The pathological overexpression of the C-X-C motif chemokine receptor 4 (CXCR4) in more than 23 human cancers designate CXCR4 as a “wide spectrum” molecular target in oncology. In nuclear medicine, radiolabeled molecules can specifically target such cell surface receptors and can be shaped for blending diagnostic and therapeutic within the same molecule (radio-theranostics). Recently, an endogenous antagonist of CXCR4, termed EPI-X4, has been identified by the applicants. EPI-X4 is a 16-mer peptide derived from human serum albumin that specifically binds CXCR4 but no other G-protein coupled receptors, without causing receptor activation. Several synthetic derivatives of EPI-X4 with increased affinity for CXCR4, resistance against proteolytic degradation in blood and high systemic retention times have been developed. Radiotheranostics based on EPI-X4 may thus offer new imaging tests and therapeutic options to patients suffering from CXCR4-expressing malignancies. The proposed research project would be the first study aiming to evaluate a new class of radiopharmaceuticals based on an endogenous peptide naturally present in the human body, which may have less off-target effects as compared to small molecules. Overall objectives: a) to develop a new radiotheranostic agent based on EPI-X4 for non-invasive molecular imaging and targeted radionuclide therapy of CXCR4-expressing malignancies, b) to identify the strengths and limitation of this radiotheranostic agent in vivo in comparison to existing CXCR4-radioligands, and c) to explore the clinical applicability of this new class of radiopharmaceuticals. Specific Aims: a) to synthesize and characterize a series of EPI-X4-based radioligands with high affinity for CXCR4, b) to evaluate them in vitro and in vivo in cancer cells and animal models, c) to optimize the radioligands in terms of affinity, biodistribution and pharmacokinetics, and d) to identify the radiotheranostic agent that fulfils the requirements for potential clinical translation. Methodology: Selected EPI-X4 derivatives will be C-terminally conjugated to the chelator DOTA and labeled with 68Ga for positron emission tomography (PET) imaging and with 177Lu for targeted radionuclide therapy. All radioligands will be characterized and screened in vitro for receptor recognition, affinity, and CXCR4 antagonism using cell lines with different receptor expression levels. The ligands that successfully pass these tests will be evaluated in vivo in CXCR4-expressing xenografts, including PET and single-photon emission computed tomography (SPECT) imaging, biodistribution, stability and pharmacokinetics. In vitro and in vivo data will be analysed for structure optimization. Comprehensive in vivo assessment and dosimetry studies will be performed with the best candidates. Expected results: The work covers the full spectrum of pre-clinical radiopharmaceutical development, from the design, evaluation, optimization and establishment of a new class of radiotheranostics for CXCR4-expressing malignancies. Based on this work, selection of a lead compound suitable for clinical translation will be possible. Also, it will provide key data that are required for submission of a regulatory dossier to national authorities and initiate a first-in-man study. Impact: This new class of radiotheranostics may offer new diagnostic and treatment options for various CXCR4-expressing malignancies, based on non-invasive and personalized selection of patients and subsequent CXCR4-targeted radionuclide therapy of potential responders. In addition, CXCR4 PET may be used as a prognostic imaging biomarker, as increased CXCR4 expression in tumors is associated with worse prognosis. Both have an impact on cancer patient management and health care systems.