Anne V. Philips

PD-L1 Expression in Triple-Negative Breast Cancer

Using tissue microarrays containing 105 triple-negative breast cancer (TNBC) specimens, Mittendorf and colleagues show that 20% of the TNBC specimens express PD-L1, half have lost PTEN, and inhibitors of PI3K pathway decrease PD-L1 expression, providing a rationale for therapeutic targeting of PD-L1 for TNBC. Early-phase trials targeting the T-cell inhibitory molecule programmed cell death ligand 1 (PD-L1) have shown clinical efficacy in cancer. This study was undertaken to determine whether PD-L1 is overexpressed in triple-negative breast cancer (TNBC) and to investigate the loss of PTEN as a mechanism of PD-L1 regulation. The Cancer Genome Atlas (TCGA) RNA sequencing data showed significantly greater expression of the PD-L1 gene in TNBC (n = 120) compared with non-TNBC (n = 716; P < 0.001). Breast tumor tissue microarrays were evaluated for PD-L1 expression, which was present in 19% (20 of 105) of TNBC specimens. PD-L1+ tumors had greater CD8+ T-cell infiltrate than PD-L1− tumors (688 cells/mm vs. 263 cells/mm; P < 0.0001). To determine the effect of PTEN loss on PD-L1 expression, stable cell lines were generated using PTEN short hairpin RNA (shRNA). PTEN knockdown led to significantly higher cell-surface PD-L1 expression and PD-L1 transcripts, suggesting transcriptional regulation. Moreover, phosphoinositide 3-kinase (PI3K) pathway inhibition using the AKT inhibitor MK-2206 or rapamycin resulted in decreased PD-L1 expression, further linking PTEN and PI3K signaling to PD-L1 regulation. Coculture experiments were performed to determine the functional effect of altered PD-L1 expression. Increased PD-L1 cell surface expression by tumor cells induced by PTEN loss led to decreased T-cell proliferation and increased apoptosis. PD-L1 is expressed in 20% of TNBCs, suggesting PD-L1 as a therapeutic target in TNBCs. Because PTEN loss is one mechanism regulating PD-L1 expression, agents targeting the PI3K pathway may increase the antitumor adaptive immune responses. Cancer Immunol Res; 2(4); 361–70. ©2014 AACR.

Breast Cancer Cell Uptake of the Inflammatory Mediator Neutrophil Elastase Triggers an Anticancer Adaptive Immune Response

There is little understanding of the impact of tumor-associated neutrophils (TAN) on adaptive immunity to tumors. In this study, we report the results of an investigation of the pathobiologic basis for the prognostic significance of neutrophil elastase, a serine protease found in neutrophil granules, in a model of cyclin E (CCNE)-overexpressing breast cancer. We established that neutrophil elastase was expressed by TAN within breast cancer tissues but not by breast cancer cells. Neutrophil elastase modulated killing of breast cancer cells by CTLs specific for CCNE-derived HLA-A2-restricted peptide (ILLDWLMEV). Breast cancer cells exhibited striking antigen-specific uptake of neutrophil elastase from the microenvironment that was independent of neutrophil elastase enzymatic activity. Furthermore, neutrophil elastase uptake increased expression of low molecular weight forms of CCNE and enhanced susceptibility to peptide-specific CTL lysis, suggesting that CCNE peptides are naturally presented on breast cancer cells. Taken together, our findings reveal a previously unknown mechanism of antitumor adaptive immunity that links cancer cell uptake of an inflammatory mediator to an effective cytolytic response against an important breast cancer antigen.

