Fucosylation Enhances Activity of Chimeric Antigen Receptor T-cells Against Lung Cancer

https://openworks.mdanderson.org/sumexp23/1060/thumbnail.jp

Pembrolizumab Enhances the Anti-Leukemia Activity of Antigen Specific Cytotoxic T Lymphocytes

https://openworks.mdanderson.org/sumexp23/1074/thumbnail.jp

Common Minor Histocompatibility Antigen Discovery Based upon Patient Clinical Outcomes and Genomic Data

BackgroundMinor histocompatibility antigens (mHA) mediate much of the graft vs. leukemia (GvL) effect and graft vs. host disease (GvHD) in patients who undergo allogeneic stem cell transplantation (SCT) [1], [2], [3], [4]. Therapeutic decision making and treatments [5] based upon mHAs will require the evaluation of multiple candidate mHAs and the selection of those with the potential to have the greatest impact on clinical outcomes. We hypothesized that common, immunodominant mHAs, which are presented by HLA-A, B, and C molecules, can mediate clinically significant GvL and/or GvHD, and that these mHAs can be identified through association of genomic data with clinical outcomes.Methodology/Principal FindingsBecause most mHAs result from donor/recipient cSNP disparities, we genotyped 57 myeloid leukemia patients and their donors at 13,917 cSNPs [6]. We correlated the frequency of genetically predicted mHA disparities with clinical evidence of an immune response and then computationally screened all peptides mapping to the highly associated cSNPs for their ability to bind to HLA molecules. As proof-of-concept, we analyzed one predicted antigen, T4A, whose mHA mismatch trended towards improved overall and disease free survival in our cohort. T4A mHA mismatches occurred at the maximum theoretical frequency for any given SCT. T4A-specific CD8+ T lymphocytes (CTLs) were detected in 3 of 4 evaluable post-transplant patients predicted to have a T4A mismatch.Conclusions/SignificanceOur method is the first to combine clinical outcomes data with genomics and bioinformatics methods to predict and confirm a mHA. Refinement of this method should enable the discovery of clinically relevant mHAs in the majority of transplant patients and possibly lead to novel immunotherapeutics [5]

A Novel T-Cell Engaging Bi-specific Antibody Targeting the Leukemia Antigen PR1/HLA-A2

Despite substantial advances in the treatment of acute myeloid leukemia (AML), only 30% of patients survive more than 5 years. Therefore, new therapeutics are much needed. Here, we present a novel therapeutic strategy targeting PR1, an HLA-A2 restricted myeloid leukemia antigen. Previously, we have developed and characterized a novel T-cell receptor-like monoclonal antibody (8F4) that targets PR1/HLA-A2 and eliminates AML xenografts by antibody-dependent cellular cytotoxicity (ADCC). To improve the potency of 8F4, we adopted a strategy to link T-cell cytotoxicity with a bi-specific T-cell-engaging antibody that binds PR1/HLA-A2 on leukemia and CD3 on neighboring T-cells. The 8F4 bi-specific antibody maintained high affinity and specific binding to PR1/HLA-A2 comparable to parent 8F4 antibody, shown by flow cytometry and Bio-Layer Interferometry. In addition, 8F4 bi-specific antibody activated donor T-cells in the presence of HLA-A2+ primary AML blasts and cell lines in a dose dependent manner. Importantly, activated T-cells lysed HLA-A2+ primary AML blasts and cell lines after addition of 8F4 bi-specific antibody. In conclusion, our studies demonstrate the therapeutic potential of a novel bi-specific antibody targeting the PR1/HLA-A2 leukemia-associated antigen, justifying further clinical development of this strategy

The Role of Antigen Cross-presentation From Leukemia Blasts on Immunity to the Leukemia-associated Antigen PR1

Cross-presentation is an important mechanism by which exogenous tumor antigens are presented to elicit immunity. Since neutrophil elastase (NE) and proteinase-3 (P3) expression is increased in myeloid leukemia, we investigated whether NE and P3 are cross-presented by dendritic cells (DC) and B-cells, and whether the NE and P3 source determines immune outcomes. We show that NE and P3 are elevated in leukemia patient serum and that levels correlate with remission status. We demonstrate cellular uptake of NE and P3 into lysosomes, ubiquitination and proteasome processing for cross-presentation. Using anti-PR1/HLA-A2 monoclonal antibody, we provide direct evidence that B-cells cross-present soluble and leukemia-associated NE and P3, while DCs cross-present only leukemia-associated NE and P3. Cross-presentation occurred at early time points but was not associated with DC or B-cell activation, suggesting that NE and P3 cross-presentation may favor tolerance. Furthermore, we show aberrant subcellular localization of NE and P3 in leukemia blasts to compartments that share common elements of the classical MHC class I antigen-presenting pathway, which may facilitate cross-presentation. Our data demonstrate distinct mechanisms for cross-presentation of soluble and cell-associated NE and P3, which may be valuable in understanding immunity to PR1 in leukemia

