CD30 as Target for Radioimmunotherapy (RIT) of Lymphoma.

Objectives: CD30 is overexpressed on the cell surface by a variety of malignant diseases including Hodgkin’s lymphoma (HL). It is cleaved from the cell surface releasing the soluble ectodomain (sCD30), which may interfere with a monoclonal antibody’s (MAb) ability to reach its target. Recently we identified two epitopes on membrane-associated CD30 that are missing on sCD30. To investigate the relevance of soluble antigen for CD30 mediated tumor targeting, in vitro, biodistribution and γ-imaging studies of three radiolabeled anti-CD30 MAbs (HeFi-1, T105, T408) targeting the CD30 antigen at different epitopes were performed.

Materials and Methods:

In vitro: HeFi-1 was labeled with ¹²⁵I and ¹¹¹In and incubated with CD30+ L540 cells. The pellet and supernatant was analyzed for cell associated radioactivity at various time points. For binding studies, HeFi-1, T105 and T408 were radiolabeled with ¹³¹I, incubated with recombinant human CD30/Fc, serum of L540 tumor bearing mice (MS) and L540 cell culture supernatant (CCS) and analyzed by HPLC-SEC.

In vivo: L540 tumor bearing nude mice were injected i.v. with dual-labeled (¹²⁵I and ¹¹¹In) HeFi-1, T105, or T408. At various timepoints tumors, blood and major organs were collected and analyzed for radioactivity. Serum samples were assayed for complex formation by HPLC-SEC. For γ-imaging studies L540 tumor bearing mice received radiolabeled HeFi–1, T105, or T408 and serial whole body gamma camera images were acquired.

Results and Conclusions:

Iodine vs. Indium:

¹¹¹In-HeFi–1 showed higher cell associated retention in vitro than ¹²⁵I-HeFi-1 as a function of time. In vivo, all anti-CD30 MAbs showed superior tumor accretion when radiolabeled with ¹¹¹In (25–35% ID/g at 72h; ¹²⁵I: 6–11%), probably due to internalization of the radioiodinated MAbs, intracellular catabolism and subsequent release of degraded products.

HPLC analyses:

HeFi-1 showed in vitro complex formation when incubated with CD30/Fc, MS and CCS, whereas T105 and T408 only bound CD30/Fc, indicating their inability to bind sCD30. In vivo, complex formation was observed for T105, although in less amounts compared to HeFi-1, suggesting that T105 binds to the membrane antigen, and the immune complex is subsequently cleaved and released from the cell surface. It also suggests that the T105 epitope may be shared between the membrane and the soluble antigen part, or undergoes conformational changes upon shedding. T408 was found to bind to the cell membrane without evidence of immune complex formation.

Biodistribution:

There were no significant differences in tumor and normal organ uptake between the three ¹¹¹In-labeled MAbs. T408 showed an accelerated blood clearance with 18% of the activity left at 72h compared to T105 (34%) and HeFi-1 (55%), probably due to the lack of complex formation, which may prolong MAb circulation. The rapid blood clearance of T408 results in a favorable tumor:blood (therapeutic) ratio at 72h with 4.3, compared to T105 (2.3) and HeFi-1 (1.9). In summary, we have shown that radiometals are more suitable for targeting CD30; we have also demonstrated the impact of soluble antigen on the pharmakokinetics of MAbs, which target CD30 at distinct epitopes. T408 with its lack of binding sCD30 and therefore rapid blood clearance and enhanced therapeutic ratio may be a promising and useful new candidate for RIT of CD30+ lymphoma.