Efficacy of [67Cu]Cu-EB-TATE Theranostic Against Somatostatin Receptor Subtype-2-Positive Neuroendocrine Tumors.

ß--emitting 177Lu-octreotate is an approved somatostatin receptor subtype 2 (SSTR2)-directed peptide receptor radionuclide therapy for the treatment of gastroenteropancreatic neuroendocrine tumors (NETs). However,177Lu-octreotate has fast pharmacokinetics, requiring up to 4 treatment doses. Moreover, 177Lu is less than ideal for theranostics because of the low branching ratio of its γ-emissions, which limits its SPECT imaging capability. Compared with 177Lu, 67Cu has better decay properties for use as a theranostic. Here, we report the preclinical evaluation of a long-lived somatostatin analog, [67Cu]Cu-DOTA-Evans blue-TATE (EB-TATE), against SSTR2-positive NETs. Methods: The in vitro cytotoxicity of [67Cu]Cu-EB-TATE was investigated on 2-dimensional cells and 3-dimensional spheroids. In vivo pharmacokinetics and dosimetry were studied in healthy BALB/c mice, whereas ex vivo biodistribution, micro-SPECT/CT imaging, and therapy studies were done on athymic nude mice bearing QGP1.SSTR2 and BON1.SSTR2 xenografts. Therapeutic efficacy was compared with [177Lu]Lu-EB-TATE. Results: Projected human effective doses of [67Cu]Cu-EB-TATE for male (0.066 mSv/MBq) and female (0.085 mSv/MBq) patients are tolerable. In vivo micro-SPECT/CT imaging of SSTR2-positive xenografts with [67Cu]Cu-EB-TATE showed tumor-specific uptake and prolonged accumulation. Biodistribution showed tumor accumulation, with concurrent clearance from major organs over a period of 72 h. [67Cu]Cu-EB-TATE was more effective (60%) at eliminating tumors that were smaller than 50 mm3 within the first 15 d of therapy than was [177Lu]Lu-EB-TATE (20%) after treatment with 2 doses of 15 MBq administered 10 d apart. Mean survival of [67Cu]Cu-EB-TATE-treated groups was 90 d and more than 90 d, whereas that of [177Lu]Lu-EB-TATE was more than 90 d and 89 d against vehicle control groups (26 d and 53 d), for QGP1.SSTR2 and BON1.SSTR2 xenografts, respectively. Conclusion: [67Cu]Cu-EB-TATE exhibited high SSTR2-positive NET uptake and retention, with favorable dosimetry and SPECT/CT imaging capabilities. The antitumor efficacy of [67Cu]Cu-EB-TATE is comparable to that of [177Lu]Lu-EB-TATE, with [67Cu]Cu-EB-TATE being slightly more effective than [177Lu]Lu-EB-TATE for complete remission of small tumors. [67Cu]Cu-EB-TATE therefore warrants clinical development.

Effectiveness of [67Cu]Cu-trastuzumab as a theranostic against HER2-positive breast cancer.

PURPOSE: To evaluate the imaging and therapeutic properties (theranostic) of 67Cu-labeled anti-human epidermal growth factor receptor II (HER2) monoclonal antibody trastuzumab against HER2-positive breast cancer (BC). METHODS: We conjugated trastuzumab with p-SCN-Bn-NOTA, 3p-C-NETA-NCS, or p-SCN-Bn-DOTA, and radiolabeled with [67Cu]CuCl2. Immunoconjugate internalization was evaluated in BT-474, JIMT-1 and MCF-7 BC cells. In vitro stability was studied in human serum (HS) and Phosphate Buffered Saline (PBS). Flow cytometry, radioligand binding and immunoreactive fraction assays were carried out. ImmunoSPECT imaging of [67Cu]Cu-NOTA-trastuzumab was done in mice bearing BT-474, JIMT-1 and MCF-7 xenografts. Pharmacokinetic was studied in healthy Balb/c mice while dosimetry was done in both healthy Balb/c and in athymic nude mice bearing JIMT-1 xenograft. The therapeutic effectiveness of [67Cu]Cu-NOTA-trastuzumab was evaluated in mice bearing BT-474 and JIMT-1 xenografts after a single intravenous (i.v.) injection of ~ 16.8 MBq. RESULTS: Pure immunoconjugates and radioimmunoconjugates (> 95%) were obtained. Internalization was HER2 density-dependent with highest internalization observed with NOTA-trastuzumab. After 5 days, in vitro stabilities were 97 ± 1.7%, 31 ± 6.2%, and 28 ± 4% in HS, and 79 ± 3.5%, 94 ± 1.2%, and 86 ± 2.3% in PBS for [67Cu]Cu-NOTA-trastuzumab, [67Cu]Cu-3p-C-NETA-trastuzumab and [67Cu]Cu-DOTA-trastuzumab, respectively. [67Cu]Cu-NOTA-trastuzumab was chosen for further evaluation. BT-474 flow cytometry showed low KD, 8.2 ± 0.2 nM for trastuzumab vs 26.5 ± 1.6 nM for NOTA-trastuzumab. There were 2.9 NOTA molecules per trastuzumab molecule. Radioligand binding assay showed a low KD of 2.1 ± 0.4 nM and immunoreactive fraction of 69.3 ± 0.9. Highest uptake of [67Cu]Cu-NOTA-trastuzumab was observed in JIMT-1 (33.9 ± 5.5% IA/g) and BT-474 (33.1 ± 10.6% IA/g) xenograft at 120 h post injection (p.i.). Effectiveness of the radioimmunoconjugate was also expressed as percent tumor growth inhibition (%TGI). [67Cu]Cu-NOTA-trastuzumab was more effective than trastuzumab against BT-474 xenografts (78% vs 54% TGI after 28 days), and JIMT-1 xenografts (90% vs 23% TGI after 19 days). Mean survival of [67Cu]Cu-NOTA-trastuzumab, trastuzumab and saline treated groups were > 90, 77 and 72 days for BT-474 xenografts, while that of JIMT-1 were 78, 24, and 20 days, respectively. CONCLUSION: [67Cu]Cu-NOTA-trastuzumab is a promising theranostic agent against HER2-positive BC.

