Celine Kerros

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.

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.

The peptide derived from the Ig-like domain of human herpesvirus 8 K1 protein induces death in hematological cancer cells

BackgroundAlthough significant progress has been made in the treatment of lymphomas, many lymphomas exhibit resistance to cell death, suggesting a defective Fas signaling, which remains poorly understood. We previously reported that cells expressing the K1 protein of human herpesvirus 8 (HHV-8) resist death through the complex formation of the Ig-like domain of K1 with Fas. Recently, we investigated whether peptides derived from the Ig-like domain of the K1 protein may affect cell death.MethodsK1 positive and negative cell lines were incubated with the K1-derived peptides, and cell death (apoptotic and necrotic) was assessed by flow cytometry and LDH assay. Activation of caspases was assessed by fluorometric assay and flow cytometry. Fas receptor-independent, peptide-mediated cell killing was tested in the Fas-resistant Daudi cell line and Jurkat cell clones deficient in caspase-8 and FADD functionality. Activation of TNF receptors I and II was blocked by pre-incubation with corresponding blocking antibodies. The effect of the K1 peptide in vivo was tested in a mouse xenograft model.ResultsWe observed that the peptide S20-3 enhanced cell death in K1-positive BJAB cells and HHV-8 positive primary effusion lymphoma (PEL) cell lines. Similar effects of this peptide were observed in B-cell lymphoma and T-lymphoblastic leukemia cells without K1 expression but not in normal human peripheral blood mononuclear cells. A single intratumoral injection of the S20-3 peptide decreased the growth of Jurkat xenografts in SCID mice. The mechanism of tumor cell death induced by the S20-3 peptide was associated with activation of caspases, but this activity was only partially inhibited by the pan-caspase inhibitor z-VAD. Furthermore, the K1 peptide also killed Fas-resistant Daudi cells, and this killing effect was inhibited by pre-incubation of cells with antibodies blocking TNFRI.ConclusionTaken together, these findings indicate that the S20-3 peptide can selectively induce the death of malignant hematological cell lines by Fas- and/or TNFRI-dependent mechanisms, suggesting the K1-derived peptide or peptidomimetic may have promising therapeutic potential for the treatment of hematological cancers.

Serine proteases enhance immunogenic antigen presentation on lung cancer cells

Lung cancer cells exposed to granulocyte serine proteases increased the presentation of both endogenous peptides and the exogenous, protease-derived, HLA-A2-restricted PR1 peptide. Circulating CTLs specific for these peptides were identified in lung cancer patients. Immunotherapies targeting immune checkpoints have proven efficacious in reducing the burden of lung cancer in patients; however, the antigenic targets of these reinvigorated T cells remain poorly defined. Lung cancer tumors contain tumor-associated macrophages (TAM) and neutrophils, which release the serine proteases neutrophil elastase (NE) and proteinase 3 (P3) into the tumor microenvironment. NE and P3 shape the antitumor adaptive immune response in breast cancer and melanoma. In this report, we demonstrate that lung cancer cells cross-presented the tumor-associated antigen PR1, derived from NE and P3. Additionally, NE and P3 enhanced the expression of human leukocyte antigen (HLA) class I molecules on lung cancer cells and induced unique, endogenous peptides in the immunopeptidome, as detected with mass spectrometry sequencing. Lung cancer patient tissues with high intratumoral TAMs were enriched for MHC class I genes and T-cell markers, and patients with high TAM and cytotoxic T lymphocyte (CTL) infiltration had improved overall survival. We confirmed the immunogenicity of unique, endogenous peptides with cytotoxicity assays against lung cancer cell lines, using CTLs from healthy donors that had been expanded against select peptides. Finally, CTLs specific for serine proteases–induced endogenous peptides were detected in lung cancer patients using peptide/HLA-A2 tetramers and were elevated in tumor-infiltrating lymphocytes. Thus, serine proteases in the tumor microenvironment of lung cancers promote the presentation of HLA class I immunogenic peptides that are expressed by lung cancer cells, thereby increasing the antigen repertoire that can be targeted in lung cancer. Cancer Immunol Res; 5(4); 319–29. ©2017 AACR.

