Rare Earth Albumin Nanoparticles Engineered To Target Cytotoxic T Cells To Evaluate Response To Immunotherapy.

Abstract Checkpoint immunotherapy, through the reversal of tumor-mediated inactivation of the immune system, has shown promise in the treatment of several types of cancer. This has culminated in the approval of seven immune checkpoint inhibitors (ICIs). However, only a small population of patients respond to these drugs. Because of the physical and economic burden of ICIs on the patient, there is a critical need to identify biomarkers that can inform on the potential response to ICIs. The presence of tumor infiltrating lymphocytes (TILs) has demonstrated good prognostic value in determining if a patient should receive ICIs. Current clinical methods to assess TILs involve invasive biopsies and immunohistochemistry, which suffer from intratumoral heterogeneity, observer variability, and a lack of real-time feedback. Here, we report on near infrared light excitable rare earth metal-based nanoparticles, termed rare earth albumin nanocomposites (ReANCs), that emit shortwave infrared (SWIR) light, allowing for deep tissue imaging and high signal-to-noise ratios compared to visible or near infrared fluorescence probes. Tumor-targeted ReANCs have been previously employed to monitor tumor progression and response to chemotherapy in mouse models of breast cancer metastasis. In this study, to target CD3+ T cells, ReANCs were conjugated using the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to a peptide derived from the sequence of the CD3-ϵ receptor sub-unit. Target specific binding was validated by flow cytometry as a measure of increased uptake of peptide-conjugated ReANCs by Jurkat cells. To specifically target cytotoxic T lymphocytes, we employed the fragment antigen binding (Fab) derived from enzymatic digestion of a CD8 antibody (clone 53-6.7) with papain. The Fab fragments were conjugated to ReANCs with sulfo-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC). Conjugation was confirmed by non-reducing gel electrophoresis and high performance liquid chromatography (HPLC). A loading efficiency of approximately 60% was achieved. Target specific binding was validated by flow cytometry as a measure of increased uptake of Fab-conjugated ReANCs by T cells isolated from splenocytes. We generated a metric for measuring immune burden around tumor spheroids by pre-labeling T cells with ReANCs and co-culturing them with tumor cell spheroids in vitro. Imaging of T cells with CD3 and CD8-targeted ReANCs provides a basis for future in vivo small animal imaging studies where we will investigate the potential of this technology to track immune cells in relation to a tumor in real time. Metrics of immune cell imaging will then inform on the potential of immunotherapy and monitor response to treatment in a longitudinal study. Citation Format: Jay V. Shah, Jake N. Siebert, Amber Gonda, Rahul Pemmaraju, Shashank Kosuri, Carolina Bobadilla Mendez, Xinyu Zhao, Shuqing He, Richard E. Riman, Mei Chee Tan, Mark C. Pierce, Edmund C. Lattime, Prabhas V. Moghe, Vidya Ganapathy. Rare earth albumin nanoparticles engineered to target cytotoxic T cells to evaluate response to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2802.