Background: PEP503 (aka NBTXR3) is a novel radio-enhancer composed of functionalized hafnium oxide nanoparticles that increases the energy deposition of radiotherapy. A phase 3 study in soft tissue sarcoma patients has significantly increased pCR and R0 when PEP503 was added to preop radiotherapy. This phase 1b/2 study was conducted with the aim to identify a recommended phase 2 dose (RP2D), and evaluate the efficacy of PEP503 intratumoral injection in combination with concurrent chemoradiation (CCRT) in patients with locally advanced rectal cancer (LARC). Methods: Patients with stage T3-T4 LARC (or with unresectable disease) suitable for neoadjuvant CCRT were enrolled. An intratumoral single administration of PEP503 (multiple punctures) was done 24 to 72 hours prior to IMRT of 50 Gy in 25 fractions of 2 Gy per fraction over 5-6 weeks with concurrent capecitabine or 5-FU. Traditional 3 + 3 design with 4 levels, 5%, 10%, 15%, and 22%, of the baseline GTV as measured by MRI, of PEP503 were assessed in Phase Ib. PEP503 nanoparticles intratumoral dispersion was analyzed by CT-scan. Surgery was performed 8 to 12 weeks after the completion of CCRT for patients with resectable tumors. Body kinetics was evaluated in Phase Ib. (ClinicalTrials.gov, NCT02465593) Results: Thirty-two (32) patients (male/female: 20/12; median age 62 years, range 38 to 76) were enrolled (1 dropped out before starting CCRT). In Phase 1b, 20 patients were treated and dose was escalated to 22%. RP2D was then determined as 22% with 6 patients treated at this dose level without DLT. Twelve (12) patients were included in the Phase 2. One (1) (3.2%) and 19 (61.3%) of evaluable patients (n = 31) had CR and PR, respectively, as the best tumor response across dose levels. No patient progressed as all evaluable patients (n = 31) achieved disease control for a DCR of 100%. Furthermore, twenty-five patients underwent surgery, of which 24 (96%) had microscopically clear resection margins and 5 of them (20%) had pathological complete response (pCR). The G3 AEs were diarrhea, ileus, thrombocytopenia, urosepsis, procedural haemorrhage, wound complication, hypokalaemia, and myalgia (all in 3.1%). No G4 AE were observed. The results of CT scans for nanoparticles dispersion demonstrated PEP503 remained within the tumor without leakage to the surrounding healthy tissues, before and after CCRT. In most patients, hafnium was not detected or below the Lower Limit of Quantification (LLOQ) in the circulation 60 minutes after PEP503 injection and was not found in urine. Conclusions: A single intratumoral injection of PEP503 in locally advanced or unresectable rectal adenocarcinoma undergoing capecitabine or 5-FU based CCRT is feasible and safe. The preliminary observed efficacy results may warrant further examination in larger clinical studies. Clinical trial information: NCT02465593.