DnaA occupies only the three highest-affinity binding sites in E. coli oriC throughout most of the cell cycle. Immediately prior to initiation of chromosome replication, DnaA interacts with additional recognition sites, resulting in localized DNA-strand separation. These two DnaA-oriC complexes formed during the cell cycle are functionally and temporally analogous to yeast ORC and pre-RC. After initiation, SeqA binds to hemimethylated oriC, sequestering oriC while levels of active DnaA are reduced, preventing reinitiation. In this paper, we investigate how resetting of oriC to the ORC-like complex is coordinated with SeqA-mediated sequestration. We report that oriC resets to ORC during sequestration. This was possible because SeqA blocked DnaA binding to hemimethylated oriC only at low-affinity recognition sites associated with GATC but did not interfere with occupation of higher-affinity sites. Thus, during the sequestration period, SeqA repressed pre-RC assembly while ensuring resetting of E. coli ORC.

Purpose: Study distribution, pharmacokinetics, and safety of intraperitoneal (IP) 212 Pb-TCMC-trastuzumab in patients with HER-2-expressing malignancy. Experimental Design: IP 212 Pb-TCMC-trastuzumab was delivered, after 4 mg/kg intravenous (IV) trastuzumab, to 3 patients with HER-2-expressing cancer who had failed standard therapies. Patients were monitored for toxicity and pharmacokinetics/dosimetry parameters. Results: Imaging studies after 0.2 mCi/m 2 (7.4 MBq/m 2 ) show little redistribution out of the peritoneal cavity and no significant uptake in major organs. Peak blood level of the radiolabeled antibody, determined by decay corrected counts, was < 23% injected dose at 63 hours; maximum blood radioactivity concentration was 6.3nCi/mL at 18 hours. Cumulative urinary excretion was £ 6% in 2.3 half-lives. The maximum external exposure rate immediately post-infusion at skin contact over the abdomen averaged 7.67 mR/h and dropped to 0.67 mR/h by 24 hours. The exposure rates at the other positions monitored (axilla, chest, and femur) decreased as a function of distance from the abdomen. The data points correlate closely with 212 Pb physical decay (T 1/2 = 10.6 hours). Follow-up >6 months showed no evidence of agent-related toxicity. Conclusions: Pharmacokinetics and imaging after 0.2 mCi/m 2 IP 212 Pb-TCMC-trastuzumab in patients with HER-2-expressing malignancy showed minimal distribution outside the peritoneal cavity, £ 6% urinary excretion, and good tolerance.

Our purpose was to study the safety, distribution, pharmacokinetics, immunogenicity and tumor response of intraperitoneal (IP) 212Pb-TCMC-trastuzumab (TCMC is S-2-(4-isothiocyantobenzl)-1, 4, 7, 10-tetraaza-1, 4, 7, 10=tetra (2-carbamoylmethl) cyclododecane) in patients with HER-2 expressing malignancy. Methods In a standard 3+3 Phase 1 design for dose escalation, 212Pb-TCMC-trastuzumab was delivered IP less than 4 hours after giving 4mg/kg IV trastuzumab to patients with peritoneal carcinomatosis who had failed standard therapies. Results Five dosage levels (7.4, 9.6, 12.6, 16.3, 21.1 MBq/m2) showed minimal toxicity at >1 year for the first group and >4 months 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 was observed in 24 hours. Maximum serum concentration of the radiolabeled antibody was 22.9% at 24h (decay corrected to injection time) and 500 Bq/mL (decay corrected to collection time). Non-decay corrected cumulative urinary excretion was ≤6% in 24h (2.3 half lives). Dose rate measurements performed at 1m from the patient registered less than 5μSv/hr (using portable detectors) in the latest cohort, significantly less than what is normally observed using nuclear medicine imaging agents. Anti-drug antibody assays performed on serum from the first 4 cohorts were all negative. Conclusions Five dose levels of IP 212Pb-TCMC-trastuzumab treatment of patients with peritoneal carcinomatosis showed little agent related toxicity, consistent with the dosimetry calculations.

