Preparation of 212Pb-labeled monoclonal antibody using a novel 224Ra-based generator solution.

INTRODUCTION: Alpha-emitting radionuclides have gained considerable attention as payloads for cancer targeting molecules due to their high cytotoxicity. One attractive radionuclide for this purpose is 212Pb, which by itself is a ß-emitter, but acts as an in vivo generator for its short-lived α-emitting daughters. The standard method of preparing 212Pb-labeled antibodies requires handling and evaporation of strong acids containing high radioactivity levels by the end user. An operationally easier and more rapid process could be useful since the 10.6h half-life of 212Pb puts time constraints on the preparation protocol. In this study, an in situ procedure for antibody labeling with 212Pb, using a solution of the generator nuclide 224Ra, is proposed as an alternative protocol for preparing 212Pb-radioimmunoconjugates. METHODS: Radium-224, the generator radionuclide of 212Pb, was extracted from its parent nuclide, 228Th. Lead-212-labeling of the TCMC-chelator conjugated monoclonal antibody trastuzumab was carried out in a solution containing 224Ra in equilibrium with progeny. Subsequently, the efficiency of separating the 212Pb-radioimmunoconjugate from 224Ra and other unconjugated daughter nuclides in the solution using either centrifugal separation or a PD-10 desalting size exclusion column was evaluated and compared. RESULTS: Radiolabeling with 212Pb in 224Ra-solutions was more than 90% efficient after only 30min reaction time at TCMC-trastuzumab concentrations from 0.15mg/mL and higher. Separation of 212Pb-labeled trastuzumab from 224Ra using a PD-10 column was clearly superior to centrifugal separation. This method allowed recovery of approximately 75% of the 212Pb-antibody-conjugate in the eluate, and the remaining amount of 224Ra was only 0.9±0.8% (n=7). CONCLUSIONS: The current work demonstrates a novel method of producing 212Pb-based radioimmunoconjugates from a 224Ra-solution, which may be simpler and less time-consuming for the end user compared with the method established for use in clinical trials of 212Pb-TCMC-trastuzumab.

Dose escalation and dosimetry of first-in-human α radioimmunotherapy with 212Pb-TCMC-trastuzumab.

UNLABELLED: Our purpose was to study the safety, distribution, pharmacokinetics, immunogenicity, and tumor response of intraperitoneal (212)Pb-TCMC-trastuzumab (TCMC is S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra(2-carbamoylmethyl)cyclododecane) in patients with human epidermal growth factor receptor type 2 (HER-2)-expressing malignancy. METHODS: In a standard 3 + 3 phase 1 design for dose escalation, (212)Pb-TCMC-trastuzumab was delivered intraperitoneally less than 4 h after administration of trastuzumab (4 mg/kg intravenously) to patients with peritoneal carcinomatosis who had failed standard therapies. RESULTS: Five dosage levels (7.4, 9.6, 12.6, 16.3, and 21.1 MBq/m(2)) showed minimal toxicity at more than 1 y for the first group and more than 4 mo for others. The lack of substantial toxicity was consistent with the dosimetry assessments (mean equivalent dose to marrow, 0.18 mSv/MBq). Radiation dosimetry assessment was performed using pharmacokinetics data obtained in the initial cohort (n = 3). Limited redistribution of radioactivity out of the peritoneal cavity to circulating blood, which cleared via urinary excretion, and no specific uptake in major organs were observed in 24 h. Maximum serum concentration of the radiolabeled antibody was 22.9% at 24 h (decay-corrected to injection time) and 500 Bq/mL (decay-corrected to collection time). Non-decay-corrected cumulative urinary excretion was 6% or less in 24 h (2.3 half-lives). Dose rate measurements performed at 1 m from the patient registered less than 5µSv/h (using portable detectors) in the latest cohort, significantly less than what is normally observed using nuclear medicine imaging agents. Antidrug antibody assays performed on serum from the first 4 cohorts were all negative. CONCLUSION: Five dose levels of intraperitoneal (212)Pb-TCMC-trastuzumab treatment of patients with peritoneal carcinomatosis showed little agent-related toxicity, consistent with the dosimetry calculations.

Gene expression profiling upon (212) Pb-TCMC-trastuzumab treatment in the LS-174T i.p. xenograft model.

Recent studies have demonstrated that therapy with (212) Pb-TCMC-trastuzumab resulted in (1) induction of apoptosis, (2) G2/M arrest, and (3) blockage of double-strand DNA damage repair in LS-174T i.p. (intraperitoneal) xenografts. To further understand the molecular basis of the cell killing efficacy of (212) Pb-TCMC-trastuzumab, gene expression profiling was performed with LS-174T xenografts 24 h after exposure to (212) Pb-TCMC-trastuzumab. DNA damage response genes (84) were screened using a quantitative real-time polymerase chain reaction array (qRT-PCR array). Differentially regulated genes were identified following exposure to (212) Pb-TCMC-trastuzumab. These included genes involved in apoptosis (ABL, GADD45α, GADD45γ, PCBP4, and p73), cell cycle (ATM, DDIT3, GADD45α, GTSE1, MKK6, PCBP4, and SESN1), and damaged DNA binding (DDB) and repair (ATM and BTG2). The stressful growth arrest conditions provoked by (212) Pb-TCMC-trastuzumab were found to induce genes involved in apoptosis and cell cycle arrest in the G2/M phase. The expression of genes involved in DDB and single-strand DNA breaks was also enhanced by (212) Pb-TCMC-trastuzumab while no modulation of genes involved in double-strand break repair was apparent. Furthermore, the p73/GADD45 signaling pathway mediated by p38 kinase signaling may be involved in the cellular response, as evidenced by the enhanced expression of genes and proteins of this pathway. These results further support the previously described cell killing mechanism by (212) Pb-TCMC-trastuzumab in the same LS-174T i.p. xenograft. Insight into these mechanisms could lead to improved strategies for rational application of radioimmunotherapy using α-particle emitters.