Abstract 4147: T cell targeted peptides for monitoring immune response in melanoma
Authors: Bauknight Dustin , Buckner Andrew , Brinton Lindsey , Bullock Timothy , Kelly Kimberly 
Published: July 2016

Abstract
Melanoma develops the ability to evade immune recognition through multiple mechanisms, despite being highly immunogenic. This is demonstrated by the effective and additive response checkpoint blockade therapies have had during combination clinical trials in melanoma [1]. While the results of these trials have been promising, large portions of patients do not respond to treatment [1,2]. Additionally, many responders take months to show a response using standard criteria. In order to monitor patient response and understand the limitations of immunotherapy, we performed phage display to discover peptides targeted to tumor infiltrating lymphocytes (TILs) in melanoma tumors.
To improve phage library characterization, we developed a method to deep sequence phage with the Ilumina MiSeq platform. Phage screens using PhD7 library (NEB) performed on HUVECS and on TILs, naïve and effector T cells, and B cells harvested from mice. The phage libraries generated from these screens were deep sequenced using the Illumina MiSeq system. The sequencing data was analyzed with an efficient, custom MATLAB script, which arranges sequences by frequency determining peptide frequencies for each library. Peptide frequency was normalized to a reference library generated by amplifying an unscreened phage library. T cell screens were compared with B cell and endothelial cell screens in sequence frequency matrices. Top TIL targeting sequences were cloned into phage, and then fluorescently labeled along with insert-less control phage. Labeled phage were incubated with TILs, or effector T cells and analyzed for specificity using flow cytometry. Four phage clones were identified with at least four fold increased binding for TILs over effector T cells isolated from the spleen. Interestingly, only subsets of the TILs were bound by the targeted phage.
We have identified and validated peptides that demonstrate specificity for CD8 TILs. These peptide sequences represent candidates for development into companion diagnostic imaging agents for use with checkpoint therapies. We are currently evaluating these peptides in an in vivo tumor model to further validate their specificity and to determine their biodistributions. Peptides that validate in vivo could be developed into useful imaging agents. After peptide sequences have been validated in vivo, we will determine the phage binding partner using a phage based pulldown. The peptide binding partners discovered through this approach will provide insight into the subset of TILs bound by the phage.
[1] Wolchok, J. D. et al., N Engl J Med 2013; 369:122-133.
[2] Drake, CG. et al., Clin Cancer Res November 15, 201117.
Citation Format: Dustin Bauknight, Andrew Buckner, Lindsey Brinton, Timothy Bullock, Kimberly Kelly. T cell targeted peptides for monitoring immune response in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4147.