Eukaryotic initiator proteins form origin recognition complexes (ORCs) that bind to replication origins during most of the cell cycle and direct assembly of prereplication complexes (pre-RCs) before the onset of S phase. In the eubacterium Escherichia coli, there is a temporally similar nucleoprotein complex comprising the initiator protein DnaA bound to three high-affinity recognition sites in the unique origin of replication, oriC. At the time of initiation, this high-affinity DnaA-oriC complex (the bacterial ORC) accumulates additional DnaA that interacts with lower-affinity sites in oriC, forming a pre-RC. In this paper, we investigate the functional role of the bacterial ORC and examine whether it mediates low-affinity DnaAoriC interactions during pre-RC assembly. We report that E. coli ORC is essential for DnaA occupation of low-affinity sites. The assistance given by ORC is directed primarily to proximal weak sites and requires oligomerization-proficient DnaA. We propose that in bacteria, DnaA oligomers of limited length and stability emerge from single highaffinity sites and extend toward weak sites to facilitate their loading as a key stage of prokaryotic pre-RC assembly.R egulating chromosome duplication requires precisely timed formation of nucleoprotein complexes that comprise initiator proteins bound to replication origins and that direct assembly of new replisomes (1-6). Among the best-studied examples of such nucleoprotein complexes are the origin recognition complexes (ORCs) bound to origins in budding yeast (7,8), and the complexes formed by DnaA binding to the unique origin of chromosomal replication, oriC, in Escherichia coli (6, 9). Yeast ORC subunits share structural motifs with DnaA as well as archeal Orc1 (9, 10), and all are members of the AAAϩ family of ATPases (11). This structural conservation among initiator proteins suggests the intriguing possibility that mechanisms used by all cell types to initiate DNA synthesis could be fundamentally similar (12).Examination of the binding patterns of initiator proteins to origins during the cell cycle (5,13,14) has revealed that in addition to structural similarities, there are temporal similarities in nucleoprotein complex formation at eukaryotic and prokaryotic replication origins. Yeast ORCs bind to replication origins throughout the cell cycle and recruit additional initiator proteins needed to form the prereplicative complexes (pre-RCs) that load helicase and unwind origin DNA before entry into S phase (7,8,14,15). In E. coli, a temporally similar nucleoprotein complex is formed by DnaA binding to three high-affinity (K d Ͻ 200 nM), 9-bp recognition sites (R1, R2, and R4) within oriC (Fig. 1); like yeast ORC, this binding persists throughout the majority of the cell cycle (13,16,17), except at the time of initiation, when additional initiator DnaA binds to lower-affinity (K d Ͼ 200 nM) sites in oriC (13, 18). The additional DnaA causes localized strand separation within an AT-rich, 13-mer repeat region that is adjacent to the left side of t...

Purpose:One-year monitoring of patients receiving intraperitoneal (IP) 212Pb-TCMC-trastuzumab to provide long-term safety and outcome data. A secondary objective was to study 7 tumor markers for correlation with outcome.Methods:Eighteen patients with relapsed intra-abdominal human epidermal growth factor receptor-2 expressing peritoneal metastases were treated with a single IP infusion of 212Pb-TCMC-trastuzumab, delivered <4 h after 4 mg/kg IV trastuzumab. Seven tumor markers were studied for correlation with outcome.Results:Six dose levels (7.4, 9.6, 12.6, 16.3, 21.1, 27.4 MBq/m2) were well tolerated with early possibly agent-related adverse events being mild, transient, and not dose dependent. These included asymptomatic, abnormal laboratory values. No late renal, liver, cardiac, or other toxicity was noted up to 1 year. There were no clinical signs or symptoms of an immune response to 212Pb-TCMC-trastuzumab, and assays to detect an immune response to this conjugate were negative for all tested. Tumor marker studies in ovarian cancer patients showed a trend of decreasing Cancer antigen 72-4 (CA 72-4) aka tumor-associated glycoprotein 72 (TAG-72) and tumor growth with increasing administered radioactivity. Other tumor markers, including carbohydrate antigen (CA125), human epididymis protein 4 (HE-4), serum amyloid A (SAA), mesothelin, interleukin-6 (IL-6), and carcinoembryonic antigen (CEA) did not correlate with imaging outcome.Conclusions:IP 212Pb-TCMC-trastuzumab up to 27 MBq/m2 seems safe for patients with peritoneal carcinomatosis who have failed standard therapies. Serum TAG-72 levels better correlated to imaging changes in ovarian cancer patients than the more common tumor marker, CA125.

SummaryPrior to initiating DNA synthesis, Escherichia coli oriC switches from ORC, comprising initiator DnaA bound at three high-affinity sites, to pre-RC, when additional DnaA molecules interact with low-affinity sites. Two types of low-affinity sites exist: R boxes that bind DnaA-ATP and DnaA-ADP with equal affinity, and I-sites with a three-to fourfold preference for DnaA-ATP. To assess the regulatory role of weak DnaA interactions during pre-RC assembly in vivo, we compared the behaviour of plasmid-borne wild-type oriC with mutants having an increased or decreased number of DnaA-ATP discriminatory I-sites. Increasing the number of discriminatory sites by replacing R5M with I2 inactivated extrachromosomal oriC function. Mutants with no discriminatory sites perturbed host growth and rapidly replaced wild-type chromosomal oriC, but normal function returned if one I-site was restored at either the I2, I3 or R5M position. These observations are consistent with assembly of E. coli pre-RC in vivo from mixtures of DnaA-ATP and DnaA-ADP, with I-site interactions coupling pre-RC assembly to DnaA-ATP levels.

