No abstract

A metal-chelating polymer (MCP) with a polyglutamide (PGlu) backbone presenting on average 13 DOTA (tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelators for complexing In orLu and 10 polyethylene glycol (PEG) chains to minimize liver and spleen uptake was conjugated to antiepidermal growth factor receptor (EGFR) monoclonal antibody (mAb), panitumumab. Because panitumumab-MCP may be dual-labeled with In andLu for SPECT, or radioimmunotherapy (RIT) exploiting the Auger electrons or β-particle emissions, respectively, we propose that panitumumab-MCP could be a useful theranostic agent for EGFR-positive PnCa. Bioconjugation was achieved by reaction of a hydrazine nicotinamide (HyNIC) group on the MCP with an aryl aromatic aldehyde introduced into panitumumab by reaction with succinimidyl-4-formylbenzamide (S-4FB). The conjugation reaction was monitored by measurement of the chromophoric bis-aryl hydrazone bond formed (ε = 24 500 M cm) to achieve two MCPs/panitumumab. Labeling of panitumumab-MCP with In orLu demonstrated that masses as small as 0.1 μg were labeled to >90% labeling efficiency (L.E.) and a specific activity (SA) of >70 MBq/μg. Panitumumab-DOTA incorporating two DOTA per mAb was labeled with In orLu to a maximum SA of 65 MBq/μg and 46 MBq/μg, respectively. Panitumumab-MCP-Lu exhibited saturable binding to EGFR-overexpressing MDA-MB-468 human breast cancer cells. The K for binding of panitumumab-MCP-Lu to EGFR (2.2 ± 0.6 nmol/L) was not significantly different than panitumumab-DOTA-Lu (1.0 ± 0.4 nmol/L). In andLu were stably complexed to panitumumab-MCP. Panitumumab-MCP-In exhibited similar whole body retention (55-60%) as panitumumab-DOTA-In in NOD-scid mice up to 72 h postinjection (p.i.) and equivalent excretion of radioactivity into the urine and feces. The uptake of panitumumab-MCP-In in most normal tissues in NOD-scid mice with EGFR-positive PANC-1 human pancreatic cancer (PnCa) xenografts at 72 h p.i. was not significantly different than panitumumab-DOTA-In, except for the liver which was 3-fold greater for panitumumab-MCP-In. Tumor uptake of panitumumab-MCP-In (6.9 ± 1.3%ID/g) was not significantly different than panitumumab-DOTA-In (6.6 ± 3.3%ID/g). Tumor uptake of panitumumab-MCP-In and panitumumab-DOTA-In were reduced by preadministration of excess panitumumab, suggesting EGFR-mediated uptake. Tumor uptake of nonspecific IgG-MCP (5.4 ± 0.3%ID/g) was unexpectedly similar to panitumumab-MCP-In. An increased hydrodynamic radius of IgG when conjugated to an MCP may encourage tumor uptake via the enhanced permeability and retention (EPR) effect. Tumor uptake of panitumumab-DOTA-In was 3.5-fold significantly higher than IgG-DOTA-In. PANC-1 tumors were imaged by microSPECT/CT at 72 h p.i. of panitumumab-MCP-In or panitumumab-DOTA-In. Tumors were not visualized with preadministration of excess panitumumab to block EGFR, or with nonspecific IgG radioimmunoconjugates. We conclude that linking panitumumab to an MCP enabled higher SA labeling with In andLu than DOTA-conjugated panitumumab, with prese...

