Neoantigen-specific T cells are increasingly viewed as important immunotherapy effectors, but physically isolating these rare cell populations is challenging. Here, we describe a sensitive method for the enumeration and isolation of neoantigen-specific CD8+ T cells from small samples of patient tumor or blood. The method relies on magnetic nanoparticles that present neoantigen-loaded major histocompatibility complex (MHC) tetramers at high avidity by barcoded DNA linkers. The magnetic particles provide a convenient handle to isolate the desired cell populations, and the barcoded DNA enables multiplexed analysis. The method exhibits superior recovery of antigen-specific T cell populations relative to literature approaches. We applied the method to profile neoantigen-specific T cell populations in the tumor and blood of patients with metastatic melanoma over the course of anti-PD1 checkpoint inhibitor therapy. We show that the method has value for monitoring clinical responses to cancer immunotherapy and might help guide the development of personalized mutational neoantigen-specific T cell therapies and cancer vaccines.

Protein catalyzed capture agents (PCCs) are synthetic antibody surrogates that can target a wide variety of biologically relevant proteins. As a step toward developing a high-throughput PCC pipeline, we report on the preparation of a barcoded rapid assay platform for the analysis of hits from PCC library screens. The platform is constructed by first surface patterning a micrometer scale barcode composed of orthogonal ssDNA strands onto a glass slide. The slide is then partitioned into microwells, each of which contains multiple copies of the full barcode. Biotinylated candidate PCCs from a click screen are assembled onto the barcode stripes using a complementary ssDNA-encoded cysteine-modified streptavidin library. This platform was employed to evaluate candidate PCC ligands identified from an epitope targeted in situ click screen against the two conserved allosteric switch regions of the Kirsten rat sarcoma (KRas) protein. A single microchip was utilized for the simultaneous evaluation of 15 PCC candidate fractions under more than a dozen different assay conditions. The platform also permitted more than a 10-fold savings in time and a more than 100-fold reduction in biological and chemical reagents relative to traditional multiwell plate assays. The best ligand was shown to exhibit an in vitro inhibition constant (IC50) of ∼24 μM.

Abstract At the heart of most cancer immunotherapies are the specific interactions between the principal cancer cell killers, T cells, and antigens presented by the tumor cells. Those interactions may be exposed through the use of checkpoint inhibitors, or they can be amplified through the use of engineered cell-based therapies. A class of antigens that has emerged as being particularly important are neoantigens, which are small fragments of mutated proteins that contain the mutation, and, for CD8+ T cell recognition, are presented by MHC Class I. In principle, neoantigens that draw T cells into a tumor can comprise personalized vaccines, and the T cell receptors (TCRs) that recognize those neoantigens can be engineered as personalized cellular therapies. However, identifying which neoantigens are drawing which T cells into the tumor and promoting cell killing has been a challenge. In this talk I will describe an approach, called nanoparticle-barcoded nucleic acid cell sorting (NP-barcoded NACS), designed for this purpose. The approach begins with a list of putative neoantigens generated from in silico analysis of the tumor genome and transcriptome. Those neoantigens are prepared as a NP-barcoded NACS library so that each neoantigen is paired with a DNA barcode. The entire library is then used to precipitate neoantigen specific CD8+ T cell populations from the patient's tumor or blood. Each cell is then individually decoded using sequential fluorescent reads to identify its cognate antigen, and the T cells may be further separated for single cell TCR sequencing. We have used this method to analyze tumor materials and blood from melanoma cancer patients responding to anti-PD-1 therapy, mouse models of immunotherapy, and tumor materials collected from various other cancer patients. The method, which is shown to be at least an order of magnitude more sensitive than alternative approaches, requires very little material, and so may be used for the analysis of non-expanded tumor infiltrating lymphocytes (TILs) or peripheral blood mononuclear cells (PBMCs). I will discuss some of the analytical details of this method, and prospects for harnessing it to develop personalized immunotherapies. I will also provide results of kinetic studies, via blood analysis, that appear to provide insights into patient responses to checkpoint inhibitor therapy. Citation Format: James R. Heath, Songming Peng, Alice Hsu, Shannon Esswein, John Heath, Won Jun Noh, Jesse Zaretsky, Toni Ribas. Technologies for personalizing cancer immunotherapies [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr IA30.

We report on the development of high affinity macrocyclic peptides as ligands for the capture and detection of PfHRP2, a protein without a defined tertiary structure. PfHRP2 is a key biomarker of the fatal falciparum strain of malaria. This intrinsically disordered protein has repeated epitopes that have inconstant occurrence over geographic regions. Thus, current antibody based diagnostic tests that target a single epitope do not account for the sequence diversity of PfHRP2 and exhibit variable performance. Ligands selected via high throughput epitope-targeted in situ click screening with combinatorial macrocylic peptide libraries yield binders with high affinity and selectivity for their protein targets. These macrocyclic peptide binders offer greater biochemical and thermal stability than antibodies. We provide a general strategy for the amplification of sensitivity in disease detection through the development of ligands that target multiple epitopes in a single and unstructured protein biomarker. We apply our macrocyclic peptide ligands towards the development of an antibody free diagnostic test for PfHRP2.