Develoment of an anti-CD3 probe for in vivo imaging of endogenous immune cells

496 Objectives The noninvasive detection and quantification of T cells is important for monitoring success of immuno-therapeutics in the fields of oncology, autoimmunity, and infection.1 Current methods for lymphocyte detection are limited to cell isolation from peripheral blood; due to the invasive nature of tissue biopsy such sampling is done less frequently.2 Thus, methods for dynamic measuring of T cell localization and migration beyond peripheral blood in vivo remains technically challenging. The objective of this study was to develop a PET probe for imaging the endogenous distribution of T cells in murine models. Methods Parental 2C11 anti-mouse CD3 antibody was enzymatically fragmented with pepsin.3 The generated F(ab’)2 fragment was modified with NOTA-NCS via non-specific lysine conjugation. Tracer binding affinity was determined via a competitive inhibition assay on isolated murine T cells.4 The biodistribution of the tracer was evaluated in immune compromised NSG and competent, Balb/c mice at 4 and 24 h p.i. Results Pepsin digestion effectively removed the Fc region of the antibody, the resulting F(ab’)2 fragment was isolated with a product yield of 31 %. Optimal Cu-64 labeling was achieved with 0.1 M NH4OAc pH 5.5, with a specific activity of 2 Ci/µmol. A three-fold decrease in CD3 binding affinity was observed as a result of fragmentation and NOTA loading [IgG Kd = 13.7; F(ab’)2-NOTA Kd = 41.3 nM]. In immune competent mice, the tracer specifically accumulated in lymphoid organs (lymph nodes and spleen). Conversely, in immune compromised NSG mice accumulation was limited to the kidneys due to tracer clearance. Conclusions The anti-mouse CD3 F(ab’)2 PET probe was successfully used to image the endogenous distribution of T cells in vivo. The development of this tracer can potentially serve as a noninvasive tool for the dynamic tracking of T cell migration in syngeneic immunotherapy models. Research Support Amgen Inc.