inaccessibility of radiolabeled antibody to poorly vascularized regions of solid tumors may reduce the therapeutic efficacy of these macromolecules. Theoretical mathematical models have predicted that increasing the protein dose administered would reduce the heterogeneity of radioantibody distribution. This investigation was undertaken to evaluate this hypothesis in experimental animal models. We have utilized the technique of macroautoradiography to demonstrate an increase in tumor penetration of the lower-affinity125I-labeled NP-4 or higher-affinity Immu-14 anti-carcinoembryonic antigen (anti-CEA) mAbs into small (60.25—0.4 g) and large (0.8–1.5 g) GW-39 and LS174T human colonic xenografts, grown subcutaneously in the nude mouse, when 400 µg unlabeled antibody is administered simultaneously with 10 µg (100 µCi) radioantibody. Further increases in protein to 800 µg result in a reduction in total tumor uptake of the antibody. These differences in mAb distribution could be visualized as early as 1 day after antibody injection. Improved mAb penetration was also achieved for the Mu-9 anti-CSAp (anti-mucin) antibody using 800 µg unlabeled antibody. An irrelevant antibody (AFP-7-31) was found to be homogeneously distributed 3 days after injection, even at a low protein dose. Attempts to improve mAb penetration by increasing the protein dose in the GS-2 colorectal tumor, a model that has low NP-4 accretion as a result physiological barriers separating antibody from antigen, were not successful. These results suggest that a more homogeneous distribution of radioantibody can be achieved by carefully selecting a dose of unlabeled antibody to coadminister. Work is currently in progress to determine the effect of improved tumor distribution of radioantibody on the therapeutic potential of a single dose of radioantibody.

Tumor targeting in a murine tumor xenograft model with the (sFv′)2 divalent form of anti-c-erbB-2 single-chain Fv

This investigation has utilized novel forms of the single-chain Fv (sFv), wherein a cysteine-containing peptide has been fused to the sFv carboxyl terminus to facilitate disulfide bonding or specific crosslinking of this sFv′ to make divalent (sFv′)2. The 741F8 anti-c-erbB-2 monoclonal antibody was used as the basis for construction of 741F8 sFv, from which the sFv′ and (sFv′)2 derivatives were prepared. Recombinant c-erbB-2 extracellular domain (ECD) was prepared in CHO cells and the bivalency of 741F8 (sFv′)2 demonstrated by its complex formation with ECD. The tumor binding properties of125I-labeled anti-c-erbB-2 741F8 sFv, sFv′, and (sFv′)2 were compared with radiolabeled antidigoxin 26-10 sFv′ and (sFv′)2 controls. Following intravenous administration of radiolabeled species to severe combined immune-deficient (SCID) mice bearing SK-OV-3 tumors (which overexpress c-erbB-2), blood and organ samples were obtained as a function of time over 24 h. Comparative analysis of biodistribution and tumor-to-organ ratios demonstrated the 741F8 sFv, sFv′, and (sFv′)2 had excellent specificity for tumors, which improved with time after injection. This contrasted with nonspecific interstitial pooling in tumors observed with the 26-10 sFv, sFv′, and (sFv′)2, which decreased with time after administration. Tumor localization was significantly better for disulfide or peptide crosslinked 741F8 (sFv′)2 having Gly4Cys tails than for monovalent 741F8 sFv′ or Fab. The superior properties of the 741F8 (sFv′)2 in targeting SK-OV-3 tumors in SCID mice suggests the importance of further investigations of divalent sFv analogs for immunotargeting.

In Vivo Radioimmunodetection of Amyloid Deposits in Experimental Amyloidosis

Two rat IgG monoclonal antibodies (MAbs) to mouse AA protein have been used as reagents for an ELISA assay for SAA, for the demonstration of tissue deposits by the immunoperoxidase method, and for the definition of AA polymorphs in solubilized amyloidotic tissue specimens run onto two-dimensional (2D) gels and probed as Western Blots. Both MAbs localize to tissue deposits in vivo when labelled (MAb*) with I125 or I123 and injected into colchicine-pretreated amyloidotic mice, assessed by (a) whole-body autoradiography (WBAR) (b) external photoscanning (c) tissue autoradiography of perfused organs. Serum t 1/2 of MAb* in amyloidotic animals was greatly accelerated compared to controls, and sustained concentration of label was found in spleen, liver, and kidney quantitated by organ counts up to 96 h post injection. MAb* label copurified with amyloid fibrils up to 10-fold over saline-soluble proteins and residue following homogenization in distilled water and acid extraction; purified fibrils contained AA protein and MAb* heavy and light chain visualized on 2D gels and by autoradiography. Animals were also injected with a mixture of the two MAbs, one labeled with Indium111-DTPA (126 μCi), the other with I125 (6 μCi) and serially scanned over a 48 h period. Fractionation of serum taken after injection showed both isotopes to be present as uncomplexed IgG in blood. WBAR was performed at 48 h and at a time at which I125/In111 radioactivity was calculated to be 127/1; autoradiograms were similar and confirmed localization of both isotopes to tissue deposits in amyloidotic animals. These studies provide evidence that MAbs to amyloid subunit proteins may be useful re-agents for the in vivo radioimmunodetection of some of the amyloid diseases.

Whole-body autoradiographic localization of [3H]phencyclidine and its metabolites in mice

When evaluated by whole-body autoradiography (WBAR) and quantitative densitometry, [3H]phencyclidine (PCP) equivalents were found to be removed rapidly from blood, after a single iv dose in mice, and avidly taken up as early as 1 min after dosage by glandular tissues including thyroid, salivary glands, pancreas, pituitary and, most prominently, by stomach mucosa. Stomach:blood [3H]PCP concentration ratios showed that rapid secretion of [3H]PCP from mucosa to the stomach contents occurred within 2 min after dosing. During early intervals, chromatographic analysis of tissue sections demonstrated that PCP was present in brain, liver, and gut primarily in its unaltered chemical form. Mice killed at 60 and 120 min showed persistently high levels of [3H]PCP equivalents within the stomach and intestines, these levels being the highest of all other tissues densitometrically measured. The early time course and magnitude of [3H]PCP uptake by stomach glandular mucosa strongly suggests that cycling of PCP occurs principally through gastroenteric recirculation. Very striking was the high concentration of [3H]PCP radioactivity observed within the adrenal as early as 5 min. The concentration of [3H]PCP equivalents in pituitary, choroid plexus, cortex, hippocampus, and thalamus was highest at 1-20 min following injection. Application of high-resolution quantitative WBAR was found to be a useful tool in the study of the biodistribution of labeled PCP, especially during very early post-treatment time points where alternative tissue counting techniques would not be feasible.