Selective Depletion of CD45RA Expressing Cells to Enrich Antigen Specific T-Cells for Immunotherapy

T-cell therapies targeting viral antigens in a range of virus-associated malignancies present challenges to successful manufacturing of virus-antigen-specific T-cells (VSTs) from patients where these VSTs potentially circulate in low-frequency and are rendered dysfunctional in the immunosuppressive tumor microenvironment.  In this study, we investigated and overcame the manufacturing hurdles encountered in the clinical trial NCT01555892 by using Epstein-Barr Virus specific T-cells (EBVSTs) targeting Epstein-Barr virus (EBV) antigens against EBV+ lymphoma. 

We identified manufacturing failures in ~25% of patients resulting from the inability to grow antigen specific T-cells and/or outgrow NK-cells. Since the majority of VSTs reside in the CD45RA negative memory fraction of PBMCs – and the CD45RA positive population includes naïve T-cells, suppressive T-regs, and NK-cells – we hypothesized that the low antigen-specificity results from the outgrowth of non-EBV specific cells and enrichment of memory T-cells prior to stimulation may increase their antigen specificity and hence the clinical potency of our EBVSTs.

Using Miltenyi Biotec CD45RA MicroBeads, human and MACS® columns, we selectively removed CD45RA expressing cells from whole (W-) PBMCs prior to stimulating the resulting CD45RA depleted (RAD)-PBMCs with EBV peptide libraries to generate derivative RAD-EBVSTs. The RAD-EBVSTs demonstrated increased proliferation, enhanced antigen-specificity, and polyfunctionality compared to W-EBVSTs including more rapid EBV+ tumor clearance in our murine model. Most importantly, RAD decreased the frequency of NK cells dominating some of the patients’ EBVSTs and overcoming our manufacturing failures by enabling the outgrowth of these cells from patient PBMCs that previously failed to. Additionally, CD45RA depletion approach enhanced the antigen specificity of VSTs to HPV, VZV, and adenoviral antigens.

Learning Objectives:

1. Classify the obstacles to successful manufacturing of antigen-specific T-cells in immunotherapy.

2. Highlight the effects of CD45RA+ subsets in expansion of antigen-specific memory T-cells and successful application of CD45RA depletion in generating virus-antigen-specific T-cells.

3. Discuss the implications of in-vitro generation and in-vivo infusions of antigen-specific T-cells