Introduction: There is a need for more nimble tools to evaluate drug activity at early stages of development, and to inform patient treatment decisions. Phase I (dose finding) and Phase II (efficacy) trials are increasingly merging, and newer “Phase IB” trials need to more rapidly assess drug activity with fewer patients. Moreover, emerging immunotherapies can induce a therapeutic effect with atypical response kinetics on imaging that differ from those classically observed with cytotoxic therapies. Noninvasive assessment of response using a blood biomarker such as cell free DNA (cfDNA) could complement tumor imaging as an early marker of response. Here we report on serial measurements of HPV16 E7 cfDNA by digital droplet PCR (ddPCR) from plasma of patients in a Phase I trial of CUE-101 in HPV16+ recurrent/metastatic head and neck squamous cell carcinoma (HNSCC). Methods/Results: 49 patients with HPV16+ HNSCC have been enrolled in the monotherapy arm of this Phase I dose escalation and expansion trial of CUE-101 monotherapy (NCT03978689). CUE-101 is a fusion protein comprised of a human leukocyte antigen (HLA) complex, HLA-A*0201; an immunodominant peptide epitope derived from the HPV16 E7 protein; 4 molecules of a reduced affinity human interleukin-2 (IL-2); and an effector attenuated- human IgG1 Fc domain. The monotherapy dose escalation arm spanned 7 dose levels, ranging from 0.06 to 8 mg/kg administered by IV infusion once every 3 weeks. Plasma samples were collected at screening and longitudinally prior to dosing in subsequent treatment cycles. HPV16 cfDNA was assayed by a previously validated ddPCR assay. Matched screening and cycle 3 plasma samples were available for 33 patients where HPV16 cfDNA was present at detectable levels. A positive plasma response, defined as a decrease of HPV cfDNA from first to second draw, was observed in 12/33 (36.5%) patients, 6 of whom (50.0%) had ≥ 50% decrease in HPV cfDNA. The most robust reductions of HPV16 cfDNA were observed at the 2 mg/kg and 4 mg/kg dose levels, with peak decreases of circulating HPV16 E7 cfDNA of 53.7% and 100%, respectively. Nevertheless, durable stable disease and clinical benefit following CUE-101 treatment was observed in patients with or without HPV cfDNA reduction at C3D1. Ongoing maturation of overall survival data in patients receiving CUE-101 monotherapy will enable exploration of correlations with changes in HPV16 cfDNA at early timepoints following initiation of treatment. Conclusion: Our data support the continued assessment of HPV16 cfDNA as an exploratory biomarker in patients receiving immunotherapy to better understand correlations with radiographic response assessments and overall clinical benefit. Citation Format: Benjamin Hanna, Grace Heavey, Apollina Goel, Steven Quayle, Yanan Kuang, Cloud Paweletz. Circulating free HPV16 DNA as a potential biomarker for immunotherapy-based trials in HPV16+ head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3365.