Targeted radioimmunotherapy of triple negative breast cancer with CSPG4-specific 212Pb-labeled monoclonal antibody.

114 Objectives The overall prognosis for patients with triple negative breast cancer (TNBC) remains poor due to the aggressiveness of this breast cancer subtype and lack of effective targeted therapies. This information prompted us to develop a novel effective radioimmunotherapy (RIT) for the treatment of TNBC. Our strategy has been guided by the cancer stem cell hypothesis, which states that to be effective a therapy must eliminate both differentiated cancer cells and cancer initiating cells (CICs). CICs are thought to be integral to metastatic spread and disease recurrence, the major causes of TNBC patients’ morbidity and mortality. The tumor antigen chondroitin sulfate proteoglycan 4 (CSPG4) was selected as a target because of its high expression on both differentiated TNBC cells and TNBC CICs and its restricted distribution in normal tissues. CSPG4-specific monoclonal antibody (mAb) 225.28 was radiolabeled with 212Pb, which acts as an in vivo generator of highly cytotoxic 212Bi alpha-particles for use in targeted therapy. The resulting RIT agent was investigated with models of TNBC in vitro and in vivo. Methods mAb 225.28 was reacted with the chelate TCMC and radiolabeled with 212Pb obtained from an 8 mCi 224Ra/212Pb generator (Oak Ridge National Laboratory). In vitro Scatchard assays assessed the binding characteristics of the radioimmunoconjugate (RIC) 212Pb-mAb 225.28 with SUM159 and 2LMP TNBC cell lines. 212Pb-mAb 225.28 and irrelevant isotype-matched 212Pb-mAb F3-C25 were used to determine the effects of targeted/non-targeted RIT on clonogenic survival in vitro with SUM159 adherent cells and non-adherent tumorspheres containing enriched CICs. Biodistribution analyses of the RIC and irrelevant isotype-matched 125I-mAb F3-C25 were performed in nude mice bearing SUM159 or 2LMP xenograft tumors to determine the specificity of the targeted RIC in vivo. 212Pb-mAb 225.28 (3.8-13.0 µCi) and 212Pb-mAb F3-C25 (9.0 µCi) were used to assess the effects of targeted/non-targeted RIT on SUM159 tumor growth in vivo following i.v. injection of the RICs. Results 212Pb-mAb 225.28 was produced in high radiolabeling yield (>90%) and purity (>95%) as indicated by ITLC analysis. Scatchard analysis showed a Kd of 0.3-0.6 nM for 212Pb-mAb 225.28 with 2LMP and SUM159 cell lines. CSPG4 expression was notably higher on SUM159 cells than on 2LMP cells (125,000 and 9,100 sites/cell, respectively). SUM159 clonogenic survival in vitro was significantly inhibited by 212Pb-mAb 225.28 (IC50 = 0.53 ± 0.08 and 0.62 ± 0.07 µCi/mL for adherent and non-adherent tumorsphere cells, respectively) compared to 212Pb-F3-C25 (3.82 ± 0.22 and 1.25 ± 0.09 µCi/mL for adherent and tumorsphere cells, respectively) (p Conclusions 212Pb-mAb 225.28 specifically binds to TNBC cells; the extent of binding depends on the level of CSPG4 expression and on the amount of mAb. Furthermore 212Pb-mAb 225.28 is effective against both differentiated TNBC cells and TNBC CICs in vitro. Lastly, 212Pb-mAb 225.28 inhibits growth of a TNBC xenograft model in vivo. These results suggest future RIT studies with 212Pb-mAb 225.28 against TNBC are warranted. This research was supported in part by NIH R21CA173120.