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Glioblastoma (GBM) is the most aggressive form of primary brain cancer, accounting for about 85% of all primary central nervous system (CNS) tumors. With standard treatment strategies like surgery, radiation, and chemotherapy, the median survival time of patients with GBM is only 12-15 months from diagnosis. The poor prognosis of GBM is due to a very high tumor recurrence rate following initial treatment, indicating a dire need for improved diagnostic and therapeutic alternatives for this disease. Antibody-based immunotheranostics holds great promise in treating GBM, combining the theranostic applications of radioisotopes and target-specificity of antibodies. In this study, we developed and validated antibody-based positron emission tomography (PET) tracers targeting the heparan sulfate proteoglycan, glypican-1 (GPC-1), for noninvasive detection of disease using diagnostic molecular imaging. GPC-1 is overexpressed in multiple solid tumor types, including GBM, and is a promising biomarker for novel immunotheranostics. Here, we investigate zirconium-89 ( 89 Zr)-conjugated Miltuximab (a clinical stage anti-GPC-1 monoclonal antibody developed by GlyTherix, Ltd.) and engineered fragments for their potential as immuno-PET tracers to detect GPC-1 positive GBM tumors in preclinical models. We explore the effects of molecular size, avidity, and Fc-domain on the pharmacokinetics and biodistribution in vivo, by comparing in parallel the full-length antibody (Miltuximab), Fab'2, Fab, and single-chain variable fragment (scFv) formats. High radiolabeling efficiency (>95%) was demonstrated by all the formats and the stability post-radiolabeling was higher for larger constructs of Miltuximab and the Fab. Receptor-mediated internalization of all 89 Zr-labeled formats was observed in a human GBM cell line in vitro, while full-length Miltuximab demonstrated the highest tumor retention (5.7 ± 0.94% ID/g, day-9 postinjection (p.i.)) and overall better tumor-to-background ratios than the smaller Fc-less formats. Results from in vivo PET image quantification and ex vivo scintillation counting were highly correlated. Altogether, 89 Zr-DFO-Miltuximab appears to be an effective immuno-PET imaging agent for detecting GPC-1 positive tumors such as GBM and the current results support utility of the Fc containing whole mAb format over smaller antibody fragments for this target.

Objectives: Miltuximab ® is a chimeric antibody targeting Glypican-1 (GPC-1), a cell surface antigen which is overexpressed in solid cancers. Miltuximab ® has shown promising safety and efficacy in radioimmunotherapy models of prostate cancer. This first in human study used Miltuximab ® radiolabelled with Gallium-67 ([ 67 Ga]Ga-DOTA-Miltuximab ® ). The primary study endpoint was to establish safety and tolerability of Miltuximab ® . Secondary endpoints were biodistribution, tumour targeting and pharmacokinetic analysis., Methods: Four cohorts of three patients (9 with advanced prostate cancer, 2 with pancreatic and 1 with bladder cancer) were dosed with 1 mg, ~250 MBq of [ 67 Ga]Ga-DOTA-Miltuximab ® . Cohort 1 received [ 67 Ga]Ga-DOTA-Miltuximab ® alone, while cohorts 2-4 were pre-infused with increasing doses (3.5, 11.5 and 24 mg, respectively) of unlabelled Miltuximab ® -DOTA 1 hour prior to [ 67 Ga]Ga-DOTA-Miltuximab ® . Safety and tolerability were assessed by clinical and standard laboratory assessments. Patients underwent whole body gamma-camera scans and SPECT/CT scans up to 144 h post-infusion. Total organ radiation exposure was determined by dosimetry of whole-body gamma scans., Results: The dosing regimen was well tolerated, with no drug-related adverse events observed. Liver and spleen uptake of [ 67 Ga]Ga-DOTA-Miltuximab ® was observed. Liver uptake was reduced by pre-infusion of unlabelled Miltuximab ® -DOTA. Dosimetry analysis showed a favorable exposure profile. [ 67 Ga]Ga-DOTA-Miltuximab ® targeting to tumour sites was observed in two prostate cancer patients who had failed enzalutamide treatment. Higher doses of unlabelled antibody achieved lower liver uptake and increased antibody serum half life., Conclusions: This study is the first in human for Miltuximab ® a first in class antibody targeting GPC-1. The trial met its primary endpoint of safety, demonstrating its potential as a safe and tolerable monoclonal antibody. This safety data, together with targeting to tumour lesions and biodistribution information supports the further clinical development of Miltuximab ® as a theranostic agent in a planned Phase I human trial., (© 2021 mums.ac.ir All rights reserved.)

Glioblastoma (GBM) is one of the most aggressive tumors and its 5-year survival is approximately 5%. Fluorescence-guided surgery (FGS) improves the extent of resection and leads to better prognosis. Molecular near-infrared (NIR) imaging appears to outperform conventional FGS, however, novel molecular targets need to be identified in GBM. Proteoglycan glypican-1 (GPC-1) is believed to be such a target as it is highly expressed in GBM and is associated with poor prognosis. We hypothesize that an anti-GPC-1 antibody, Miltuximab ® , conjugated with the NIR dye, IRDye800CW (IR800), can specifically accumulate in a GBM xenograft and provide high-contrast in vivo fluorescent imaging in rodents following systemic administration. Miltuximab ® was conjugated with IR800 and intravenously administered to BALB/c nude mice bearing a subcutaneous U-87 GBM hind leg xenograft. Specific accumulation of Miltuximab ® -IR800 in subcutaneous xenograft tumor was detected 24 h later using an in vivo fluorescence imager. The conjugate did not cause any adverse events in mice and caused strong fluorescence of the tumor with tumor-to-background ratio (TBR) reaching 10.1 ± 2.8. The average TBR over the 10-day period was 5.8 ± 0.6 in mice injected with Miltuximab ® -IR800 versus 2.4 ± 0.1 for the control group injected with IgG-IR800 ( p = 0.001). Ex vivo assessment of Miltuximab ® -IR800 biodistribution confirmed its highly specific accumulation in the tumor. The results of this study confirm that Miltuximab ® -IR800 holds promise for intraoperative fluorescence molecular imaging of GBM and warrants further studies.