[en] Introduction: Alpha particles possess an exquisite degree of cytotoxicity when employed for targeted α-particle therapy (TAT) or radioimmunotherapy (RIT). ²¹²Pb, which acts as an in vivo generator of the α-emitting nuclide ²¹²Bi has shown great promise in pre-clinical studies when used to label the HER2 binding antibody, trastuzumab. Currently, the first RIT clinical trial employing ²¹²Pb radiolabeled trastuzumab is in progress. This report provides detailed current protocol operations and steps that were generated for use in the clinical trial as well as the relevant pre-clinical experimentation, and describes in detail the labeling of proteins or peptides with ²¹²Pb as provided via a ²²⁴Ra based generator system. Methods: ²¹²Pb was eluted from the ²²⁴Ra/²¹²Pb generator using hydrochloric acid (2 M). The generator eluate was evaporated and digested with nitric acid (8 M) followed by extraction of the ²¹²Pb with dilute nitric acid (0.1 M). The dilute nitric acid solution of ²¹²Pb was used to label the immunoconjugate Trastuzumab-TCMC (2-(4-isothiocyanatobenzyl-1,4,7,10-tetraaza-1,4,7,10, tetra-(2-carbamonylmethyl)-cyclododecane) at pH 5.5. Results: Elution of ²¹²Pb from the generator was efficient yielding > 90% of available ²¹²Pb. Trastuzumab-TCMC was efficiently labeled with a radiochemical yield of 94% ± 4% (n = 7) by ITLC and an isolated yield of 73% ± 3% (n = 7). Conclusions: The results show the feasibility of generating radioimmunoconjugates and peptide conjugates for use as in vivo α generator systems in the clinic. The technology holds promise in applications involving the treatment of minimal disease such as micrometastases and residual tumor after surgical debulking, hematological cancers, infections, and compartmental cancers, such as ovarian cancer

[en] Radioisotopes of Pb(II) have been of some interest in radioimmunotherapy and radioimmunoimaging (RII). However, the absence of a kinetically stable bifunctional chelating agent for Pb(II) has hampered its use for these applications. ²⁰³Pb (T_1/2=52.02 h) has application potential in RII, with a γ-emission that is ideal for single photon emission computerized tomography, whereas ²¹²Pb (T_1/2=10 h) is a source of highly cytotoxic α-particles via its decay to its ²¹²Bi (T_1/2=60 min) daughter. The synthesis of the novel bifunctional chelating agent 2-(4-isothiocyanotobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra-(2-carbamoyl methyl)-cyclododecane (4-NCS-Bz-TCMC) is reported herein. The Pb[TCMC]²⁺ complex was less labile to metal ion release than Pb[DOTA]^2- at pH 3.5 and below in isotopic exchange experiments. In addition to increased stability to Pb²⁺ ion release at low pH, the bifunctional TCMC ligand was found to have many other advantages over the bifunctional 1,4,7,10-tetraazacyclodocane-1,4,7,10-tetraacetic acid (DOTA) ligand. These include a shorter and more straightforward synthetic route, a more efficient conjugation reaction to a monoclonal antibody (mAb), with a higher chelate to protein ratio, a higher percent immuroreactivity, and a more efficient radiolabeling reaction of the mAb-ligand conjugate with ²⁰³Pb

[en] A simple and rapid procedure was developed for purification of cyclotron produced ⁸⁶Y via the ⁸⁶Sr(p,n) ⁸⁶Y reaction. A commercially available Sr(II) selective resin was used to separate ⁸⁶Y from the cyclotron irradiated Sr(II) target with a recovery of the enriched Sr(II) target while yielding a 75-80% recovery of ⁸⁶Y suitable for radiolabeling either proteins or peptides. To demonstrate the utility of this methodology, the anti-HER2 monoclonal antibody Herceptin^TM was radiolabeled with the purified ⁸⁶Y and compared to ¹¹¹In labeled Herceptin^TM. The biodistribution study demonstrated that ¹¹¹In-Herceptin^TM, while a suitable surrogate for ⁹⁰Y in the major organs, did not parallel the uptake of ⁸⁶Y-Herceptin^TM in the bone, and thus may not accurately predict the level of ⁹⁰Y accumulation in the bone for clinical RIT applications. This result exemplifies the requirement of employing appropriate matched pair isotopes for imaging and therapy to insure that dosimetry considerations may be addressed accurately