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.

Membrane-Associated Proteinase 3 on Granulocytes and Acute Myeloid Leukemia Inhibits T Cell Proliferation

Proteinase 3 (P3), a serine protease expressed by myeloid cells, localized within azurophil granules, and also expressed on the cellular membrane of polymorphonuclear neutrophils (PMN), is the target of autoimmunity in granulomatosis with polyangiitis. PR1, an HLA-A2 restricted nonameric peptide derived from P3, has been targeted effectively in myeloid leukemia. We previously showed (Molldrem et al. 2003. J. Clin. Invest. 111: 639–647) that overexpression of P3 in chronic myeloid leukemia induces apoptosis of high-affinity PR1-specific T cells, leading to deletional tolerance and leukemia outgrowth. In this study, we investigated the effect of membrane P3 (mP3)–expressing PMN and acute myeloid leukemia (AML) blasts on the proliferation of CD4 and CD8 T cells in vitro. We demonstrate that mP3-expressing PMN significantly inhibits autologous healthy donor T cell proliferation but does not affect cytokine production in activated T cells and that this effect requires cell proximity and was abrogated by P3 blockade. This inhibition required P3 enzyme activity. However, suppression was not reversed by either the addition of catalase or the inhibition of arginase I. In addition to P3 blockade, anti–low density lipoprotein receptor-related protein 1 (LRP1) Ab also restored T cells’ capacity to proliferate. Last, we show dose-dependent inhibition of T cell proliferation by mP3-expressing AML blasts. Together, our findings demonstrate a novel mechanism whereby PMN- and AML-associated mP3 inhibits T cell proliferation via direct LRP1 and mP3 interaction, and we identify P3 as a novel target to modulate immunity in myeloid leukemia and autoimmune disease.

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 3984: Neuropilin-1 mediates neutrophil elastase uptake and antigen cross-presentation in breast cancer cells

We previously showed that soluble neutrophil elastase (NE) is rapidly taken up by tumor cells that lack endogenous NE expression, including breast cancer (BrCa). NE uptake led to cross-presentation of PR1, an NE-derived HLA-A2-restricted peptide that is an immunotherapeutic target in hematologic malignancies. The mechanism of NE uptake, however, remains unknown. Here, we demonstrated that NE internalization was temperature- and time-sensitive, dose-saturation was observed, and uptake was partially blocked by chlorpromazine (CPZ) and wortmannin, supporting a receptor-mediated process and suggesting a role for clathrin- and PI3K-dependent mechanisms. To identify a candidate NE receptor, we performed mass spectrometry (MS) of proteins after anti-NE co-immunoprecipitation of NE-pulsed MDA-MB-231 BrCa cells and we identified neuropilin-1 (NRP1) as an NE co-associated protein. Binding of NE to NRP1 was confirmed by ELISA, and peptide epitope mapping studies indicated NE bound via it consensus sequence RRXR, which is also known to bind the b1b2 domain of NRP1. NE bound to NRP1 with high affinity (Kd=38.7 nM) as measured with biolayer interferometry. To confirm the role of NRP1 in cellular uptake of NE, we transfected MDA-MB-231 BrCa cells with siRNA or shRNA against NRP1 and showed that NRP1 knockdown resulted in a 2-fold decrease in NE uptake as determined with flow cytometry. Similarly, blocking with an NRP1-neutralizing antibody decreased NE uptake by 60% vs. isotype control antibody, an effect that was also observed in other breast cancer cell lines that expressed NRP1. Conversely, transient expression of NRP1 in the NRP1-deficient T47D BrCa cell line was sufficient to induce uptake of NE. Importantly, knockdown of NRP1 expression in MDA-231 cells also prevented PR1 cross-presentation as determined with the anti-PR1/HLA-A2 monoclonal antibody 8F4. This was confirmed by the loss of susceptibility of NE-pulsed MDA-MB-231 cells to lysis by PR1-specific cytotoxic T cells following knockdown of NRP1 expression. Our data support a novel function of NRP1 in the uptake and cross-presentation of neutrophil-derived proteins by non-hematopoietic cancer cells. Because 8F4 mediates killing of PR1/HLA-A2+ leukemia and NRP1 is broadly expressed on many tumors, our results suggest a role for immunotherapy strategies that target NE-derived peptides on NRP1+ tumors. Citation Format: Celine Kerros, Satyendra C. Tripathi, Dongxing Zha, Sergeeva Anna, Haley L. Peters, Hiroyuki Katayama, Pariya Sukhumalchandra, Kathryn R. Cox, Alexander A. Perakis, Lisa S. St John, Gheath Alatrash, Elizabeth A. Mittendorf, Karen C. Dwyer, Samir M. Hanash, Jeffrey J. Molldrem. Neuropilin-1 mediates neutrophil elastase uptake and antigen cross-presentation in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3984. doi:10.1158/1538-7445.AM2017-3984

