Design de miniproteínas com propriedades farmacocinéticas melhoradas para teranóstica de cancro com recurso a técnicas de imagiologia molecular

OB 1 - Miniproteins (up to 100) will be generated with the Rosetta software (collaboration with David Baker and Derrick R Hicks from UW and Rita Melo IST-C2TN). A preliminary de novo construct with nM affinity for PD-L1 is already available. The top 6 more negatively charged sequences having a single cysteine will be expressed and purified. Two minibinders, one neutral and one positively charged, will be used as control. Outcome 1 – “PD-L1 miniproteins” with different net charges at physiologic pH will be developed to study the effect of electrostatic interactions between positively charged residues on the miniproteins and the negatively charged surface of renal tubule cells in the overall in vivo biodistribution of the probes, namely, tumor and kidney uptake. OB 2 – In parallel with OB1, miniproteins with an engineered single cysteine will be modified under established conditions by reaction with a carbonyl acrylamide reagent conjugated to a macrocyclic chelator (e.g. NODAGA), a fluorophore (e.g. Cy3) or a drug (Val-Cit-PAB-MMAE)(with Gonçalo Bernardes from University of Cambridge, please see Letter of Support). Outcome 2 – Miniproteins targeting PD-L1 with different charges and carrying different pendant tags (radionuclide, fluorophore or drug) will be obtained for upcoming studies. OB 3 – “PD-L1 miniproteins” conjugated to a bifunctional chelator will be radiolabeled with an imaging radionuclide (e.g. Ga-67, Ga-68, or Cu-64) (with João Galamba Correia from IST-C2TN) and evaluated in tumor cell lines expressing different levels of the receptor to confirm recognition of the target. Fluorescent miniproteins will be evaluated using 3D cancer spheroids to assess penetration in the deepest regions of the tumor by confocal microscopy (with Filipa Mendes from IST-C2TN). Comparative studies with a fluorescent antibody will be also performed. Outcome 3 – Small penetrant miniproteins that bind selectively to PD-L1 expressing tumors will be identified. OB 4 – Evaluation of the pharmacokinetics of the radiolabeled proteins (up to 6) will be performed in mice bearing A20 tumors. Uptake in main tissues and tumor will be measured and expressed at different times points as % of injected activity per gram of tissue (IA/g tissue) (with Lurdes Gano from IST-C2TN). Biodistribution of two minibinders with neutral and positive charges will be also evaluated. Radiotracers with the best target to non-target ratios will be further evaluated by PET imaging. Outcome 4 – The influence of the overall charge of the miniproteins in their in vivo profile will be assessed. Probes with the best tumor to blood and tumor to kidney ratios will be explored for drug delivery. OB 5 - The therapeutic effect of the optimized miniproteins conjugated to the drug MMAE will be evaluated in cells and animals bearing A20 tumors by analysis of the tumor growth and survival rates (with Raquel Coelho from IMM). PET scans using the corresponding 68Ga surrogates before and after treatment will be acquired to evaluate response to therapy. Lastly, the miniproteins with the best efficacy based on previous studies will be conjugated to a therapeutic isotope and their efficacy for radiotherapy assessed. Outcome 5 – A miniprotein with improved pharmacokinetics (high tumor uptake and reduced kidney accumulation) will be discovered and used for immunotherapy (PD-1/PD-L1 blocking therapy), chemotherapy (MMAE drug) and radiotherapy (Lu-177).