From these facilities, coded, linked samples will be sent to the designated labs for analysis upon request. 5.1.1 Immune Phenotyping Exploratory immunologic studies will be conducted to evaluate the study drug’s effect on the immune response before and after treatment, to gain insight into potential biomarkers, and help improve the administered therapy. Blood will be collected as per Study Calendar 3.5. The following immune assays may be performed at the Laboratory of Tumor Immunology and Biology (LTIB) at the NCI’s Center for Cancer Research (CCR) in select participants where adequate samples are available: 1. PBMCs may be analyzed for changes in standard immune cell types (CD4 and CD8 T cells, natural killer [NK] cells, regulatory T cells [Tregs], myeloid -derived suppressor cells [MDSCs], and dendritic cells) as well as 123 immune cell subsets, using multi-color flow cytometry. 2. PBMCs from selected subjects may be analyzed for function of specific immune cell subsets, including CD4 and CD8 T cells, NK cells, Tregs, and MDSCs using flow- based assays. 3. PBMCs may be analyzed for tumor antigen- specific immune responses using an intracellular cytokine staining assay. PBMCs will be stimulated in vitro with overlapping 15- mer peptide pools encoding the tumor -associated antigens such as CEA, MUC -1 and Brachyury; control peptide pools will involve the use of human leukocyte antigen peptide as a negative control and CEFT peptide mix as a positive control. CEFT is a mixture of peptides of CMV, Epstein- Barr virus, influenza, and tetanus toxin. Post -stimulation a nalyses of CD4 and CD8 T cells will involve the production of IFN -γ, IL -2, TNF, and the degranulation marker CD107a. If sufficient PBMCs are available, assays may also be performed for the development of T cells to other tumor -associated antigens. 57 Abbreviated Title: Combo Immunotx in HPV Cancers Version Date: 05.12.2023 5.1.2 Soluble F actors Samples for soluble factor analysis will be collected as per Study Calendar 3.5. -Sera may be analyzed pre- and post -therapy for the following soluble factors: sCD27, sCD40 ligand using commercial ELISA kits. -Sera may be analyzed for changes in cytokines (IFN -γ, IL -10, IL -12, IL -2, IL -4, etc.), chemokines, antibodies, tumor -associated antigens, and/or other markers using ELISA or multiplexed assays (e.g., Mesoscale, Luminex, cytokine bead array). 5.1.3 Analyses of Tumor Tissue for Immune Markers Study of immune infiltration as well as PD -L1 status within the tumor microenvironment pre vs. post treatment by IHC and/or multiplex immunofluorescence may be performed by the GMB TIME Lab . Where available, archival tumor samples will be requested (preferably tissue samples from the last 6 months) . For participants with lesions amenable to biopsy, two optional biopsies may be performed at baseline and at first restaging. Tumor samples will be sent to the Laboratory of Pathology for disease evaluation; remaining samples will be used for research. Tissue samples for research may also be stored in CSP as described in S ection 5.3.1. 5.1.4 Assessment of HPV status In participants with available tumor tissue (either archival or by optional biopsy), HPV testing will be performed using the Roche Cobas or Becton Dickinson HPV PCR based DNA assay , if no prior HPV testing of the tumor has been performed. This will be done through collaboration with Dr.