Enrichment and Expansion with Nanoscale Artificial Antigen Presenting Cells for Adoptive Immunotherapy

Adoptive immunotherapy (AIT) can mediate durable regression of cancer, but widespread adoption of AIT is limited by the cost and complexity of generating tumor-specific T cells. Here we develop an Enrichment + Expansion strategy using paramagnetic, nanoscale artificial antigen presenting cells (aAPC) to rapidly expand tumor-specific T cells from rare naïve precursors and predicted neo-epitope responses. Nano-aAPC are capable of enriching rare tumor-specific T cells in a magnetic column and subsequently activating them to induce proliferation. Enrichment + Expansion resulted in greater than 1000-fold expansion of both mouse and human tumor-specific T cells in 1 week, with nano-aAPC based enrichment conferring a proliferation advantage during both <i>in vitro</i> culture and after adoptive transfer <i>in vivo</i>. Robust T cell responses were seen not only for shared tumor antigens, but also for computationally predicted neo-epitopes. Streamlining the rapid generation of large numbers of tumor-specific T cells in a cost-effective fashion through Enrichment + Expansion can be a powerful tool for immunotherapy

Screening of immunogenic yellow fever peptides.

<p>Blood samples were obtained from 220 vaccinees. 653 peptides from Env and non-structural proteins were organized in pools and tested by ELISPOT assay. Then, 108 immunogenic peptides were selected from the pools and tested individually. In total, 16 peptides were positive in at least 10% the subjects. Statistical association showed that nine immunogenic peptides were associated with HLA class-I. Seven from these peptides could activate CD4⁺ depleted PBMCs from YF-17DD vaccinees <i>ex vivo</i>. In addition, 9–10 mers peptides covering the NS4b_77–91 were cultured in CD8⁺ T-cells isolated from HLA-A*02:01-positive individuals. Four epitopes (NS4b_77–85, NS4b_75–83, NS4b_75–84, NS4b_76–84) were able to induce a specific response. Biochemical binding assays indicated that the most prevalent immunogenic peptides could bind multiple HLA-II molecules.</p

Binding affinity in nanomols of the most frequent peptides that activated T-cells.

<p>Numbers in bold: values of IC₅₀ below the cutoff of 1000 nmol by which determine that the peptide has affinity to the HLA molecule.</p><p>(−): not determined.</p

Summary of screening peptide pools (first round).

<p>The table shows a list of all yellow fever proteins mapped, total number of peptides tested for each protein (No. of peptides), volunteers (No. vaccinees) and peptides into a pool that were positive at least one subject (No. of peptides recognized).</p>*<p>Env peptide pools. Twenty peptide pools were positive at least one subject.</p

YF-17DD Immunodominant HLA-A*0201 restricted epitope in the NS4b protein.

<p><b>A</b>. Sequences of the peptides used to identify the immunogenic nonamer epitope in NS4b. IC₅₀ values for IEDB-AR prediction were calculated; <b>B</b>. PBMCs from HLA-A2 vaccinees were cultured in the presence or absence of 9–10mer (NS4b_77–85, NS4b_76–84, NS4b_75–83, NS4b_75–84) peptides. ELISPOT assay; <b>C.</b> Intracellular cytokine staining were performed. All peptides analyzed could activate CD8⁺ T-cells and the expansion of NS4b_76–84-specific CD8⁺ T-cells was the highest among the four peptides tested. Plots gated on CD8⁺ T-cells for one representative donor are shown.</p