Effectiveness of 225Ac-Labeled Anti-EGFR Radioimmunoconjugate in EGFR-Positive Kirsten Rat Sarcoma Viral Oncogene and BRAF Mutant Colorectal Cancer Models.

Eighty percent of colorectal cancers (CRCs) overexpress epidermal growth factor receptor (EGFR). Kirsten rat sarcoma viral oncogene (KRAS) mutations are present in 40% of CRCs and drive de novo resistance to anti-EGFR drugs. BRAF oncogene is mutated in 7%-10% of CRCs, with even worse prognosis. We have evaluated the effectiveness of [225Ac]Ac-macropa-nimotuzumab in KRAS mutant and in KRAS wild-type and BRAFV600E mutant EGFR-positive CRC cells in vitro and in vivo. Anti-CD20 [225Ac]Ac-macropa-rituximab was developed and used as a nonspecific radioimmunoconjugate. Methods: Anti-EGFR antibody nimotuzumab was radiolabeled with 225Ac via an 18-membered macrocyclic chelator p-SCN-macropa. The immunoconjugate was characterized using flow cytometry, radioligand binding assay, and high-performance liquid chromatography, and internalization was studied using live-cell imaging. In vitro cytotoxicity was evaluated in 2-dimensional monolayer EGFR-positive KRAS mutant DLD-1, SW620, and SNU-C2B; in KRAS wild-type and BRAFV600E mutant HT-29 CRC cell lines; and in 3-dimensional spheroids. Dosimetry was studied in healthy mice. The in vivo efficacy of [225Ac]Ac-macropa-nimotuzumab was evaluated in mice bearing DLD-1, SW620, and HT-29 xenografts after treatment with 3 doses of 13 kBq/dose administered 10 d apart. Results: In all cell lines, in vitro studies showed enhanced cytotoxicity of [225Ac]Ac-macropa-nimotuzumab compared with nimotuzumab and controls. The inhibitory concentration of 50% in the DLD-1 cell line was 1.8 nM for [225Ac]Ac-macropa-nimotuzumab versus 84.1 nM for nimotuzumab. Similarly, the inhibitory concentration of 50% was up to 79-fold lower for [225Ac]Ac-macropa-nimotuzumab than for nimotuzumab in KRAS mutant SNU-C2B and SW620 and in KRAS wild-type and BRAFV600E mutant HT-29 CRC cell lines. A similar trend was observed for 3-dimensional spheroids. Internalization peaked 24-48 h after incubation and depended on EGFR expression. In the [225Ac]Ac-macropa-nimotuzumab group, 3 of 7 mice bearing DLD-1 tumors had complete remission. Median survival was 40 and 34 d for mice treated with phosphate-buffered saline and [225Ac]Ac-macropa-rituximab (control), respectively, whereas it was not reached for the [225Ac]Ac-macropa-nimotuzumab group (>90 d). Similarly, median survival of mice bearing HT-29 xenografts was 16 and 12.5 d for those treated with [225Ac]Ac-macropa-rituximab and phosphate-buffered saline, respectively, and was not reached for those treated with [225Ac]Ac-macropa-nimotuzumab (>90 d). One of 7 mice bearing HT-29 xenografts and treated using [225Ac]Ac-macropa-nimotuzumab had complete remission. Compared with untreated mice, [225Ac]Ac-macropa-nimotuzumab more than doubled (16 vs. 41 d) the median survival of mice bearing SW620 xenografts. Conclusion: [225Ac]Ac-macropa-nimotuzumab is effective against KRAS mutant and BRAFV600E mutant CRC models.