[en] Copper offers a unique selection of radioisotopes (⁶⁰Cu, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, and ⁶⁷Cu) with half-lives ranging from 9.8 min to 61.9 h suitable for imaging and/or radiotherapy. In peptide/antibody targeted radiotherapy one of the most studied chelating agents for copper, 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), has been employed in clinical trials, but transchelation to ceruloplasmin and/or superoxide dismutase in vivo has been noted. In this study, a series of novel hexadentate chelating agents based on N,N',N''-tris(2-pyridylmethyl)-1,3,5-cis,cis,-triaminocyclohexane (tachpyr) have been synthesized and the serum stability of their copper complexes was evaluated as compared to TETA. Copper complexes of tachpyr modified at the 3, 4, or 5 position or with replacement of pyridine by imidazole have serum stability comparable to Cu[TETA]. When the complexes were cross-challenged, Cu[TETA] versus tachpyr or 1,3,5-cis,cis,-triaminocyclohexane- N,N',N''-tris-(2-methyl-(N-methylimidazole)) (IM), tachpyr and IM appear to have superior copper chelation ability to TETA. When challenged by a large excess of non-radioactive copper, copper exchange with the tachpyr radio-copper complex was observed. However, tachpyr clearly exhibited a significant preference for Cu(II) over Zn(II) or Fe(III). Therefore, tachpyr, 1,3,5-cis,cis,-triaminocyclohexane-N,N',N''-tri-(3-methyl -2-methylpyridineimine) (tachpyr(3-Me)), 1,3,5-cis,cis,-triaminocyclohexane-N,N',N''-tri-(4-methyl -2-methylpyridineimine) (tachpyr(4-Me)), 1,3,5-cis,cis,-triaminocyclohexane-N,N',N''-tri-(5-methyl -2-methylpyridineimine) (tachpyr(5-Me)) and IM easily form copper complexes with high stability. These novel chelating agents provide an attractive lead for creation of new copper radiopharmaceuticals for diagnosis and therapy applications

[en] A simple, non-radioactive method for the determination of ligand-to-protein ratio (L/P) for novel ligand-antibody conjugates has been developed based on an exchange equilibrium with the purple Cu(II) complex of arsenazo III. The method requires a UV/Vis spectrometer and has been verified for monoclonal antibody Herceptin conjugates of a variety of ligand modalities, including common macrocyclic compounds NOTA and TETA, and with a new bifunctional tachpyridine (1H-Pyrrole-1-butanamide,N-[4-[[(1α,3α,5α)-3,5-bis[(2-pyridinylmethyl) amino]cyclohexyl](2-pyridinylmethyl)amino]butyl]-2,5-dihydro-2, 5-dioxo-(9CI)). The spectroscopically derived values for L/P were verified by titration of the ligand-antibody conjugate with ⁶⁴Cu. In each case, the value obtained by UV/Vis spectroscopy matches that found by radiolabeling. The method is rapid, taking less than 30 minutes with each ligand in this study.

