Radioimmunotherapy of human pancreatic cancer xenografts in NOD-scid mice with [64Cu]Cu-NOTA-panitumumab F(ab')2 alone or combined with radiosensitizing gemcitabine and the PARP inhibitor, rucaparib.

INTRODUCTION: Our objective was to determine the feasibility of extending our previously reported PET imaging study of pancreatic cancer (PnCa) with [64Cu]Cu-NOTA-panitumumab F(ab')2 to radioimmunotherapy (RIT) by exploiting the ß-particle and Auger electron emissions of 64Cu (PET theranostic concept). To enhance the effectiveness of [64Cu]Cu-NOTA-panitumumab F(ab')2, we further combined RIT with radiosensitizing gemcitabine (GEM) and the poly(ADP)ribose polymerase inhibitor (PARPi), rucaparib. METHODS: Normal tissue toxicity was assessed in non-tumor-bearing NOD-scid mice injected i.v. with [64Cu]Cu-NOTA-panitumumab F(ab')2 (1.85-9.25 MBq; 10 µg) or [64Cu]Cu-NOTA-anti-mouse EGFR Ab30 F(ab')2 (12.95 MBq). Body weight was monitored, and hematopoietic (CBC), liver (ALT) and kidney [creatinine (SCr)] toxicity were assessed. RIT studies were performed in NOD-scid mice with s.c. OCIP23 human PnCa patient-derived xenografts (PDX) administered [64Cu]Cu-NOTA-panitumumab F(ab')2 (3.7 MBq; 10 µg), unlabeled panitumumab F(ab')2 (10 µg) or normal saline every two weeks. Subsequent studies evaluated RIT with [64Cu]Cu-NOTA-panitumumab F(ab')2 (12.95 MBq; 10 µg) administered alone or combined with GEM and the PARPi, rucaparib administered on a 14-day treatment cycle for up to 6 cycles in NOD-scid mice with s.c. PANC-1 human PnCa xenografts. The radiation absorbed dose in PANC-1 tumors and normal organs in mice after a single i.v. injection of [64Cu]Cu-NOTA-panitumumab F(ab')2 (12.95 MBq; 10 µg) was estimated based on previously reported biodistribution studies of [64Cu]Cu-NOTA-panitumumab F(ab')2. RESULTS: No normal tissue toxicity was observed in non-tumor-bearing NOD-scid mice administered up to 3.7 MBq (10 µg) of [64Cu]Cu-NOTA-panitumumab F(ab')2 but slightly increased ALT was noted at 9.25 MBq. Administration of [64Cu]Cu-NOTA-anti-mouse EGFR Ab30 F(ab')2 (12.95 MBq; 10 µg) caused some hematopoietic toxicity but no increase in ALT or SCr or decreased body weight. A slight tumor growth delay and increased survival was noted in NOD-scid mice with s.c. OCIP23 PDX treated with [64Cu]Cu-NOTA-panitumumab F(ab')2 (3.7 MBq; 10 µg) or unlabeled panitumumab F(ab')2 (10 µg) compared to normal saline treated mice. RIT with [64Cu]Cu-NOTA-panitumumab F(ab')2 (12.95 MBq; 10 µg) combined with GEM + PARPi for up to 6 cycles was most effective for the treatment of PANC-1 tumors. Tumor doubling time increased to 13.3 ± 0.9 days vs. 7.8 ± 3.7 days for RIT alone and 9.3 ± 2.2 days for normal saline treatment. Median survival was significantly longer (P < 0.05) than in mice treated with normal saline (35 days) for RIT + GEM + PARPi (71 days), GEM + PARPi (44 days) and RIT + GEM (43 days) but not for RIT alone (25 days). RIT + GEM + PARPi provided a longer median survival than RIT (P < 0.01), GEM + PARPi (P = 0.01) but not RIT + GEM (P = 0.23). Nonetheless, PANC-1 tumors grew exponentially in all treatment groups. The absorbed dose in PANC-1 tumors after a single i.v. injection of [64Cu]Cu-NOTA-panitumumab F(ab')2 (12.85 MBq; 10 µg) was 0.8 Gy, while the dose in normal organs ranged from 0.6-1.2 Gy. CONCLUSIONS: We conclude that RIT with [64Cu]Cu-NOTA-panitumumab F(ab')2 did not cause significant normal tissue toxicity but was not effective when administered alone for treatment of PnCa xenografts in NOD-scid mice. Combining RIT with GEM and the PARPi, rucaparib enhanced its effectiveness but tumors continued to grow exponentially. Our results suggest that 64Cu is not feasible for RIT of PnCa due to low tumor absorbed doses. 177Lu which has a higher abundance of moderate energy ß-particle emissions may be more effective than 64Cu. The hematopoietic toxicity of [64Cu]Cu-NOTA-anti-mouse EGFR Ab30 F(ab')2 may be mediated by binding to mouse EGFR expressed on some hematopoietic stem cells. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: Direct extension of PET with 64Cu(Cu)-NOTA-panitumumab F(ab')2 to RIT exploiting the ß-particle and Auger electron emissions of 64Cu is not feasible. Theranostic approaches that combine PET with RIT employing 177Lu may be more promising and should be explored.