Abstract IA17: T cell receptor-like antibody 8F4 targets leukemia and non-hematopoietic cancer

Adaptive anti-tumor immune surveillance of leukemia, called the graft-versus-leukemia (GVL) effect in the setting of allogeneic stem cell transplantation (alloSCT), is responsible for inducing and maintaining long term remission. However, although donor T cells mediate GVL, they also mediate graft-versus-host disease (GVHD), which results in significant morbidity and can be fatal, and is thus a substantial barrier to the more widespread application of alloSCT for many patients with hematological malignancies. CD8 T cells are activated by cognate peptide/MHC-I target cell antigens through binding of clonally unique αβ-heterodimer T cell receptors (TCRs) on the cell surface. Thus, because GVL and GVHD target antigens can be unique, immunotherapy strategies that target GVL antigens such as adoptively transferred T cells could be developed to mediate GVL without GVHD. Likewise, TCR-like monoclonal antibodies (mAbs) that similarly bind to peptide/MHC-I also could be useful as novel immunotherapeutic agents to mediate GVL in the absence of cellular therapies. Moreover, if potent anti-tumor activity was mediated by such TCR-like mAbs, they could have advantages over adoptively transferred donor T cells, including easier standardized manufacturing, ease of dose and scheduled administration, and they could be given to patients in the absence of an alloSCT. We identified PR1, an HLA-A2-restricted 9-mer peptide a peptide derived from aberrantly expressed proteinase 3 (P3) and neutrophil elastase (NE) in myeloid leukemia, as a leukemia-associated target antigen for CD8 T cells that mediate GVL. PR1-specific CD8 T cells target HLA-A2+ AML, CML and MDS but not normal hematopoietic cells and they contribute to cytogenetic and molecular remission of myeloid leukemia. We developed a murine TCR-like mAb that binds to PR1/HLA-A2 (8F4) and showed that 8F4 mediated complement-dependent cytotoxicity (CDC) of AML, CML and MDS progenitor cells and blasts, but not normal bone marrow cells. Because 8F4 did not eliminate normal human hematopoietic cells in a xenograft model, we humanized 8F4 to an IgG1 isotype (h8F4). In AML PDX models, 8F4 and h8F4 eliminated HLA-A2+ AML and leukemia stem cells (LSCs). In preclinical safety models, only mild reversible blood cytopenia was observed after multiple-dose IV administration of high-dose 8F4. Based on these findings, h8F4 is being developed for a first-in-human phase I safety study in HLA-A2+ patients with high-risk AML. In addition, 8F4 also mediated lysis of many non-hematopoietic cancers due to PR1 cross-presentation on surface HLA-A2, which increased cancer cells susceptibility to 8F4-mediated lysis. Therefore, 8F4 also could be tested for the treatment of non-hematopoietic HLA-A2+ cancers such as breast cancer, non-small cell lung cancer, and colon cancer. Citation Format: Jeffrey J. Molldrem, Anna Sergeeva, Hong He, Amanda C. Herrmann, Tian-Hui Yang, Celine Kerros, Haley Peters, Jin Seon Im, Sapna Parshottam, Sijie Lu, Qing Ma, Karen C. Dwyer, Elizabeth Mittendorf, Gheath Alatrash. T cell receptor-like antibody 8F4 targets leukemia and non-hematopoietic cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr IA17.

