Generation and Expansion of PRAME-Specific Cytotoxic T-Lymphocytes for Adoptive T-Cell Therapy of Hematological Malignancies.
Authors: Quintarelli Concetta , Dotti Gianpietro , Pane Fabrizio , Rooney Cliona M. , Brenner Malcolm K. , Savoldo Barbara 
Published: October 2019

Abstract
The cancer testis antigen PRAME is a potential target for T-cell based adoptive immunotherapy for many myeloid and lymphoid malignancies, since these cells frequently either overexpress the antigen constitutively, or following induction with demethylating agents. Although several HLA class I A*0201 PRAME-derived T cell epitopes have been previously identified, the ex-vivo generation of PRAME-specific cytotoxic T lymphocytes (CTLs) has proved to be a major challenge. We have now optimized a method that consistently and reproducibly generates peptide tumor-specific CTLs. CD8+ cells selected from peripheral blood mononuclear cells. HLA A*0201 donors were first primed with autologous CD40L-activated B blasts loaded with A2-restricted peptides in the presence of low doses of IL-2, IL-7 and IL-15. Following this initial priming, we re-stimulated the T cells with an artificial antigen presenting cell line (AAPC), consisting of the human chronic myelogenous leukemia cell line K562 genetically modified to stably express the HLA-A*0201 molecule and the CD80 co-stimulatory molecule (K562/A*0201/CD80). These AAPC were loaded with HLA-A2 restricted peptides and used to stimulate CTLs in the presence of IL-2. Table 1 shows that this method consistently generated CTLs recognizing multiple HLA-A2 restricted peptides derived from well-characterized tumor-associated antigens including hTERT and PR1. The frequency of expanded T-lymphocytes was evaluated by IFNγ Elispot assay.
Table 1. Irrelevant peptide Mart1-ELA Mage3-KVA PR1-VLQ WT1-RMF hTERT-RLV hTERT-ILA Tyr-YMD Number of IFNγ Spot Forming Cells (SFC)/10^5 cells (4 donors) 63±12 895±116 676±37 325±71 560±54 650±70 1019±240 897±127 8±3 810±30 488±19 15±10 48±18 13±7 101±12 503±20 7±2 1379±105 54±11 140±10 631±19 744±33 465±8 1103±97 3±2 1430±52 2±0 3±1 3±1 20±18 376±11 632±121
The specificity of the response was confirmed by tetramer analysis and cytotoxic activity with 51Cr release assay on peptide-loaded PHA blasts. We then evaluated whether this approach could be used to generate and expand PRAME-specific CTLs. In 5 HLA A*0201 healthy donors, after priming with B-blasts and 4 stimulations with K562/A*0201/CD80 cells loaded with the PRAME-derived peptide ALY, we obtained 22±7 fold T-cell expansion. T cells were PRAME specific, as the frequency of IFNγ+ T cells after exposure to the ALY peptide was significantly higher compared to exposure to irrelevant peptide (511±260 IFNg SFC/105 cells vs. 30±10 IFNg SFC/105 cells) in 4 of the 5 donors tested. In addition, CTLs significantly lysed autologous-PHA blasts loaded with ALY (84±12% at 20:1 E:T ratio) while lysis of blasts loaded with the irrelevant peptide was <10%. We then tested whether PRAME-CTLs recognized primary tumor cells, using CD33+ blast cells selected from the peripheral blood of 2 HLA A*0201 patients with PRAME expressing Chronic Myelogenous Leukemia (as assessed by Real Time PCR). The frequency of IFNγ+ T cells was 320±31 SFC/105 when ALY-CTLs were used as effector cells, but only 6±2 SFC/105 for CTLs expanded from the same donors using an irrelevant antigen. In conclusion, our data show that we can efficiently stimulate and expand PRAME-specific CTLs, and suggest that our approach could be developed for clinical application.