A novel TCR-like CAR with specificity for PR1/HLA-A2 effectively targets myeloid leukemia in vitro when expressed in human adult peripheral blood and cord blood T cells

Background aims The PR1 peptide, derived from the leukemia-associated antigens proteinase 3 and neutrophil elastase, is overexpressed on HLA-A2 in acute myeloid leukemia (AML). We developed a T-cell receptor (TCR)-like monoclonal antibody (8F4) that binds the PR1/HLA-A2 complex on the surface of AML cells, efficiently killing them in vitro and eliminating them in preclinical models. Humanized 8F4 (h8F4) with high affinity for the PR1/HLA-A2 epitope was used to construct an h8F4- chimeric antigen receptor (CAR) that was transduced into T cells to mediate anti-leukemia activity. Methods Human T cells were transduced to express the PR1/HLA-A2-specific CAR (h8F4-CAR-T cells) containing the scFv of h8F4 fused to the intracellular signaling endo-domain of CD3 zeta chain through the transmembrane and intracellular costimulatory domain of CD28. Results Adult human normal peripheral blood (PB) T cells were efficiently transduced with the h8F4-CAR construct and predominantly displayed an effector memory phenotype with a minor population (12%) of central memory cells in vitro. Umbilical cord blood (UCB) T cells could also be efficiently transduced with the h8F4-CAR. The PB and UCB-derived h8F4-CAR-T cells specifically recognized the PR1/HLA-A2 complex and were capable of killing leukemia cell lines and primary AML blasts in an HLA-A2-dependent manner. Conclusions Human adult PB and UCB-derived T cells expressing a CAR derived from the TCR-like 8F4 antibody rapidly and efficiently kill AML in vitro. Our data could lead to a new treatment paradigm for AML in which targeting leukemia stem cells could transfer long-term immunity to protect against relapse

Third-party umbilical cord blood-derived regulatory T cells prevent xenogenic graft-versus-host disease

Background aims: Naturally occurring regulatory T cells (Treg) are emerging as a promising approach for prevention of graft-versus-host disease (GvHD), which remains an obstacle to the successful outcome of allogeneic hematopoietic stem cell transplantation. However, Treg only constitute 1-5% of total nucleated cells in cord blood (CB) (<3× 106 cells), and therefore novel methods of Treg expansion to generate clinically relevant numbers are needed. Methods: Several methodologies are currently being used for ex vivo Treg expansion. We report a new approach to expand Treg from CB and demonstrate their efficacy in vitro by blunting allogeneic mixed lymphocyte reactions and in vivo by preventing GvHD through the use of a xenogenic GvHD mouse model. Results: With the use of magnetic cell sorting, naturally occurring Treg were isolated from CB by the positive selection of CD25+ cells. These were expanded to clinically relevant numbers by use ofCD3/28 co-expressing Dynabeads and interleukin (IL)-2. Ex vivo-expanded Treg were CD4+25+FOXP3+127lo and expressed a polyclonal T-cell receptor, Vβ repertoire. When compared with conventional T-lymphocytes (CD4+25- cells), Treg consistently showed demethylation of the FOXP3 TSDR promoter region and suppression of allogeneic proliferation responses in vitro. Conclusions: In our NOD-SCID IL-2Rγnull xenogeneic model of GvHD, prophylactic injection of third-party, CB-derived, ex vivo-expanded Treg led to the prevention of GvHD that translated into improved GvHD score, decreased circulating inflammatory cytokines and significantly superior overall survival. This model of xenogenic GvHD can be used to study the mechanism of action of CB Treg as well as other therapeutic interventions

High avidity cyclin E1-derived peptide-specific CTL kill lymphoid leukemia cells and cross-recognize a homologous cyclin E2-derived peptide

Abstract Using a similar strategy that successfully identified PR1 as a leukemia-associated antigen (LAA), we identified two homologous HLA-A2-restricted peptides from cyclin E1 (CCNE1) and cyclin E2 (CCNE2) that could be used to elicit peptide-specific CTL from healthy donors in vitro. Two homologous nonameric peptides from CCNE1 (CCNE1144–152) and CCNE2 (CCNE2144–152), which differ by a single amino acid at position 7, have equal binding affinity for HLA-A2 and each elicited peptide-specific CTL with equal efficiency, as measured by specific lysis of T2 cells pulsed with either peptide (CCNE1 59.7% vs CCNE2 72.6% specific lysis, respectively, at E: T 10:1). TCR-Vβ spectratype analysis showed CCNE1-CTL clones to be derived from 3 Vβ families, while CCNE2-CTL clones were derived from a single Vβ family. The CCNE1-CTL and the CCNE2-CTL bound to each of the CCNE1/A2 and CCNE2/A2 tetramers, but staining intensity was greater for the CCNE1-CTL, suggesting greater TCR avidity of the CCNE1-CTL for both peptides. Because each clone cross-recognized the other homologous peptide, we hypothesized that each clone would efficiently kill leukemia that over-expressed either or both CCNE1 and CCNE2 proteins. FACsorted high avidity CTL showed higher specific lysis of peptide-pulsed T2 than did low avidity CTL (38.8% vs 31.9% specific lysis, respectively, at E: T 10:1, p = 0.02). The fluorescence decay of tetramer dissociation (ln (peptide/HLA-A2 tetramer)) over time was linear for each clone, suggesting that avidity was proportional to TCR affinity and tetramer dissociation t1/2 was determined based on first order kinetics. CCNE1-CTL had higher affinity for CCNE1144–152/HLA-A2 (CCNE1/A2, t1/2=84.5min; CCNE2/A2, t1/2=25.3min) and preferentially killed CCNE1144–152-pulsed T2 cells (CCNE1, 56.9% vs CCNE2, 38%, respectively, at E: T 10:1, p = 0.004). Interestingly, CCNE2-CTL also had higher TCR affinity for CCNE1144–152/HLA-A2 (CCNE1/A2, t1/2=29.5min; CCNE2/A2, t1/2=10.7min), but showed only slightly higher specific lysis of CCNE1144–152-pulsed T2 cells (CCNE1 = 49.3% vs CCNE2 = 44.2% specific lysis, respectively, at E: T 10:1, p = 0.33). Each clone specifically lysed HLA-A2+ T-ALL leukemia cells in proportion to both CCNE1 and CCNE2 protein overexpression assessed by Western blot (CCNE1-CTL, R2=0.89; CCNE2-CTL, R2=0.88). In contrast, healthy HLA-A2+ BM cells, which do not overexpress CCNE1 or CCNE2, and control HLA-A2− CML cells that overexpress both proteins, were not lysed. Both the high and low affinity clones showed equal lysis of T-ALL cells that expressed large amounts of each protein (specific lysis = 24.3% by CCNE1-CTL, vs lysis = 23.8% by CCNE2-CTL, at E: T 10:1). However, high affinity CCNE1-CTL killed T-ALL cells significantly better than low affinity CCNE2-CTL (16.8% vs 6.6% lysis, respectively, at E: T 10:1, p =0.02) when the T-ALL expressed a 2.5-fold lower amount of both CCNE1 and CCNE2 proteins. We conclude that the CCNE1 and CCNE2 homologous self-peptides are lymphoid leukemia-associated antigens. Furthermore, while the higher TCR affinity of CCNE1-CTL suggests that the CCNE1 peptide is the more dominant epitope, ultimate target susceptibility is enhanced due to degeneracy of the resulting CTL clones against homologous peptide epitopes