Whole genomes define concordance of matched primary, xenograft, and organoid models of pancreas cancer.

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis among solid malignancies and improved therapeutic strategies are needed to improve outcomes. Patient-derived xenografts (PDX) and patient-derived organoids (PDO) serve as promising tools to identify new drugs with therapeutic potential in PDAC. For these preclinical disease models to be effective, they should both recapitulate the molecular heterogeneity of PDAC and validate patient-specific therapeutic sensitivities. To date however, deep characterization of the molecular heterogeneity of PDAC PDX and PDO models and comparison with matched human tumour remains largely unaddressed at the whole genome level. We conducted a comprehensive assessment of the genetic landscape of 16 whole-genome pairs of tumours and matched PDX, from primary PDAC and liver metastasis, including a unique cohort of 5 'trios' of matched primary tumour, PDX, and PDO. We developed a pipeline to score concordance between PDAC models and their paired human tumours for genomic events, including mutations, structural variations, and copy number variations. Tumour-model comparisons of mutations displayed single-gene concordance across major PDAC driver genes, but relatively poor agreement across the greater mutational load. Genome-wide and chromosome-centric analysis of structural variation (SV) events highlights previously unrecognized concordance across chromosomes that demonstrate clustered SV events. We found that polyploidy presented a major challenge when assessing copy number changes; however, ploidy-corrected copy number states suggest good agreement between donor-model pairs. Collectively, our investigations highlight that while PDXs and PDOs may serve as tractable and transplantable systems for probing the molecular properties of PDAC, these models may best serve selective analyses across different levels of genomic complexity.

EGFR-Targeted Metal Chelating Polymers (MCPs) Harboring Multiple Pendant PEG2K Chains for MicroPET/CT Imaging of Patient-Derived Pancreatic Cancer Xenografts.

In this study, we developed a new generation of metal chelating polymer (MCP) reagents that carry multiple polyethylene glycol (PEG) pendant groups to provide stealth to MCP-based radioimmunoconjugates (RICs). We describe the MCP synthesis for covalent attachment to panitumumab F(ab')2 fragments (pmabF(ab')2) in which different numbers of pendant methoxy-PEG chains [M = 2000, ∼45 ethylene glycol (EG) repeat units, referred to as PEG2K] are incorporated into the polymer backbone. The pendant PEG2K chains were designed to provide a protein-repellant corona so that metal chelators attached closer to the polymer backbone will be less apparent to the physiological environment. DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) groups to chelate 64Cu were installed on these conjugates to be employed for PET imaging. The conjugation of MCPs to pmabF(ab')2 was based on a UV quantifiable bis-aromatic hydrazone formation under mild conditions (pH 5-6) between an aromatic aldehyde introduced on ε-NH2 groups of lysines in the F(ab')2 fragments and a hydrazinonicotinamide (HyNic) group installed on the initiating end of the MCP. Three MCPs with 17 polyglutamide (PGlu) repeat units, DOTA chelators and with an average of 2, 4, and 8 pendant PEG2K chains were studied to examine their in vitro and in vivo characteristics, as well as their potential for PET/CT imaging. A pmabF(ab')2-MCP conjugate carrying 2 PEG2K and one carrying 8 PEG2K pendant chains in the polymer were selected for microPET/CT imaging and biodistribution studies in tumor-bearing mice. Orthotopic pancreatic patient-derived xenografts tumors were visualized by PET/CT imaging. These RICs showed low levels of liver and spleen uptake along with even lower levels of kidney uptake. These encouraging results confirm the stealth properties of the MCPs with pendant PEG2K chains.

MicroPET/CT imaging of patient-derived pancreatic cancer xenografts implanted subcutaneously or orthotopically in NOD-scid mice using (64)Cu-NOTA-panitumumab F(ab')2 fragments.

