ESTRO 2020 Abstract book

S113 ESTRO 2020

Maastricht Comprehensive Cancer Center- Maastricht University Medical Center- Maastricht- The Netherlands, Department of Precision Medicine- M-Lab, Maastricht, The Netherlands ; 2 Convert, Pharmaceuticals, Liege, Belgium ; 3 University of Auckland, Auckland Cancer Society Research Centre- School of Medical Science, Auckland, New Zealand Purpose or Objective Immunotherapy (IO) has become an important therapy for cancer. The immunocytokine L19-IL2 successfully attracts and activates immune cells at the tumour site by binding to the tumour vasculature and providing an IL2 stimuli. However, immunotherapies such as L19-IL2 are often not reaching its full potential due to an immune suppressive tumour microenvironment (TME). A key factor accountable for treatment resistance and immunosuppression is hypoxia. Specifically targeting hypoxia could hence improve the infiltration and function of immune cells. CP- 506 is a drug activated in hypoxic tumour regions where it has the ability of cell kill due to the induction of DNA damage. In addition, immune checkpoint inhibitor antibody anti-PD-L1 (aPD-L1) will be used due to the current standard of care status for a variety of cancers and the ability to overcome immune exhaustion. We hypothesise that targeting tumour hypoxia using CP-506 combined with immunotherapy increases therapeutic outcome. Material and Methods Balb/c mice were injected unilaterally with CT26 or C51 colon carcinoma cells and randomized at an average tumour volume of 200 mm 3 . Treatment groups (n=6 to 8) consist of a control group, single agents, CP-506 + L19-IL2 or aPD-L1 and CP-506 + L19-IL2 + aPD-L1. L19-IL2 (1 µg/g, i.v.) was administered on day 1,3 and 5, aPD-L1 (10 µg/g, ip) at day 1,3,5,8 and 10 and CP-506 (600 mg/kg, i.p.) was administered for five consecutive days starting at day 1. Outcome was determined by growth delay of tumours followed-up until 4 times start volume was reached. In an additional experiment, tumours were taken out at day 4 and 6 of treatment for immunohistochemistry staining and flow cytometry analysis. Results Growth delay was significantly enhanced by CP-506 in combination with L19-IL2 compared to treatment with single agents (p< 0.05). Immunohistochemistry staining indicated an increased necrotic fraction in the combination group. Triple combination (CP-506 + L19-IL2 + aPD-L1) further prolonged survival. Flow cytometry analysis demonstrated increased intratumoural immune infiltration in the combination groups containing L19-IL2 with in the initial treatment response an increase in dendritic cells and a gradual increase in CD8 cytotoxic T cells in ratio to CD4 T regulatory cells. Conclusion Our study indicates that the use of the hypoxia-activated prodrug CP-506 enhances efficacy of L19-IL2 and L19-IL2 + aPD-L1. Triple combination therapy was most beneficial. Not only was tumour growth delayed, tumours were also more necrotic due to the synergy between CP-506 and L19- IL2. Intratumoural and systemically, innate and adaptive immune systems were upregulated upon treatment with L19-IL2 in double and triple combination treatment. Taken together, based on these results CP-506 appears to be an effective combination strategy with IO. OC-0204 Molecular imaging of the endogenous hypoxia related marker CA IX with 111In-labeled VHH B9 S. Van Lith 1 , F. Huizing 2 , B. Hoeben 2 , S. Doulkeridou 3 , M. Gotthardt 1 , P. Van Bergen en Henegouwen 3 , S. Heskamp 1 , J. Bussink 2 1 Radboud University Medical Center, Radiology and Nuclear Medicine, Nijmegen, The Netherlands ; 2 Radboud University Medical Center, Radation Oncology, Nijmegen, The Netherlands ; 3 Utrecht University, Cell Biology-

Department of Biology- Science FacultyUtrecht, Utrecht, The Netherlands Purpose or Objective Hypoxia is present in the majority of solid tumors and is associated with poor outcome and radioresistance. Carbonic anhydrase IX (CAIX) is a transmembrane enzyme that is upregulated by cells under hypoxic conditions. Therefore, non-invasive imaging of CAIX could be of prognostic value and it may allow radiotherapy planning and treatment effect monitoring. The aim of this study was to validate and optimize SPECT imaging of CAIX expression in a head-and-neck squamous cell carcinoma model using an anti-CAIX variable domain of heavy chain antibody (VHH, nanobody). Material and Methods VHH B9 was conjugated site-specifically with maleimide- DTPA and labeled with 111 In. The binding affinity and internalization of [ 111 In]In-DTPA-B9 was analyzed using SKRC-52 cells, which ubiquitously express CAIX. Subsequently, a dose-escalation study was performed in athymic nude mice with subcutaneous SCCNij153 head- and-neck cancer xenografts. Targeting specificity was determined by blocking specific uptake with unlabeled VHH B9, and by analyzing tumor uptake of an 111 In-labeled irrelevant VHH R2. A subgroup of mice was co-injected with plasma expander gelofusin, to reduce renal retention of [ 111 In]In-DTPA-B9. Tracer uptake at 4 hours after injection was determined by ex vivo radioactivity counting and SPECT/CT scans. Furthermore, spatial correlation between tracer uptake on autoradiography images and immunohistochemical staining of CAIX expression was analyzed. Results DTPA-B9 was radiolabeled with 111 In at a specific activity of 4 MBq/µg. [ 111 In]In-DTPA-B9 bound to SKRC-52 cells with high affinity (IC 50 = 11.3 nm, Kd = 27.2 nM) and the internalization rate of the tracer was relatively low (K e = 0.01). A protein dose of 5 µg resulted in the highest uptake in SCCNij153 tumors in vivo (1.05±0.14%ID/g), with tumor- to-blood and tumor-to-muscle ratios of 11.5 and 24.7, respectively. Unlabeled B9 reduced tumor uptake to 0.30±0.03%ID/g and irrelevant [ 111 In]In-DTPA-R2 showed tumor uptake of 0.20±0.14%ID/g. Gelofusin did not significantly alter kidney uptake of [ 111 In]In-DTPA-B9. Immunohistochemistry and autoradiography images showed co-localization of [ 111 In]In-DTPA-B9 and expression of CAIX. Conclusion [ 111 In]In-DTPA-B9 VHH shows specific targeting of CAIX expression in head-and-neck cancer xenografts. In follow- up studies we will compare this tracer to other CAIX imaging tracers and we will determine its potential for treatment selection and monitoring of hypoxia responses to therapy. OC-0205 NOTCH inhibition promotes stem cell renewal and epithelial integrity in irradiated bronchial cells L. Giuranno 1 , E. Moreno Roig 2 , C. Wansleeben 2 , M. Vooijs 2 1 Maastricht Radiation Oncology MAASTRO, Radiotherapy, Maastricht, The Netherlands ; 2 Maastricht University, Radiotherapy, Maastricht, The Netherlands Purpose or Objective Tumor control by radiotherapy is limited by dose-limiting side-effects on normal tissues which negatively affect quality of life. The NOTCH signaling pathway plays a key role in lung cell differentiation, and regeneration of the airway epithelium. However, the mechanism through which NOTCH inhibition integrates with airway repair and cellular differentiation is not fully understood. We hypothesized that NOTCH inhibition has a protective effect in cells exposed to radiotherapy and may represent a potential target for intervention to modulate normal tissue toxicity.

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