Broad Cross-Presentation of the Hematopoietically Derived PR1 Antigen on Solid Tumors Leads to Susceptibility to PR1-Targeted Immunotherapy

PR1 is a HLA-A2–restricted peptide that has been targeted successfully in myeloid leukemia with immunotherapy. PR1 is derived from the neutrophil granule proteases proteinase 3 (P3) and neutrophil elastase (NE), which are both found in the tumor microenvironment. We recently showed that P3 and NE are taken up and cross-presented by normal and leukemia-derived APCs, and that NE is taken up by breast cancer cells. We now extend our findings to show that P3 and NE are taken up and cross-presented by human solid tumors. We further show that PR1 cross-presentation renders human breast cancer and melanoma cells susceptible to killing by PR1-specific CTLs (PR1-CTL) and the anti-PR1/HLA-A2 Ab 8F4. We also show PR1-CTL in peripheral blood from patients with breast cancer and melanoma. Together, our data identify cross-presentation as a novel mechanism through which cells that lack endogenous expression of an Ag become susceptible to therapies that target cross-presented Ags and suggest PR1 as a broadly expressed tumor Ag.

Neutrophil elastase enhances antigen presentation by upregulating human leukocyte antigen class I expression on tumor cells

Neutrophil elastase (NE) is an innate immune cell-derived inflammatory mediator that we have shown increases the presentation of tumor-associated peptide antigens in breast cancer. In this study, we extend these observations to show that NE uptake has a broad effect on enhancing antigen presentation by breast cancer cells. We show that NE increases human leukocyte antigen (HLA) class I expression on the surface of breast cancer cells in a concentration and time-dependent manner. HLA class I upregulation requires internalization of enzymatically active NE. Western blots of NE-treated breast cancer cells confirm that the expression of total HLA class I as well as the antigen-processing machinery proteins TAP1, LMP2, and calnexin does not change following NE treatment. This suggests that NE does not increase the efficiency of antigen processing; rather, it mediates the upregulation of HLA class I by stabilizing and reducing membrane recycling of HLA class I molecules. Furthermore, the effects of NE extend beyond breast cancer since the uptake of NE by EBV–LCL increases the presentation of HLA class I-restricted viral peptides, as shown by their increased sensitivity to lysis by EBV-specific CD8+ T cells. Together, our results show that NE uptake increases the responsiveness of breast cancer cells to adaptive immunity by broad upregulation of membrane HLA class I and support the conclusion that the innate inflammatory mediator NE enhances tumor cell recognition and increases tumor sensitivity to the host adaptive immune response.

Trastuzumab increases HER2 uptake and cross-presentation by dendritic cells

Early-phase clinical trials evaluating CD8+ T cell-eliciting, HER2-derived peptide vaccines administered to HER2+ breast cancer patients in the adjuvant setting suggest synergy between the vaccines and trastuzumab, the mAb targeting the HER2 protein. Among 60 patients enrolled in clinical trials evaluating the E75 + GM-CSF and GP2 + GM-CSF vaccines, there have been no recurrences in patients vaccinated after receiving trastuzumab as part of standard therapy in the per treatment analyses conducted after a median follow-up of greater than 34 months. Here, we describe a mechanism by which this synergy may occur. Flow cytometry showed that trastuzumab facilitated uptake of HER2 by dendritic cells (DC), which was mediated by the Fc receptor and was specific to trastuzumab. In vitro, increased HER2 uptake by DC increased cross-presentation of E75, the immunodominant epitope derived from the HER2 protein, an observation confirmed in two in vivo mouse models. This increased E75 cross-presentation, mediated by trastuzumab treatment, enabled more efficient expansion of E75-specific cytotoxic T cells (E75-CTL). These results demonstrate a mechanism by which trastuzumab links innate and adaptive immunity by facilitating activation of antigen-specific T cells. On the basis of these data, we conclude that HER2-positive breast cancer patients that have been treated with trastuzumab may experience a more robust antitumor immune response by restimulation of T cells with the E75 peptide vaccine, thereby accounting for the improved disease-free survival observed with combination therapy. Cancer Res; 77(19); 5374-83. ©2017 AACR.

