Background: Treatment options are limited for patients with recurrent or metastatic human papillomavirus 16 positive (HPV16+) cancers. Generation and maintenance of HPV16+ cancers requires stable expression of HPV16-specific E7 and E6 oncoproteins, which are also a source of tumor-specific immunogenic neoantigens. HB-201 and HB-202 are replicating live-attenuated vectors based on lymphocytic choriomeningitis virus and Pichinde virus, respectively, which express the same non-oncogenic HPV16 E7E6 fusion protein and infect antigen presenting cells to induce tumor-specific T cell responses. The Phase 1 part of this study of HB-200 therapy (HB-201 single-vector therapy and HB-202/HB-201 two-vector alternating therapy) was conducted to determine RP2D for further exploration alone or in combination with pembrolizumab. Methods: The Phase 1 part used a 3+3 dose escalation design with up to 3 dose levels (DLs) of HB-201 and 4 DLs of HB-202/HB-201 explored. Patients with HPV16+ head and neck squamous cell carcinoma (HNSCC) or with other HPV16+ cancers were evaluated. Safety, tolerability, immunogenicity, and preliminary antitumor activity by Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 or immune RECIST were assessed to determine RP2D. Results: As of January 2022, 65 patients with a median of 3 prior anticancer treatments have been enrolled in the Phase 1 part of the study. All had HPV16+ confirmed genotype; the most common primary site was oropharynx, followed by anal and cervix. Adverse events were generally mild or moderate. For HB-201, 3 DLs, 2 dosing schedules and 2 administration routes were assessed across 40 patients. At DL3 of HB-201 administered intravenously (IV), dose-limiting toxicity (DLT) occurred in 1/6 patients in the HNSCC group (Grade 4 encephalopathy, fully recovered) and 1/2 patients in the non-HNSCC group (Grade 3 rash, fully recovered). Preliminary safety, efficacy, and immunogenicity data support IV injection of DL3 (5 × 10⁷ units) every 3 weeks (Q3W) as the RP2D for HB-201 single-vector therapy. For HB-202/HB-201, 4 DLs and 2 administration routes were assessed across 25 patients. At DL4 of HB-202/HB-201 IV, 1/5 subjects in the HNSCC group reported a DLT (Grade 4 hepatitis, recovering at time of discontinuation). RP2D for HB-202/HB-201 will be determined in the very near future. Tumor control, including partial response, have been observed in subjects treated with either HB-201 or HB-202/HB-201 as monotherapy. Conclusions: HB-201 and HB-202/HB-201 were generally well tolerated and showed preliminary antitumor activity in heavily pre-treated patients with HPV16+ solid tumors. DL3 was selected as RP2D for HB-201 monotherapy. In the Phase 2 part of the study a combination of HB-201 at 5 × 10⁶ units IV Q3W with pembrolizumab is being tested in HPV16+ HNSCC patients. Clinical trial information: NCT04180215.