[en] The studies reported herein present the first in vitro and in vivo comparison of radioimmunoconjugates (RIC) radiolabeled with ¹⁷⁷Lu using the acyclic CHX-A''-DTPA ligand and the macrocyclic ligands, C-DOTA and PA-DOTA. The in vivo studies include pharmacokinetics and biodistribution of the formed ¹⁷⁷Lu-labeled immunoconjugates in a tumor bearing murine model with engineered monoclonal antibody HuCC49ΔCH2. The in vitro analysis indicated that the CHX-A'' RIC was superior with respect to immunoreactivity, radiolabeling with ¹⁷⁷Lu, and specific activity. The in vivo pharmacokinetic data by itself indicated that the Lu(III)-PA-DOTA complex may not be as stable as Lu(III) complexes with CHX-A'' or C-DOTA. All three RICs demonstrated tumor targeting of human colon carcinoma xenografts in athymic mice. In these biodistribution studies, there appears to be no overall pattern or trend of one RIC over the other two. Based on these in vitro and in vivo studies, the CHX-A'' DTPA ligand should be considered a suitable bifunctional chelate for the radiolabeling of monoclonal antibodies with ¹⁷⁷Lu for radioimmunotherapy applications

[en] Recent studies have demonstrated that therapy with ²¹²Pb-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 ²¹²Pb-TCMC-trastuzumab, gene expression profiling was performed with LS-174T xenografts 24 h after exposure to ²¹²Pb-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 ²¹²Pb-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 ²¹²Pb-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 ²¹²Pb-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 ²¹²Pb-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. The apoptotic response and associated gene modulations have not been clearly defined following exposure of cells to α-particle radioimmunotherapy (RIT). Gene expression profiling was performed with LS-174T i.p. (intraperitoneal) xenografts after exposure to ²¹²Pb-TCMC-trastuzumab. Differentially regulated 22 genes were identified following the stressful growth arrest conditions provoked by ²¹²Pb-TCMC-trastuzumab, providing an informative approach toward understanding the molecular basis of tumor biology in response to α-particle radiation and leading to improved strategies for RIT using α-particle emitters. This study provides data which is among the first to describe in detail, the cellular response to α-particle irradiation in vivo

[en] Purpose: To elucidate the mechanism of the therapeutic efficacy of targeted α-particle radiation therapy using ²¹²Pb-TCMC-trastuzumab together with gemcitabine for treatment of disseminated peritoneal cancers. Methods and Materials: Mice bearing human colon cancer LS-174T intraperitoneal xenografts were pretreated with gemcitabine, followed by ²¹²Pb-TCMC-trastuzumab and compared with controls. Results: Treatment with ²¹²Pb-TCMC-trastuzumab increased the apoptotic rate in the S-phase-arrested tumors induced by gemcitabine at earlier time points (6 to 24 hours). ²¹²Pb-TCMC-trastuzumab after gemcitabine pretreatment abrogated G2/M arrest at the same time points, which may be associated with the inhibition of Chk1 phosphorylation and, in turn, cell cycle perturbation, resulting in apoptosis. ²¹²Pb-TCMC-trastuzumab treatment after gemcitabine pretreatment caused depression of DNA synthesis, DNA double-strand breaks, accumulation of unrepaired DNA, and down-regulation of Rad51 protein, indicating that DNA damage repair was blocked. In addition, modification in the chromatin structure of p21 may be associated with transcriptionally repressed chromatin states, indicating that the open structure was delayed at earlier time points. Conclusion: These findings suggest that the cell-killing efficacy of ²¹²Pb-TCMC-trastuzumab after gemcitabine pretreatment may be associated with abrogation of the G2/M checkpoint, inhibition of DNA damage repair, and chromatin remodeling