Biparatopic anti-HER2 drug radioconjugates as breast cancer theranostics.

BACKGROUND: HER2 is overexpressed in 25-30% of breast cancer. Multiple domains targeting of a receptor can have synergistic/additive therapeutic effects. METHODS: Two domain-specific ADCs trastuzumab-PEG6-DM1 (domain IV) and pertuzumab-PEG6-DM1 (domain II) were developed, characterised and radiolabeled to obtain [89Zr]Zr-trastuzumab-PEG6-DM1 and [67Cu]Cu-pertuzumab-PEG6-DM1 to study their in vitro (binding assay, internalisation and cytotoxicity) and in vivo (pharmacokinetics, biodistribution and immunoPET/SPECT imaging) characteristics. RESULTS: The ADCs had an average drug-to-antibody ratio of 3. Trastuzumab did not compete with [67Cu]Cu-pertuzumab-PEG6-DM1 for binding to HER2. The highest antibody internalisation was observed with the combination of ADCs in BT-474 cells compared with single antibodies or ADCs. The combination of the two ADCs had the lowest IC50 compared with treatment using the single ADCs or controls. Pharmacokinetics showed biphasic half-lives with fast distribution and slow elimination, and an AUC that was five-fold higher for [89Zr]Zr-trastuzumab-PEG6-DM1 compared with [67Cu]Cu-pertuzumab-PEG6-DM1. Tumour uptake of [89Zr]Zr-trastuzumab-PEG6-DM1 was 51.3 ± 17.3% IA/g (BT-474), and 12.9 ± 2.1% IA/g (JIMT-1) which was similarly to [67Cu]Cu-pertuzumab-PEG6-DM1. Mice pre-blocked with pertuzumab had [89Zr]Zr-trastuzumab-PEG6-DM1 tumour uptakes of 66.3 ± 33.9% IA/g (BT-474) and 25.3 ± 4.9% IA/g (JIMT-1) at 120 h p.i. CONCLUSION: Using these biologics simultaneously as biparatopic theranostic agents has additive benefits.

Engineering of a Fully Human Anti-MUC-16 Antibody and Evaluation as a PET Imaging Agent.

Antibodies that recognize cancer biomarkers, such as MUC16, can be used as vehicles to deliver contrast agents (imaging) or cytotoxic payloads (therapy) to the site of tumors. MUC16 is overexpressed in 80% of epithelial ovarian cancer (EOC) and 65% of pancreatic ductal adenocarcinomas (PDAC), where effective 'theranostic' probes are much needed. This work aims to develop fully human antibodies against MUC16 and evaluate them as potential immuno-PET imaging probes for detecting ovarian and pancreatic cancers. We developed a fully human monoclonal antibody, M16Ab, against MUC16 using phage display. M16Ab was conjugated with p-SCN-Bn-DFO and radiolabeled with 89Zr. 89Zr-DFO-M16Ab was then evaluated for binding specificity and affinity using flow cytometry. In vivo evaluation of 89Zr-DFO-M16Ab was performed by microPET/CT imaging at different time points at 24−120 h post injection (p.i.) and ex vivo biodistribution studies in mice bearing MUC16-expressing OVCAR3, SKOV3 (ovarian) and SW1990 (pancreatic) xenografts. 89Zr-DFO-M16Ab bound specifically to MUC16-expressing cancer cells with an EC50 of 10nM. 89Zr-DFO-M16Ab was stable in serum and showed specific uptake and retention in tumor xenografts even after 120 h p.i. (microPET/CT) with tumor-to-blood ratios > 43 for the SW1990 xenograft. Specific tumor uptake was observed for SW1990/OVCAR3 xenografts but not in MUC16-negative SKOV3 xenografts. Pharmacokinetic study shows a relatively short distribution (t1/2α) and elimination half-life (t1/2ß) of 4.4 h and 99 h, respectively. In summary, 89Zr-DFO-M16Ab is an effective non-invasive imaging probe for ovarian and pancreatic cancers and shows promise for further development of theranostic radiopharmaceuticals.

