A Novel HLA-A*0201 Restricted Peptide Derived from Cathepsin G Is an Effective Immunotherapeutic Target in Acute Myeloid Leukemia

Abstract

Purpose Immunotherapy targeting aberrantly expressed leukemia associated antigens (LAA) has shown promise in the management of acute myeloid leukemia (AML). However, because of the heterogeneity and clonal evolution that is a feature of myeloid leukemia, targeting single peptide epitopes has had limited success, highlighting the need for novel antigen discovery. In this study, we characterize the role of the myeloid azurophil granule protease cathepsin G (CG) as a novel target for AML immunotherapy. Experimental Design We used Immune Epitope Database and in vitro binding assays to identify immunogenic epitopes derived from CG. Flow cytometry, immunoblotting and confocal microscopy were used to characterize the expression and processing of CG in AML patient samples, leukemia stem cells and normal neutrophils. Cytotoxicity assays determined the susceptibility of AML to CG-specific cytotoxic T lymphocytes (CTL). Dextramer staining and cytokine flow cytometry were performed to characterize the immune response to CG in patients. Results CG was highly expressed and ubiquitinated in AML blasts, and was localized outside granules in compartments that facilitate antigen presentation. We identified five HLA-A*0201 binding nonameric peptides (CG1-CG5) derived from CG, and demonstrated immunogenicity of the highest HLA-A*0201 binding peptide, CG1. We showed killing of primary AML by CG1-CTL, but not normal bone marrow. Blocking HLA-A*0201 abrogated CG1-CTL mediated cytotoxicity, further confirming HLA-A*0201 dependent killing. Finally, we demonstrated functional CG1-CTLs in peripheral blood from AML patients following allogeneic stem cell transplantation. Conclusion CG is aberrantly expressed and processed in AML and is a novel immunotherapeutic target that warrants further development.

Peptide/MHC Tetramer–Based Sorting of CD8+ T Cells to a Leukemia Antigen Yields Clonotypes Drawn Nonspecifically from an Underlying Restricted Repertoire

“…Samples were washed and analyzed on a MACSQuant flow cytometer. Tetramer-positive cells were enumerated in the live (far red negative), lymphocyte (FSC/SSC), “lineage” (Pacific Blue) negative, CD8 (FITC) positive gate (9-11). A negative tetramer and/or a fluorescence minus one (FMO) sample was used to set the tetramer gate.…”

Cathepsin G is broadly expressed in acute myeloid leukemia and is an effective immunotherapeutic target

“…However, CG is expressed later in myeloid differentiation, resides under a different promoter than NE and P3, and demonstrates a distinct pattern of expression, suggesting that targeting both PR1 and CG could be synergistic. 6 In our prior work 4 , we demonstrated that CG is highly expressed in primary patient AML blasts, AML cell lines, and, critically, in leukemia stem cells (LSCs). We showed that CG is localized outside azurophil granules and is ubiquitinated, favoring antigen presentation.…”

“…4 CG is largely restricted to the myeloid lineage and like proteinase 3 (P3) and neutrophil elastase (NE), the sources of the well-established HLA-A2 (i.e., HLA-A*0201) LAA PR1, 5 CG is contained within the primary granules of maturing and mature neutrophils. However, CG is expressed later in myeloid differentiation, resides under a different promoter than NE and P3, and demonstrates a distinct pattern of expression, suggesting that targeting both PR1 and CG could be synergistic.…”

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

“…The humanized antibody targeting the PR1/HLA-A2 combined epitope produced by the 8F4 hybridoma was cloned as a single chain (scFv) [18]. The genes coding the VH and VL chains of the monoclonal antibody were cloned by RT-PCR using murine variable domain-specific primers modified to generate SfiI restriction sites at the 5′ end of the amplified VL and 3′ end of the amplified VH.26.…”

“…Target cells were stained with 5 μg/ml of Calcein AM (Thermo Fisher) for 15 minutes at 37°C, washed, and resuspended at 2×10 5 /ml in culture media. Two-thousand target cells in 10 μl were incubated with effector cells (CAR-transduced or control cytotoxic T-lymphocytes (CTL)) at varying E:T ratios with 5 μl of 0.4% Trypan Blue added into each well of a Terasaki plate to quench the reaction [18, 21]. The BioTek FLx800 Microplate fluorescence reader was used to read the plate.…”