Abstract LB-163: Development of an immunoPET tracer for imaging human CD8+ T cells
Authors: Olafsen Tove , Ho David , Epin Eric , Zhang Green , Torgov Michael , Beigarten Charlie , Agahi Giti , Cabral Edward , Wu Anna M. , Gudas Jean M. 
Published: May 2015

Abstract
Background: Infiltration of CD8+ T cells into human tumors is associated with an increased disease-specific survival in many cancers. A better understanding of T cell trafficking and characterization of the subset of CD8+ T cells with the highest localized activity would improve the ability to schedule and select patients for chemo- and immuno-therapy regimens. To facilitate this objective, we have engineered a humanized anti-CD8 antibody fragment and used it to image human CD8+ T cells in a tumor xenograft and humanized mouse model.
Methods: The VH and VL sequences of a murine anti-CD8 antibody were cloned by RT-PCR, engineered into minibody (Mb) fragments (scFv-CH3 dimer) of 80 kDa in size and humanized by CDR grafting onto a human germline framework. Multiple humanized variants were evaluated and the lead Mb candidate was selected based on ELISA, flow cytometry and SPR binding properties. The lead Mb, IAb22G3M1 was transiently expressed in CHO cells and purified by Protein L chromatography. PET imaging was performed with desferrioxamine (Df) conjugated IAb22G3M1 radiolabeled with Zr-89 (T1/2 3.3 d). SCID mice bearing subcutaneous HPB-ALL (CD8+ve) or Daudi (CD8-ve) xenografts were serially imaged at 4, 24 and 41 h after i.v. administration of 89Zr-IAb22G3M2 and tissues harvested and counted to determine the biodistribution at the time of sacrifice. The Mb was also evaluated in NOD-SCID-Gamma (NSG) mice that were engrafted with 20 million human PBMCs. In this latter study IAb22G3M1 was radiolabeled with Cu-64 (T1/2 12.7 hrs) following NODAGA conjugation. Mice were imaged at 4 and 7 hrs followed by tissue collection for biodistribution. NSG mice that were not grafted with PBMCs served as experimental controls.
Results: Purification of IAb22G3M1 yielded a product that migrated at the expected MW of 80 kDa with very low (&lt;1%) HMW aggregate. Cell based binding to human CD8 expressing T cells demonstrated a relative affinity of &lt;0.1 nM. Following conjugation and radiolabeling, immunoreactivity was preserved. PET imaging of HPB-ALL xenografts showed specific tumor targeting at 24 and 41 h. The uptake in the CD8 positive HPB-ALL tumors was ∼6-fold higher (10.0±2.4%ID/g) than in the negative Daudi tumors (1.6±0.4%ID/g). Positive tumor to blood ratio was 13.5 for the positive vs. 3.5 for the negative tumor. In mice engrafted with human PBMCs, spleen uptake was 35.8±9.2%ID/g compared to 16.8±3.2%ID/g in control mice. The apparent splenic antigen sink in PBMC grafted mice likely explains the rapid blood clearance and the different tissue uptakes observed between the two groups.
Conclusion: We have successfully generated a functional anti-CD8 imaging agent that can be used to detect and monitor T cell trafficking and expansion in vivo. Pre-clinical PET imaging studies suggest that IAb22G3M1 is a promising tracer for detecting CD8 positive immune cells in vivo in suitable models paving the way for clinical translation.
Citation Format: Tove Olafsen, David Ho, Eric Epin, Green Zhang, Michael Torgov, Charlie Beigarten, Giti Agahi, Edward Cabral, Anna M. Wu, Jean M. Gudas. Development of an immunoPET tracer for imaging human CD8+ T cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-163. doi:10.1158/1538-7445.AM2014-LB-163