Simultaneous Imaging and Therapy Using Epitope-Specific Anti-Epidermal Growth Factor Receptor (EGFR) Antibody Conjugates.

Matuzumab and nimotuzumab are anti-EGFR monoclonal antibodies that bind to different epitopes of domain III of EGFR. We developed 89Zr-matuzumab as a PET probe for diagnosis/monitoring of response to treatment of a noncompeting anti-EGFR nimotuzumab antibody drug conjugate (ADC) using mouse colorectal cancer (CRC) xenografts. We developed 89Zr-matuzumab and performed quality control in EGFR-positive DLD-1 cells. The KD of matuzumab, DFO-matuzumab and 89Zr-matuzumab in DLD-1 cells was 5.9, 6.2 and 3 nM, respectively. A competitive radioligand binding assay showed that 89Zr-matuzumab and nimotuzumab bound to noncompeting epitopes of EGFR. MicroPET/CT imaging and biodistribution of 89Zr-matuzumab in mice bearing EGFR-positive xenografts (HT29, DLD-1 and MDA-MB-231) showed high uptake that was blocked with pre-dosing with matuzumab but not with the noncompeting binder nimotuzumab. We evaluated nimotuzumab-PEG6-DM1 ADC in CRC cells. IC50 of nimotuzumab-PEG6-DM1 in SNU-C2B, DLD-1 and SW620 cells was dependent on EGFR density and was up to five-fold lower than that of naked nimotuzumab. Mice bearing the SNU-C2B xenograft were treated using three 15 mg/kg doses of nimotuzumab-PEG6-DM1, and 89Zr-matuzumab microPET/CT was used to monitor the response to treatment. Treatment resulted in complete remission of the SNU-C2B tumor in 2/3 mice. Matuzumab and nimotuzumab are noncompeting and can be used simultaneously.

Palladium Probe Consisting of Naphthalimide and Ethylenediamine for Selective Turn-On Sensing of CO and Cell Imaging.

An assay to detect carbon monoxide (CO), one of the gaseous signaling molecules, has been prepared using a new palladium complex probe. The ethylenediamine group linked to the naphthalimide fluorophore coordinates to Pd(II) which intramolecularly quenches the emission. Upon treatment with CO, the absorbance of the turn-on fluorescent sensor changes due to the formation of a complex between Pd(II) and CO at room temperature in a phosphate buffer. As the concentration of CO increases, the probe peak emission intensity at 527 nm gradually increases. Other analyte controls, such as K+, Mg2+, Al3+, Zn2+, Cr3+, Hg2+, Fe3+, alanine, glycine, leucine, lysine, serine, threonine, tyrosine, F-, Cl-, Br-, NO, NO2-, NO3-, HCO3-, CH3COO-, H2O2, •OH, and tBuOO•, exhibit no significant effect on emission intensity. The response time of the probe to CO was quite fast because of the relatively weak coordination of Pd(II) to the pendent ethylenediamine group. The Pd probe is capable of detecting CO in aqueous buffer as well as in living cells with high selectivity and stability, providing a potential real-time indicator for studying CO-involved reactions in biological systems.

Reduced Graphene Oxide-Oligonucleotide Interfaces: Understanding Based on Electrochemical Oxidation of Guanines.

Investigation into the interactions between biomolecules DNA/RNA and carbon nanomaterials is very important for applications in bioassays and bioanalysis. Graphene and graphene oxide (GO) have been successfully adopted by exploiting the binding affinity difference between single-stranded oligonucleotides (ssDNA) and double-stranded oligonucleotides (dsDNA) to graphene sheets. In this work, we describe the electrochemical DNA oxidation with [Ru(bpy)3]2+ to understand the interaction between dsDNA (and corresponding ssDNA) and reduced graphene oxide (rGO). The electrochemical oxidation rate of guanine bases of ssDNA bound to rGO by electrochemically generated [Ru(bpy)3]3+ was much slower than those unbound to rGO. Our study revealed that ssDNA constrained on rGO was significantly protected from the electron transfer to [Ru(bpy)3]3+ because of π,π-stacking interaction between nucleobases and rGO. On the other hand, the oxidation rates of 11-, 20-, and 27-mer dsDNA bound to rGO increased relative to those of dsDNA alone, demonstrating that the guanine bases of dsDNA on the interaction with rGO became more accessible to [Ru(bpy)3]3+. Our electrochemical data illustrated that dsDNA could be totally or partially dehybridized and bind to rGO to form ssDNA/rGO. Furthermore, absorption, circular dichroism spectra, and fluorescence measurements of ethidium bromide using ssDNA and dsDNA with rGO supported the dehybridization of dsDNA in the presence of rGO.