Neuropilin-1 mediates Neutrophil Elastase uptake and cross-presentation in breast cancer cells

Neutrophil elastase (NE) can be rapidly taken up by tumor cells that lack endogenous NE expression, including breast cancer, which results in cross-presentation of PR1, an NE-derived HLA-A2-restricted peptide that is an immunotherapy target in hematological and solid tumor malignancies. The mechanism of NE uptake, however, remains unknown. Using the mass spectrometry-based approach, we identify neuropilin-1 (NRP1) as a NE receptor that mediates uptake and PR1 cross-presentation in breast cancer cells. We demonstrated that soluble NE is a specific, high-affinity ligand for NRP1 with a calculated Kd of 38.7 nm. Furthermore, we showed that NRP1 binds to the RRXR motif in NE. Notably, NRP1 knockdown with interfering RNA or CRISPR-cas9 system and blocking using anti-NRP1 antibody decreased NE uptake and, subsequently, susceptibility to lysis by PR1-specific cytotoxic T cells. Expression of NRP1 in NRP1-deficient cells was sufficient to induce NE uptake. Altogether, because NRP1 is broadly expressed in tumors, our findings suggest a role for this receptor in immunotherapy strategies that target cross-presented antigens.

Cathepsin G is expressed by acute lymphoblastic leukemia and is a potential immunotherapeutic target

Cathepsin G (CG) is a myeloid azurophil granule protease that is highly expressed by acute myeloid leukemia (AML) blasts and leukemia stem cells. We previously identified CG1 (FLLPTGAEA), a human leukocyte antigen-A2-restricted nonameric peptide derived from CG, as an immunogenic target in AML. In this report, we aimed to assess the level of CG expression in acute lymphoid leukemia (ALL) and its potential as an immunotherapeutic target in ALL. Using RT-PCR and western blots, we identified CG mRNA and protein, respectively, in B-ALL patient samples and cell lines. We also examined CG expression in a large cohort of 130 patients with ALL via reverse-phase protein array (RPPA). Our data show that CG is widely expressed by ALL and is a poor prognosticator. In addition to endogenous expression, we also provide evidence that CG can be taken up by ALL cells. Finally, we demonstrate that patient ALL can be lysed by CG1-specific cytotoxic T lymphocytes in vitro. Together, these data show high expression of CG by ALL and implicate CG as a target for immunotherapy in ALL.

Targeting the Leukemia Antigen PR1 with Immunotherapy for the Treatment of Multiple Myeloma

Purpose: PR1 is a human leukocyte antigen (HLA)-A2 nonameric peptide derived from neutrophil elastase (NE) and proteinase 3 (P3). We have previously shown that PR1 is cross-presented by solid tumors, leukemia, and antigen-presenting cells, including B cells. We have also shown that cross-presentation of PR1 by solid tumors renders them susceptible to killing by PR1-targeting immunotherapies. As multiple myeloma is derived from B cells, we investigated whether multiple myeloma is also capable of PR1 cross-presentation and subsequently capable of being targeted by using PR1 immunotherapies. Experimental Design: We tested whether multiple myeloma is capable of cross-presenting PR1 and subsequently becomes susceptible to PR1-targeting immunotherapies, using multiple myeloma cell lines, a xenograft mouse model, and primary multiple myeloma patient samples. Results: Here we show that multiple myeloma cells lack endogenous NE and P3, are able to take up exogenous NE and P3, and cross-present PR1 on HLA-A2. Cross-presentation by multiple myeloma utilizes the conventional antigen processing machinery, including the proteasome and Golgi, and is not affected by immunomodulating drugs (IMiD). Following PR1 cross-presentation, we are able to target multiple myeloma with PR1-CTL and anti-PR1/HLA-A2 antibody both in vitro and in vivo. Conclusions: Collectively, our data demonstrate that PR1 is a novel tumor-associated antigen target in multiple myeloma and that multiple myeloma is susceptible to immunotherapies that target cross-presented antigens. Clin Cancer Res; 24(14); 3386–96. ©2018 AACR.

