Abstract
Background:Harnessing the cytotoxic properties of T cells to destroy tumor cells has been the central goal of anti-cancer immunotherapy. One approach to achieve this goal has been the use adoptive cell transfer (ACT). This method involves the isolation, in vitro manipulation and re-infusion of antigen-specific T cells into cancer patients. This approach has been used to mediate durable responses in a number of malignancies. Despite its significant therapeutic potential, the routine use of ACT has been limited. This has been due, in large part, to the high cost, intricate and labor-intensive methods required to successfully generate autologous antigen-specific T cells. There is a clear need for less expensive more efficient and streamlined approaches for generation of antigen-specific T cells for use in ACT. To this end, we have developed a T cell Enrichment+Expansion strategy using paramagnetic, Nano scale artificial Antigen Presenting Cells (nano-aAPCs), which are capable of enriching rare tumor-specific T cells in a magnetic column and activating them. Nano-aAPCs, are not only more biocompatible than traditional aAPC, but by manipulating paramagnetic nanoparticle based aAPC with magnetic fields, we propose a method to quickly generate large numbers of high frequency tumor-specific T cells. Our hypothesis is that magnetic enrichment and expansion with nano-aAPC can reduce time in culture and increase frequency of antigen-specific cells, two factors that we predict will improve T cell expansion and persistence after adoptive transfer.
Methods:HLA-Ig dimer was loaded with tumor antigen peptides for MART1, NY-ESO and WT1. To manufacture nano-APCs, magnetic nanoparticles (size 50-100nm) were decorated with loaded HLA-Ig (signal 1) and anti-CD28 antibody (signal 2). Peripheral blood mononuclear cells (PBMCs) were collected from normal donors and CD8+ T cells were isolated. CD8+ T cells were subsequently incubated with nano-APCs. The T cell/nano-APCs mixtures were then pass through a magnetic column. The relevant T cells were bound to the nano-APCs and therefore were attached to the column while the irrelevant T cells passed through the column. Relevant T cells were then washed off the column and cultured using standard T cell culture techniques for 7 days. Cell number and specificity were evaluated on Day 0 and day 7.
Results: CD8+ T cells specific for the tumor antigens MART1, NYESO and WT1 were successfully expanded using our nano-APCs. After one week, we saw fold expansions of >100-fold for all 3 antigens studied. T cell specificity increased from <1% for all 3 antigens on day 0 to 27% for NY-ESO, 16% for MART1 and 4% for WT1 on day 7. These numbers are comparable, and in most cases a vast improvement, to the numbers of antigen specific cells obtained using conventional methods in 7 days (i.e. dendritic cells as APCs).
Conclusions: The initial data presented here represent proof of principle evidence of the feasibility and efficacy of our proposed streamlined and cost effective enrichment and expansion approach for the generation of antigen specific T cells. Here we expanded significant numbers of CD8+ T cells targeting the tumor antigens MART1 NY-ESO and WT1 in just 7 days using our nano-APCs. While MART1 was used a model antigen to develop or system, both NY-ESO and WT1 are tumor antigens that are relevant for several hematological malignancies. Streamlining the generation of large numbers of high-frequency tumor-specific T cells in a cost effective, reproducible fashion through Enrichment+Expansion could be a powerful addition to current tumor immunotherapy protocols.
Disclosures
Oelke: NextImmune: Equity Ownership. Schneck:NextImmune: Equity Ownership.
High frequency of antitumor T cells in the blood of melanoma patients before and after vaccination with tumor antigens