(P 172) DEVELOPMENT OF A TARGETED DUAL-MODALITY RADIOIMMUNOCONJUGATE FOR IMAGE GUIDED SARCOMA SURGERY

Objective: Fluorescence guided surgery (FGS) for bone and soft tissue sarcoma resection is an evolving field. This technology provides surgeons with a visual tool to identify tumour from normal tissue during surgical resection, with the aim of reducing the positive margin rate and improving oncological outcomes for patients. Although FGS has shown promise, currently only non-targeted agents such as indocyanine green have been adopted with known limitations, and therefore our objective was to develop and test a sarcoma specific agent for this purpose. Membrane type-1 matrix metalloproteinase (MT1-MMP) is a cell surface proteinase overexpressed in sarcoma tissue and cell lines, and its function and structure are well understood. We therefore selected MT1-MMP as a suitable molecular target for our antibody-based imaging agent. Our aim was to develop a targeted tracer that can be used for both pre- and intra-operative imaging of sarcoma tumours. We proposed the development of a dual-modality imaging agent that contains both a positron emitting radioisotope, allowing the use of positron emission tomography (PET) for pre-operative imaging, and a near infrared (NIR) fluorescent probe for intraoperative FGS. To overcome issues associated with a non-site selective antibody conjugation method, such as potential blocking of antigen binding sites, we sought to implement a reliable site-selective conjugation method. We wanted to evaluate the radioimmunoconjugate (RIC) both in vitro and in vivo to determine its target specificity, binding properties and biodistribution, to assess whether this probe has potential as a pre- and intra-operative imaging tool for sarcoma.

Methods: We report the preclinical development and evaluation of a dual-modal (PET/NIR) radioimmunoconjugate ([89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW) that is targeted against the matrix metalloprotease MT1-MMP. [89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW and a non-specific IgG control were synthesised through a chemoenzymatic site-selective conjugation method (figure 1) and characterised by UV/Vis spectroscopy, radio-SDS-PAGE and LCMS. The stability of the RIC was evaluated in PBS, mouse serum and human serum over 7 days. The RIC was analysed using saturation binding and immunoreactivity assays to determine binding properties with HT1080-WT (MT1-MMP positive) or HT1080-KO (MT1-MMP negative) dedifferentiated chondrosarcoma cell lines. The HT1080-KO cell line was generated via CRISPR Cas9 knockout of the MMP14 gene encoding for MT1-MMP. A novel dedifferentiated chondrosarcoma mouse model based on intra-femoral inoculation of HT1080-WT or HT1080-KO cells in NSG mice was used to evaluate target binding and biodistribution of our RIC (figure 3A). Mice were imaged for fluorescence and Cerenkov luminescence using IVIS imaging at 24, 48 and 72 h post-injection of the RIC.

Results: [89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW and an IgG control were synthesised (IRDye800CW degree of labelling = 1.74 +/- 0.47 and 1.64 +/- 0.78 respectively), radiolabelled and isolated (radiochemical purity = 99.38 +/- 0.88 % and 99.71 +/- 0.25 % respectively), as per figure 1. Radio-SDS-PAGE and LCMS show site-selective conjugation of IRDye800CW and DFO to the heavy chain of the antibodies (figure 2). Saturation binding and immunoreactivity assays indicated desirable binding properties, and the ability of the RIC to selectively bind MT1-MMP was further confirmed via radio-immunostaining of biopsied osteosarcoma human and mouse tissues. Using an orthotopic mouse model (figure 3A), fluorescence and Cerenkov radiation images acquired 24, 48, and 72 h post injection of [89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW indicated preferential uptake in the femora of NSG mice bearing HT1080-WT tumours (figures 3B and 4), and ex vivo gamma counting revealed significantly higher uptake in the targeted group (17.64 +/- 3.84 %ID/g) compared to controls (p = 0.0006). This was supported by autoradiography images of sections of invaded thigh muscle, which showed higher fluorescence and autoradiography signal in the targeted group compared to controls.

Conclusion: This preclinical study supports [89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW as a promising dual-modal imaging agent for potential use in pre-operative imaging, surgical planning and intra-operative fluorescence guided surgery for bone and soft tissue sarcomas. High target specificity of the RIC to MT1-MMP has been demonstrated both in vitro and in vivo using a novel mouse model of dedifferentiated chondrosarcoma. The future development of targeted RICs is an important step to overcome the current limitations of non-targeted probes for FGS in sarcoma, to obtain better resection margins and improve patient outcomes. Taken together, the results from this study support the future development of [89Zr]Zr-DFO-anti-MT1-MMP-IRDye800CW for this application.

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