Abstract 3243: Neutrophil elastase regulates PD-L1 expression in breast cancer

Introduction: We have previously reported that breast cancer cells take up the inflammatory mediator neutrophil elastase (NE) from tumor-associated neutrophils (TANs). NE uptake leads to: 1) increased cleavage of cyclin E (CCNE) to its low molecular weight isoforms and generation of the CCNE-derived immunogenic epitope CCNE144-152, 2) cross-presentation of the NE-derived epitope PR1, and 3) increased human leukocyte antigen (HLA) class I expression. NE is therefore a link between innate and adaptive immune responses in breast cancer. The current study was undertaken to investigate the effects of NE uptake on PD-L1 expression as another mechanism impacting adaptive immune responses in breast cancer. Methods: The breast cancer cell line MDA-MB-231 was maintained in standard media or media supplemented with NE. The effect of NE on cell surface expression of PD-L1 was determined using flow cytometry. Total PD-L1 protein expression was determined using western blot analysis performed on whole cell lysates. NE was inhibited with either elafin or alpha-1-antitrypsin to assess the requirement of enzymatic activity for the observed effects. Transcriptional regulation of PD-L1 expression by NE was assessed by qPCR. To evaluate specific transcription factors (TFs) involved in PD-L1 regulation, nuclear protein was isolated and analyzed for the activity of 16 TFs using a commercial plate array. The impact of NE uptake on TFs identified on the array was confirmed by western blot analysis. The functional effects of changes in PD-L1 expression were evaluated using an annexin V assay to assess T-cell apoptosis. Results: Addition of NE to breast cancer cells resulted in a reduction in PD-L1 surface expression as determined by flow cytometry and in total PD-L1 expression as shown by western blot analysis. The effect was reversed after removal of the cells from NE-supplemented media. The addition of elafin to inhibit NE enzymatic activity led to increased NE uptake by MDA-MB-231 cells with no change in PD-L1 cell surface expression. Treatment with alpha-1-antitrypsin prevented NE uptake and also abrogated its effect on PD-L1 expression. Together, these results suggest that uptake of enzymatically active NE is required to decrease PD-L1 expression. qPCR showed a decrease in the PD-L1 transcript suggesting that the effect of NE on PD-L1 is transcriptionally regulated. NE treatment resulted in decreased expression of TFs known to be involved in PD-L1 regulation, including STAT1, STAT3, and JUN. NE-treated breast cancer cells induced less apoptosis of T-cells compared with untreated breast cancer cells. Conclusions: Uptake of enzymatically active NE by breast cancer results in decreased cell surface and total PD-L1 expression, an effect that is in part transcriptionally regulated. These findings suggest another mechanism whereby the innate inflammatory mediator NE may impact adaptive immune responses in breast cancer. Citation Format: Victor Gall, Anne V. Philips, Akhil Chawla, Na Qiao, Lisa S. St. John, Pariya Sukhumalchandra, Mao Zhang, Jeffrey J. Molldrem, Gheath Alatrash, Elizabeth A. Mittendorf. Neutrophil elastase regulates PD-L1 expression in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3243.

Fucosylation enhances activity of cytotoxic T lymphocytes against breast cancer

Fucosylation is a process by which fucose sugar groups are added to cell surface receptors. This process is mediated by fucosyl transferases that attach terminal fucose groups to acceptor molecules on the cell surface. Fucosylation of cord blood stem cells and human regulatory T cells (Treg) were shown to enhance cord blood engraftment and Treg homing to inflamed tissues. Since tumor tissues have a component of inflammation, we hypothesized that fucosylation of antigen specific cytotoxic T lymphocytes (CTL) ex vivo will enhance their migration into tumors and subsequent killing of tumor cells. Specifically we tested whether ex vivo fucosylation of CTL that target the HLA-A2 restricted HER2/neu-derived peptide, E75, enhances E75-CTL migration and cytotoxic functions.