Abstract 2351: Characterization of Neutrophil elastase uptake in breast cancer: implications for immunotherapy

Neutrophil Elastase (NE)_a serine protease released by neutrophils in the tumor microenvironment_induces invasion and metastasis. We demonstrated that while NE is not endogenously expressed in breast cancer cells, breast cancer cells (BrCA) take up exogenous NE derived from tumor-associated neutrophils. NE uptake results in the expression of the HLA-A2-restricted peptides CCNE1 and PR1, derived from cyclin E and NE, respectively, on the surface of triple-negative (TN) BrCA cells (MDA-MB-231). Expression of these peptides/HLA-A2 molecules induces BrCA cell susceptibility to cytolysis by CCNE1- and PR1-specific cytotoxic T lymphocytes (CTLs), and to 8F4, a monoclonal antibody that binds specifically to the PR1/HLA-A2 complex. We hypothesize that NE uptake is a receptor-mediated process that results in cross-presentation of NE-derived peptides on HLA molecules of BrCA cells. In this study, we found that NE uptake in BrCA is specific, time and dose-dependent, and become saturated, suggesting a receptor-mediated uptake mechanism. We showed that MDA-MB-231 did not take up cathepsin G, a related serine protease, suggesting specificity of NE receptor uptake. NE internalization was partially blocked by chlorpromazine and by wortmannin, implicating clathrin-dependent uptake and PI3Kinase-dependence, respectively. Confocal microscopy showed that NE was co-localized with the early endosome marker, EEA-1, as early as 10 minutes after uptake. Flow cytometry indicated that surface-bound NE on MDA-MB-231 cells decreased after five minutes, and Western blot showed a simultaneous decrease of MAPKinases phosphorylation (p-Erk, p-p38) and loss of IRS-1 signaling. Inhibition of NE enzyme activity by serine proteases inhibitors, such as elafin or PMSF, potentiated NE uptake in BrCA cells, indicating that enzyme activity is not required for uptake. Conclusion: The results support a novel mechanism of rapid receptor-mediated uptake of soluble exogenous NE by TN BrCA. NE uptake is efficient, PI3 kinase-dependent, sensitive to clathrin inhibition, and associated with down-regulation of Erk phosphorylation and IRS-1. In addition, uptake does not require NE enzyme activity. Following uptake, NE co-localizes to an early endosomal compartment, an organelle associated with HLA class I peptide loading. Since we previously linked NE uptake to CCNE1 and PR1 peptide presentation on MDA-MB-231 cells, which enhanced BrCA susceptibility to immunotherapies that target CCNE1 and PR1, our results here strongly suggest receptor-mediated NE uptake that could regulate HLA class I peptide presentation. Understanding the mechanisms that regulate NE uptake and peptide presentation on the surface of BrCA will help us develop new strategies to enhance peptide presentation by BrCA, potentially improving the response of BrCA and other tumor types to antigen-specific immunotherapies. Citation Format: Celine Kerros, Satyendra C. Tripathi, Anne V. Philips, Gheath Al-Atrash, Kathryn E. Ruisaard, Karen C. Dwyer, Elizabeth A. Mittendorf, Samir Hanash, Jeffrey J. Molldrem. Characterization of Neutrophil elastase uptake in breast cancer: implications for immunotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2351. doi:10.1158/1538-7445.AM2015-2351