Novel Immunotherapy Targets Cathepsin G in Solid Tumors

https://openworks.mdanderson.org/sumexp21/1221/thumbnail.jp

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

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

Peripheral Tolerance to PR1 Is Maintained After Uptake of Soluble Proteinas-3 and Neutrophil Elastase

Abstract Abstract 4301 The leukemia associated antigens (LAA) proteinase-3 (P3) and neutrophil elastase (NE) are serine proteases found within neutrophil azurophil granules and are aberrantly expressed by leukemia. The human leukocyte antigen (HLA)-A2 restricted nonomeric peptide PR1, derived from P3 and NE, has been detected on leukemia cell surface in association with HLA-A2. PR1-specific cytotoxic T lymphocytes (CTL) were detected in patients following hematopoietic stem cell transplantation and were associated with clinical remission. Furthermore, in a phase I/II clinical trial, PR1-vaccine showed immunologic and clinical responses in patients with MDS and AML. Antigen cross presentation is a mechanism by which exogenous antigens are taken up by antigen presenting cells (APC) and presented in association with major histocompatibility (MHC)-I molecule. Cross presentation is thought to be the primary mechanism by which tumor antigens are presented to elicit tumor specific immune responses. Cross presentation by dendritic cells (DC), which express co-stimulatory molecules, leads to immune priming, while antigen presentation by B cells is associated with immune tolerance. Since cross presentation of leukemia antigens has been associated with anti-leukemia immune responses, we investigated whether P3 and NE are cross presented by DCs and B cells, and whether the source of P3 and NE (soluble vs. leukemia-cell associated) determines priming of the anti-PR1 immune response. We have previously reported that serum P3 levels are significantly higher in leukemia patients, and we now also report higher serum NE levels in patients with AML (median±standard deviation (SD)= 1236 ±572 ng/ml; n=15) and CML (median ± SD = 941 ± 494 ng/ml; n=15), in comparison with healthy donor blood (median ± SD = 65 ± 177 ng/ml; n=5) (p<0.05). Using flow cytometry and confocal microscopy, we demonstrate uptake of soluble P3 and NE into lysosomes by B cells as well as normal and leukemia-derived DCs. Using western blots (WB), we show that leukemia-cell associated P3 and NE are found in nuclear, membrane, cytosolic and skeletal compartments, unlike neutrophils where they are primarily limited to the granule-containing compartment. Moreover, immunoprecipitation and WB assays showed that intracellular P3 and NE are ubiquitinated, suggesting that they are processed by proteasomes for subsequent PR1 presentation. Using BrDU uptake assay, we show that cross presentation of PR1 by HMY.CIR-A2 APC cell line leads to a 2-fold increase in proliferation of in vitro generated PR1-specific CTLs through a proteasomal dependent pathway. Using the monoclonal antibody 8F4 (anti-PR1-HLA-A2), we show that PR1 is cross presented by normal B cells from soluble P3 and NE (1.6 fold increase in PR1 expression vs. unpulsed B cells). Furthermore, we show that B cells cross present PR1 from cell-associated NE and P3 from irradiated HLA-A2+ and HLA-A2- primary leukemia and leukemia cell lines (1.7 fold increase in PR1 expression vs. unpulsed B cells). In contrast to B cells, normal DCs cross presented cell-associated, but not soluble, P3 and NE (1.6-2.0 fold increase in PR1 expression vs. unpulsed or soluble NE/P3 pulsed DCs). Furthermore, cell-associated NE and P3 increased the co-stimulatory receptors CD83, CD86, and HLA-DR in normal DCs, while soluble NE and P3 did not affect, and in some cases decreased, expression of co-stimulatory DC receptors. In conclusion, this study shows that 1) soluble and cell-associated P3 and NE are cross presented by B cells, 2) only cell-associated P3 and NE are cross presented by DCs, and 3) cell-associated, but not soluble, P3 and NE increased co-stimulatory receptors in DCs, all suggesting that cross presentation of soluble P3 and NE leads to tolerance to PR1. Since serum P3 and NE are increased in leukemia, this may be a mechanism by which leukemia evades the anti-PR1 immune response. Our findings may also be applicable not only to the anti-leukemia immune response, but also to autoimmune diseases where serum P3 and NE were shown to be increased and to inflammatory states that are common to many cancers. Disclosures: No relevant conflicts of interest to declare

Addition of anti-4-1BB antibody to the REP led to increased post-REP TIL tumor antigen-specific CTL activity.

<p>Melanoma TIL from HLA-A0201⁺ patients with a significant population of CD8⁺MART-1 tetramer⁺ cells were rapidly expanded with or without anti-4-1BB as before. The post-REP TIL were sorted by FACS for CD8⁺ T cells and assayed for tumor antigen-specific CTL activity using a flow-cytometry-based assay that measures caspase-3 cleavage in target cells. The results of three different patient TIL lines are shown. The top panels (<b>A</b>) show the CTL activity of TIL #2292 using the melanoma cell line 624 (HLA-A0201⁺) and the control HLA-A-unmatched line 938 as targets (left side), or MART-1 peptide-pulsed T2 target cells as targets (right side). The bottom panels (<b>B</b>) show the CTL activity of two other HLA-A0201⁺ TIL lines (#2276 and #2122) against 624 or 938 cells with similar results. In all cases (<b>A</b> and <b>B</b>) the levels of non-specific killing were markedly lower.</p