Our aim was to evaluate the effectiveness and normal tissue toxicity of radioimmunotherapy (RIT) of s.c. PANC-1 human pancreatic cancer (PnCa) xenografts in NRG mice using anti-EGFR panitumumab linked to metal-chelating polymers (MCPs) that present 13 DOTA chelators to complex the β-emitter, 177 Lu. The clonogenic survival (CS) of PANC-1 cells treated in vitro with panitumumab-MCP-177 Lu (0.3−1.2 MBq) and DNA double-strand breaks (DSBs) in the nucleus of these cells were measured by confocal immunofluorescence microscopy for γ-H2AX. Subcellular distribution of radioactivity for panitumumab-MCP-177 Lu was measured, and absorbed doses to the cell nucleus were calculated. Normal tissue toxicity was assessed in non tumor-bearing NRG mice by monitoring body weight, complete blood cell counts (CBC), serum alanine aminotransferase (ALT), and creatinine (Cr) after i.v. injection of 6 MBq (10 μg) of panitumumab-MCP-177 Lu. RIT was performed in NRG mice with s.c. PANC-1 tumors injected i.v. with 6 MBq (10 μg) of panitumumab-MCP-177 Lu. Control mice received nonspecific human IgG-MCP-177 Lu (6 MBq; 10 μg), unlabeled panitumumab (10 μg), or normal saline. The tumor growth index (TGI) was compared. Tumor and normal organ doses were estimated based on biodistribution studies. Panitumumab-MCP-177 Lu reduced the CS of PANC-1 cells in vitro by 7.7-fold at the highest amount tested (1.2 MBq). Unlabeled panitumumab had no effect on the CS of PANC-1 cells. γ-H2AX foci were increased by 3.8-fold by panitumumab-MCP-177 Lu. Panitumumab-MCP-177 Lu deposited 3.84 Gy in the nucleus of PANC-1 cells. Administration of panitumumab-MCP-177 Lu (6 MBq; 10 μg) to NRG mice caused no change in body weight, CBC, or ALT and only a slight increase in Cr compared to NRG mice treated with normal saline. Panitumumab-MCP-177 Lu strongly inhibited tumor growth in NRG mice (TGI = 2.3 ± 0.2) compared to normal saline-treated mice (TGI = 5.8 ± 0.5; P < 0.01). Unlabeled panitumumab had no effect on tumor growth (TGI = 6.0 ± 1.6; P > 0.05). The absorbed dose of PANC-1 tumors was 12.3 Gy. The highest normal organ doses were absorbed by the pancreas, liver, spleen, and kidneys. We conclude that EGFR-targeted RIT with panitumumab-MCP-177 Lu was able to overcome resistance to panitumumab in KRAS mutant PANC-1 tumors in NRG mice and may be a promising approach to treatment of PnCa in humans.

Background Epidermal growth factor receptors (EGFR) are overexpressed on > 90% of pancreatic cancers (PnCa) and represent an attractive target for the development of novel therapies, including radioimmunotherapy (RIT). Our aim was to study RIT of subcutaneous (s.c.) PANC-1 human PnCa xenografts in mice using the anti-EGFR monoclonal antibody, panitumumab labeled with Auger electron (AE)-emitting, 111In or β-particle emitting, 177Lu at amounts that were non-toxic to normal tissues. Results Panitumumab was conjugated to DOTA chelators for complexing 111In or 177Lu (panitumumab-DOTA-[111In]In and panitumumab-DOTA-[177Lu]Lu) or to a metal-chelating polymer (MCP) with multiple DOTA to bind 111In (panitumumab-MCP-[111In]In). Panitumumab-DOTA-[177Lu]Lu was more effective per MBq exposure at reducing the clonogenic survival in vitro of PANC-1 cells than panitumumab-DOTA-[111In]In or panitumumab-MCP-[111In]In. Panitumumab-DOTA-[177Lu]Lu caused the greatest density of DNA double-strand breaks (DSBs) in the nucleus measured by immunofluorescence for γ-H2AX. The absorbed dose in the nucleus was 3.9-fold higher for panitumumab-DOTA-[177Lu]Lu than panitumumab-DOTA-[111In]In and 7.7-fold greater than panitumumab-MCP-[111In]In. No normal tissue toxicity was observed in NOD/SCID mice injected intravenously (i.v.) with 10.0 MBq (10 μg; ~ 0.07 nmoles) of panitumumab-DOTA-[111In]In or panitumumab-MCP-[111In]In or in NRG