[en] Cetuximab is a recombinant, human/mouse chimeric IgG₁ monoclonal antibody that binds to the epidermal growth factor receptor (EGFR/HER1). Cetuximab is approved for the treatment of patients with HER1-expressing metastatic colorectal cancer. Limitations in currently reported radiolabeled cetuximab for PET applications prompted the development of ⁸⁶Y-CHX-A''-DTPA-cetuximab as an alternative for imaging HER1-expressing cancer. ⁸⁶Y-CHX-A''-DTPA-cetuximab can also serve as a surrogate marker for ⁹⁰Y therapy. Bifunctional chelate, CHX-A''-DTPA was conjugated to cetuximab and radiolabeled with ⁸⁶Y. In vitro immunoreactivity was assessed in HER1-expressing A431 cells. In vivo biodistribution, PET imaging and noncompartmental pharmacokinetics were performed in mice bearing HER1-expressing human colorectal (LS-174T and HT29), prostate (PC-3 and DU145), ovarian (SKOV3) and pancreatic (SHAW) tumor xenografts. Receptor blockage was demonstrated by coinjection of either 0.1 or 0.2 mg cetuximab. ⁸⁶Y-CHX-A''-DTPA-cetuximab was routinely prepared with a specific activity of 1.5-2 GBq/mg and in vitro cell-binding in the range 65-75%. Biodistribution and PET imaging studies demonstrated high HER1-specific tumor uptake of the radiotracer and clearance from nonspecific organs. In LS-174T tumor-bearing mice injected with ⁸⁶Y-CHX-A''-DTPA-cetuximab alone, ⁸⁶Y-CHX-A''-DTPA-cetuximab plus 0.1 mg cetuximab or 0.2 mg cetuximab, the tumor uptake values at 3 days were 29.3 ± 4.2, 10.4 ± 0.5 and 6.4 ± 0.3%ID/g, respectively, demonstrating dose-dependent blockage of the target. Tumors were clearly visualized 1 day after injecting 3.8-4.0 MBq ⁸⁶Y-CHX-A''-DTPA-cetuximab. Quantitative PET revealed the highest tumor uptake in LS-174T (29.55 ± 2.67%ID/cm³) and the lowest tumor uptake in PC-3 (15.92 ± 1.55%ID/cm³) xenografts at 3 days after injection. Tumor uptake values quantified by PET were closely correlated (r ² = 0.9, n = 18) with values determined by biodistribution studies. This study demonstrated the feasibility of preparation of high specific activity ⁸⁶Y-CHX-A''-DTPA-cetuximab and its application for quantitative noninvasive PET imaging of HER1-expressing tumors. ⁸⁶Y-CHX-A''-DTPA-cetuximab offers an attractive alternative to previously labeled cetuximab for PET and further investigation for clinical translation is warranted. (orig.)

[en] Introduction: Despite the great potential of targeted α-radioimmunotherapy (RIT) as demonstrated by pre-clinical and clinical trials, limited progress has been made on the improvement of chelation chemistry for ²¹²Bi and ²¹³Bi. A new bifunctional ligand 3p-C-NETA was evaluated for targeted α RIT using ²¹²Bi and ²¹³Bi. Methods: Radiolabeling of 3p-C-NETA with ^205/6Bi, a surrogate of ²¹²Bi and ²¹³Bi, was evaluated at pH 5.5 and room temperature. In vitro stability of the ^205/6Bi-3p-C-NETA-trastuzumab conjugate was evaluated using human serum (pH 7, 37 °C). Immunoreactivity and specific activity of the ^205/6Bi-3p-C-NETA-trastuzumab conjugate were measured. An in vivo biodistribution study was performed to evaluate the in vivo stability and tumor targeting properties of the ^205/6Bi-3p-C-NETA-trastuzumab conjugate in athymic mice bearing subcutaneous LS174T tumor xenografts. Result: The 3p-C-NETA-trastuzumab conjugate was extremely rapid in complexing with ^205/6Bi, and the corresponding ^205/6Bi-3p-C-NETA-trastuzumab was stable in human serum. ^205/6Bi-3p-C-NETA-trastuzumab was prepared with a high specific activity and retained immunoreactivity. ^205/6Bi-3p-C-NETA-trastuzumab conjugate displayed excellent in vivo stability and targeting as evidenced by low normal organ and high tumor uptake. Conclusion: The results of the in vitro and in vivo studies indicate that 3p-C-NETA is a promising chelator for RIT applications using ²¹²Bi and ²¹³Bi. Further detailed in vivo evaluations of 3p-C-NETA for targeted α RIT are warranted