Peptide receptor radiotherapy (PRRT) with somatostatin analogs has been successfully utilized as a treatment for somatostatin overexpressing tumors for years. Treatment of neuroendocrine tumors (NETs) with the beta particle emitter 177 Lu-DOTATATE is currently considered the standard of care for subjects with gastroenteropancreatic NETs (GEP-NETs). Despite the success of 177 Lu-DOTATATE, there remains significant room for improvement in terms of both safety and efficacy. Targeted alpha-emitter therapy with isotopes such as lead-212 ( 212 Pb) has the potential to improve both. Herein, we present the preliminary results of the phase 1 first-in-human dose-escalation trial evaluating 212 Pb-DOTAMTATE in patients with somatostatin receptor positive NETs.Methods: A total of 20 subjects with histologically confirmed NETs, prior positive somatostatin analogue scans, and no prior history of 177 Lu/ 90 Y/ 111 In PRRT, with different primary sites of the disease, were enrolled. Treatment began with single ascending doses of 212 Pb-DOTAMTATE, with subsequent cohorts receiving an incremental 30% dose increase, which was continued until a tumor response or a dose-limiting toxicity was observed. This was followed by a multiple ascending dose regimen. The recommended phase 2 dose (RP2D) regimen consisted of 4 cycles of 2.50 MBq/kg (67.6 µCi/kg) of 212 Pb-DOTAMTATE administrated at 8-week intervals, intravenously.Results: Ten subjects received the highest dose of 2.50 MBq/kg/cycle (67.6 µCi/kg/cycle). Treatment was well tolerated, with the most common treatment-emergent adverse events (TEAEs) being nausea, fatigue, and alopecia.No serious TEAEs were related to the study drug, and no subjects required treatment delay or a dose reduction. An objective radiological response (ORR) of 80% was observed for the first 10 subjects treated at the RP2D. Conclusion:Targeted alpha therapy with 212 Pb-DOTAMTATE has been shown to be well-tolerated.Preliminary efficacy results are highly promising. If these results are confirmed in a larger, multicenter clinical trial, it would provide a substantial benefit over currently FDA approved therapies for patients with metastatic or inoperable SSTR-expressing NETs regardless of the grade and location of the primary tumor.

Background and PurposeWe assessed the contribution of antibody internalization in the efficacy and toxicity of intraperitoneal α-radioimmunotherapy (RIT) of small volume carcinomatosis using 212Pb-labeled monoclonal antibodies (mAbs) that target HER2 (internalizing) or CEA (non-internalizing) receptors.Materials and MethodsAthymic nude mice bearing 2–3 mm intraperitoneal tumor xenografts were intraperitoneally injected with similar activities (370, 740 and 1480 kBq; 37 MBq/mg) of 212Pb-labeled 35A7 (anti-CEA), trastuzumab (anti-HER2) or PX (non-specific) mAbs, or with equivalent amounts of unlabeled mAbs, or with NaCl. Tumor volume was monitored by bioluminescence and survival was reported. Hematologic toxicity and body weight were assessed. Biodistribution of 212Pb-labeled mAbs and absorbed dose-effect relationships using MIRD formalism were established.ResultsTransient hematological toxicity, as revealed by white blood cells and platelets numbering, was reported in mice treated with the highest activities of 212Pb-labeled mAbs. The median survival (MS) was significantly higher in mice injected with 1.48 MBq of 212Pb-35A7 (non-internalizing mAbs) (MS = 94 days) than in animals treated with the same activity of 212Pb-PX mAbs or with NaCl (MS = 18 days). MS was even not reached after 130 days when follow-up was discontinued in mice treated with 1.48 MBq of 212Pb-trastuzumab. The later efficacy was unexpected since final absorbed dose resulting from injection of 1.48 MBq, was higher for 212Pb-35A7 (35.5 Gy) than for 212Pb-trastuzumab (27.6 Gy). These results also highlight the lack of absorbed dose-effect relationship when mean absorbed dose was calculated using MIRD formalism and the requirement to perform small-scale dosimetry.ConclusionsThese data indicate that it might be an advantage of using internalizing anti-HER2 compared with non-internalizing anti-CEA 212Pb-labeled mAbs in the therapy of small volume xenograft tumors. They support clinical investigations of 212Pb-mAbs RIT as an adjuvant treatment after cytoreductive surgery in patients with peritoneal carcinomatosis.