mice injected i.v. with 6.0 MBq (10 μg; ~ 0.07 nmoles) of panitumumab-DOTA-[177Lu]Lu. There was no decrease in complete blood cell counts (CBC) or increased serum alanine aminotransferase (ALT) or creatinine (Cr) or decreased body weight. RIT inhibited the growth of PANC-1 tumours but a 5-fold greater total amount of panitumumab-DOTA-[111In]In or panitumumab-MCP-[111In]In (30 MBq; 30 μg; ~ 0.21 nmoles) administered in three fractionated amounts every three weeks was required to achieve greater or equivalent tumour growth inhibition, respectively, compared to a single amount of panitumumab-DOTA-[177Lu]Lu (6 MBq; 10 μg; ~ 0.07 nmoles). The tumour doubling time (TDT) for NOD/SCID mice with s.c. PANC-1 tumours treated with panitumumab-DOTA-[111In]In or panitumumab-MCP-[111In]In was 51.8 days and 28.1 days, respectively. Panitumumab was ineffective yielding a TDT of 15.3 days vs. 15.6 days for normal saline treated mice. RIT of NRG mice with s.c. PANC-1 tumours with 6.0 MBq (10 μg; ~ 0.07 nmoles) of panitumumab-DOTA-[177Lu]Lu increased the TDT to 20.9 days vs. 11.5 days for panitumumab and 9.1 days for normal saline. The absorbed doses in PANC-1 tumours were 8.8 ± 3.0 Gy and 2.6 ± 0.3 Gy for panitumumab-DOTA-[111In]In and panitumumab-MCP-[111In]In, respectively, and 11.6 ± 4.9 Gy for panitumumab-DOTA-[177Lu]Lu. Conclusion RIT with panitumumab labeled with Auger electron-emitting, 111In or β-particle-emitting, 177Lu inhibited the growth of s.c. PANC-1 tumours in NOD/SCID or NRG mice, at administered amounts that caused no normal tissue toxicity. We conclude that EGFR-targeted RIT is a promising approach to treatment of PnCa.

The development of resistance and unwanted harmful interaction with other biomolecules instead of DNA are the major drawbacks for application of platinum (Pt) complexes in cancer chemotherapy. To conquer these problems, much works have been done so far to discover innovative Pt complexes. The objective of the current study was to evaluate the anti cancer activities of a series of four and five-coordinated Pt(II) complexes, having deprotonated 2-phenyl pyridine (abbreviated as C^N), biphosphine moieties, i.e., dppm = bis(diphenylphosphino) methane (Ph(2)PCH(2)PPh(2)) and dppa = bis(diphenylphosphino)amine (Ph(2)PNHPPh(2)), as the non-leaving carrier groups. The growth inhibitory effect of the Pt complexes [Pt(C^N)(dppm)]PF(6): C(1), [Pt(C^N)(dppa)]PF(6): C(2), and [Pt(C^N)I(dppa)]: C(3), toward the cancer cell lines was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. In addition, the florescence quenching experiments of the interaction between human serum albumin (HSA) and the Pt complexes were performed in order to obtain the binding parameters and to evaluate the denaturing properties of these complexes upon binding to the general carrier protein of blood stream. The structure-activity relationship studies reveal that four-coordinated Pt complexes C(1) and C(2) with both significant hydrophobic and charge characteristics, not only exhibit strong antiproliferation activity toward the cancer cell lines, but also they display lower denaturing effect against carrier protein HSA. On the other hand, five-coordinated C(3) complex with the unusual intermolecular NH…Pt hydrogen binding and the intrinsic ability for oligomerization, exhibits poor anticancer activity and strong denaturing property. The current study reveals that the balance between charge and hydrophobicity of the Pt complexes, also their hydrogen binding abilities and coordination mode are important for their anticancer activities. Moreover, this study may suggest C(1) and C(2) as the potential template structures for synthesis of new generation of four-coordinated Pt complexes with strong anticancer activities and weak denaturing effects against proteins.