The Development of Peptide-Based Tools for the Analysis of Angiogenesis

SummaryLimitations to the application of molecularly targeted cancer therapies are the inability to accurately match patient with effective treatment and the absence of a prompt readout of posttreatment response. Noninvasive agents that rapidly report vascular endothelial growth factor (VEGF) levels using positron emission tomography (PET) have the potential to enhance anti-angiogenesis therapies. Using phage display, two distinct classes of peptides were identified that bind to VEGF with nanomolar affinity and high selectivity. Co-crystal structures of these different peptide classes demonstrate that both bind to the receptor-binding region of VEGF. 18F-radiolabelling of these peptides facilitated the acquisition of PET images of tumor VEGF levels in a HM7 xenograph model. The images obtained from one 59-residue probe, 18F-Z-3B, 2 hr postinjection are comparable to those obtained with anti-VEGF antibody B20 72 hr postinjection. Furthermore, VEGF levels in growing SKOV3 tumors were followed using 18F-Z-3B as a PET probe with VEGF levels increasing with tumor size

Evaluation of a 3-hydroxypyridin-2-one (2,3-HOPO) Based Macrocyclic Chelator for 89 Zr 4+ and Its Use for ImmunoPET Imaging of HER2 Positive Model of Ovarian Carcinoma in Mice

Abstract A novel octadentate 3-hydroxypyridin-2-one (2,3-HOPO) based di-macrocyclic ligand was evaluated for chelation of 89 Zr; subsequently, it was used as a bi-functional chelator for preparation of 89 Zr-labeled antibodies. Quantitative chelation of 89 Zr 4+ with the octadentate ligand forming 89 ZrL complex was achieved under mild conditions within 15 minutes. The 89 Zr-complex was stable in vitro in presence of DTPA, but a slow degradation was observed in serum. In vivo, the hydrophilic 89 Zr-complex showed prevalently renal excretion; and an elevated bone uptake of radioactivity suggested a partial release of 89 Zr 4+ from the complex. The 2,3-HOPO based ligand was conjugated to the monoclonal antibodies, HER2-specific trastuzumab and an isotypic anti-gD antibody, using a p-phenylene bis-isothiocyanate linker to yield products with an average loading of less than 2 chelates per antibody. Conjugated antibodies were labeled with 89 Zr under mild conditions providing the PET tracers in 60-69% yield. Despite the limited stability in mouse serum; the PET tracers performed very well in vivo. The PET imaging in mouse model of HER2 positive ovarian carcinoma showed tumor uptake of 89 Zr-trastuzumab (29.2 ± 12.9 %ID/g) indistinguishable (p = 0.488) from the uptake of positive control 89 Zr-DFO-trastuzumab (26.1 ± 3.3 %ID/g). In conclusion, the newly developed 3-hydroxypyridin-2-one based di-macrocyclic chelator provides a viable alternative to DFO-based heterobifunctional ligands for preparation of 89 Zr-labeled monoclonal antibodies for immunoPET studies

ImmunoPET helps predicting the efficacy of antibody-drug conjugates targeting TENB2 and STEAP1

The efficacy of antibody-drug conjugates (ADCs) targeted to solid tumors depends on biological processes that are hard to monitor in vivo. Zr-89-immunoPET of the ADC antibodies could help understand the performance of ADCs in the clinic by confirming the necessary penetration, binding, and internalization. This work studied monomethyl auristatin E (MMAE) ADCs against two targets in metastatic castration-resistant prostate cancer, TENB2 and STEAP1, in four patient-derived tumor models (LuCaP35V, LuCaP70, LuCaP77, LuCaP96.1). Three aspects of ADC biology were measured and compared: efficacy was measured in tumor growth inhibition studies; target expression was measured by immunohistochemistry and flow cytometry; and tumor antibody uptake was measured with In-111-mAbs and gamma counting or with Zr-89-immunoPET. Within each model, the mAb with the highest tumor uptake showed the greatest potency as an ADC. Sensitivity between models varied, with the LuCaP77 model showing weak efficacy despite high target expression and high antibody uptake. Ex vivo analysis confirmed the in vivo results, showing a correlation between expression, uptake and ADC efficacy. We conclude that Zr-89-immunoPET data can demonstrate which ADC candidates achieve the penetration, binding, and internalization necessary for efficacy in tumors sensitive to the toxic payload

Evaluation of 2-[18F]fluoroacetate Kinetics in Rodent Models of Cerebral Hypoxia–Ischemia

Glia account for 90% of human brain cells and have a significant role in brain homeostasis. Thus, specific in vivo imaging markers of glial metabolism are potentially valuable. In the brain, 2-fluoroacetate is selectively taken up by glial cells and becomes metabolically trapped in the tricarboxylic acid cycle. Recent work in rodent brain injury models demonstrated elevated lesion uptake of 2-[(18)F]fluoroacetate ([(18)F]FACE), suggesting possible use for specifically imaging glial metabolism. To assess this hypothesis, we evaluated [(18)F]FACE kinetics in rodent models of cerebral hypoxia-ischemia at 3 and 24 hours post insult. Lesion uptake was significantly higher at 30 minutes post injection (P<0.05). An image-based method for input function estimation using cardiac blood was validated. Analysis of whole blood showed no significant metabolites and plasma activity concentrations of ∼50% that of whole blood. Kinetic models describing [(18)F]FACE uptake were developed and quantitatively compared. Elevated [(18)F]FACE uptake was found to be driven primarily by K₁/k₂ rather than k₃, but changes in the latter were detectable. The two-tissue irreversible uptake model (2T3k) was found to be necessary and sufficient for modeling [(18)F]FACE uptake. We conclude that kinetic modeling of [(18)F]FACE uptake represents a potentially useful tool for interrogation of glial metabolism

Preclinical Efficacy of an Antibody-Drug Conjugate Targeting Mesothelin Correlates with Quantitative Zr-89-ImmunoPET

Antibody-drug conjugates (ADC) use monoclonal antibodies (mAb) as vehicles to deliver potent cytotoxic drugs selectively to tumor cells expressing the target. Molecular imaging with zirconium-89 (Zr-89)-labeled mAbs recapitulates similar targeting biology and might help predict the efficacy of these ADCs. An anti-mesothelin antibody (AMA, MMOT0530A) was used to make comparisons between its efficacy as an ADC and its tumor uptake as measured by Zr-89 immunoPET imaging. Mesothelin-targeted tumor growth inhibition by monomethyl auristatin E (MMAE), ADC AMA-MMAE (DMOT4039A), was measured in mice bearing xenografts of ovarian cancer OVCAR-3 x 2.1, pancreatic cancers Capan-2, HPAC, AsPC-1, and HPAF-II, or mesothelioma MSTO-211H. Ex vivo analysis of mesothelin expression was performed using immunohistochemistry. AMA-MMAE showed the greatest growth inhibition in OVCAR-3 x 2.1, Capan-2, and HPAC tumors, which showed target-specific tumor uptake of Zr-89-AMA. The less responsive xenografts (AsPC-1, HPAF-II, and MSTO-211H) did not show Zr-89-AMA uptake despite confirmed mesothelin expression. ImmunoPET can demonstrate the necessary delivery, binding, and internalization of an ADC antibody in vivo and this correlates with the efficacy of mesothelin-targeted ADC in tumors vulnerable to the cytotoxic drug delivered. (C) 2016 AACR