INTRODUCTION: Our objective was to study microPET/CT imaging of patient-derived pancreatic cancer xenografts in NOD-scid mice using F(ab')2 fragments of the fully-human anti-EGFR monoclonal antibody, panitumumab (Vectibix) labeled with (64)Cu. More than 90% of pancreatic cancers are EGFR-positive. METHODS: F(ab')2 fragments were produced by proteolytic digestion of panitumumab IgG or non-specific human IgG, purified by ultrafiltration then modified with NOTA chelators for complexing (64)Cu. Panitumumab IgG and Fab fragments were similarly labeled with (64)Cu. EGFR immunoreactivity was determined in competition and direct (saturation) cell binding assays. The biodistribution of (64)Cu-labeled panitumumab IgG, F(ab')2 and Fab was compared in non-tumor-bearing Balb/c mice. MicroPET/CT and biodistribution studies were performed in NOD-scid mice engrafted subcutaneously (s.c.) or orthotopically with patient-derived OCIP23 pancreatic tumors, or in NOD-scid with s.c. PANC-1 human pancreatic cancer xenografts. RESULTS: Panitumumab F(ab')2 fragments were produced in high purity (>90%), derivitized with 3.2±0.7 NOTA/F(ab')2, and labeled with (64)Cu (0.3-3.6MBq/µg). The binding of (64)Cu-NOTA-panitumumab F(ab')2 to OCIP23 or PANC-1 cells was decreased significantly by an excess of panitumumab IgG. The Kd for binding of (64)Cu-NOTA-panitumumab F(ab')2 to EGFR on PANC-1 cells was 0.14±0.05nmol/L. F(ab')2 fragments exhibited more suitable normal tissue distribution for tumor imaging with (64)Cu than panitumumab IgG or Fab. Tumor uptake at 48h post injection (p.i.) of (64)Cu-NOTA-panitumumab F(ab')2 was 12.0±0.9% injected dose/g (ID/g) in s.c. and 11.8±0.9% ID/g in orthotopic OCIP23 tumors vs. 6.1±1.1% ID/g in s.c. PANC-1 xenografts. Tumor/Blood (T/B) ratios were 5:1 to 9:1 for OCIP23 and 2.4:1 for PANC-1 tumors. Tumor uptake of (64)Cu-NOTA-non-specific F(ab')2 in OCIP23 xenografts was 5-fold lower than (64)Cu-panitumumab F(ab')2. All tumor xenografts were clearly imaged by microPET/CT at 24 or 48h p.i. of (64)Cu-NOTA-panitumumab F(ab')2. CONCLUSIONS: (64)Cu-panitumumab F(ab')2 fragments bound with high affinity to EGFR on pancreatic cancer cells in vitro and localized specifically in patient-derived pancreatic cancer xenografts in mice in vivo, allowing tumor visualization by microPET/CT at 24 or 48h p.i.

Activation of Src and Src-associated signaling pathways in relation to hypoxia in human cancer xenograft models.

The hypoxic response in vitro involves alterations in signaling proteins, including Src, STAT3 and AKT that are considered to be broadly pro-survival. The involvement of these signaling proteins in the hypoxic microenviroments that occur in solid tumors was investigated by the use of multicolor fluorescence image analysis to colocalize signaling proteins and regions of hypoxia in 4 human tumor xenografts, pancreatic carcinoma BxPC3 and PANC1 and cervical squamous cell carcinoma ME180 and SiHa. Expression levels of total Src protein (mean intensity x labeled region fraction) were higher in hypoxic regions, identified using the nitroimidazole probe EF5, relative to non-EF5 regions in all 4 tumor models. This was associated with higher levels of phosphorylated (p-) Y419p-Src and its substrate Y861p-FAK in EF5 positive regions of BxPC3 tumors. This effect was also seen in tumor-bearing mice continuously breathing 7% oxygen for 3 hr which markedly increased the extent of EF5 positive labeling. In contrast, the hypoxia treatment resulted in a significant decrease in S727p-STAT3 in BxPC3 xenografts and suggested that STAT3 activity is responsive to acute hypoxia, whereas Src-FAK signaling is associated with predominantly chronically hypoxic EF5 positive regions. Src activity in both hypoxic and nonhypoxic BxPC3 tumor regions was suppressed when mice were treated with the Src inhibitor AZD0530 (25 mg/kg/day, 5 days), suggesting that both hypoxic and normoxic tumor regions are accessible to pharmacological Src inhibition. These results show that signaling pathways are responsive to tumor hypoxia in vivo, although the effects appear to differ between individual tumor types.