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

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

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

Testing of T cell-based cancer therapeutics often involves measuring cancer antigen-specific T-cell populations with the assumption that they arise from in vivo clonal expansion. This analysis, using peptide/MHC tetramers, is often ambiguous

PR1-Specific T Cells Are Associated with Unmaintained Cytogenetic Remission of Chronic Myelogenous Leukemia After Interferon Withdrawal

Interferon-alpha (IFN) induces complete cytogenetic remission (CCR) in 20-25% CML patients and in a small minority of patients; CCR persists after IFN is stopped. IFN induces CCR in part by increasing cytotoxic T lymphocytes (CTL) specific for PR1, the HLA-A2-restricted 9-mer peptide from proteinase 3 and neutrophil elastase, but it is unknown how CCR persists after IFN is stopped.We reasoned that PR1-CTL persist and mediate CML-specific immunity in patients that maintain CCR after IFN withdrawal. We found that PR1-CTL were increased in peripheral blood of 7/7 HLA-A2+ patients during unmaintained CCR from 3 to 88 months after IFN withdrawal, as compared to no detectable PR1-CTL in 2/2 IFN-treated CML patients not in CCR. Unprimed PR1-CTL secreted IFNgamma and were predominantly CD45RA+/-CD28+CCR7+CD57-, consistent with functional naïve and central memory (CM) T cells. Similarly, following stimulation, proliferation occurred predominantly in CM PR1-CTL, consistent with long-term immunity sustained by self-renewing CM T cells. PR1-CTL were functionally anergic in one patient 6 months prior to cytogenetic relapse at 26 months after IFN withdrawal, and in three relapsed patients PR1-CTL were undetectable but re-emerged 3-6 months after starting imatinib.These data support the hypothesis that IFN elicits CML-specific CM CTL that may contribute to continuous CCR after IFN withdrawal and suggest a role for T cell immune therapy with or without tyrosine kinase inhibitors as a strategy to prolong CR in CML

Aberrant Subcellular Localization of Azurophil Granule Proteins in Myeloid Leukemia Favors Peptide Antigen Presentation on MHC-I and Susceptibility to Killing by Cytotoxic T Lymphocytes

Abstract The recognition of leukemia-associated antigens (LAA) by immune cells is essential for generating anti-leukemia responses. Whether elicited by vaccine or hematopoietic stem cell transplantation (HSCT), anti-leukemia immunity requires that cytotoxic T lymphocytes (CTL) recognize and mount an effective response against leukemia cells. PR1 is a nonomeric HLA-A2-restricted peptide (VLQELNVTV) derived from the myeloid-restricted serine proteases neutrophil elastase (ELA2) and proteinase-3 (PRTN3), recognized azurophil granule proteins. PR1-specific cytotoxic T lymphocytes (PR1-CTL) have been shown to preferentially recognize and kill myeloid leukemia cells. We hypothesized that aberrant protein expression in target leukemia cells predisposes to CTL killing. To determine the subcellular localization of ELA2 and PRTN3 in leukemia and in healthy donor (HD) neutrophils, high-speed ultracentrifugation of leukapheresed cells from patients with AML and CML in blast crises yielded 4 fractions: cytoplasmic, nuclear, membrane and golgi/endoplasmic reticulum (ER). Western blot analyses identified higher levels of ELA2 and PRTN3 in cytoplasmic, nuclear, and golgi/ER fractions in leukemia, compared to HD granulocytes. Confocal microscopy of leukapheresed myeloid blasts and HD granulocytes confirmed these findings. We further hypothesized that cross-presentation of PR1 is necessary for priming anti-leukemia immunity. Confocal microscopy of the human B-cell line HMy.CIR-A2, co-incubated with either purified or recombinant ELA2 and PRTN3 demonstrated co-localization of both proteins with LAMP-2, a marker of lysosomes and early endosomes, and small distribution in cytoplasm after 2 hours. Uptake of ELA2 and PRTN3 by HMy.CIR-A2 cells was additionally confirmed by Western blots. Priming of naive PR1-CTL by co-culture of HD lymphocytes with PRTN3 or ELA2-transfected HMy.CIR-A2 cells was confirmed by cell proliferation assays using BrDU. This could be abrogated by addition of lactacystin and cyclohexamide to cell culture. Together, these results demonstrate that ELA2 and PRTN3 are aberrantly localized preferentially in gogli/ER and cytoplasmic fractions in leukemia, rather than azurophil granules, which are often absent or deficient in myeloid leukemia. Similar forced subcellular localization of these proteins in B cells leads to their susceptibility to PR1-CTL killing. Therefore, we conclude that the natural mistrafficking of PRTN3 and ELA2 in myeloid leukemia is necessary to render cells susceptible to CTL attack and that cross-presentation of ELA2 and PRTN3 contributes to priming of a PR1-CTL response