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 vivoConclusions: 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.

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.

Reduced graft-versus-host disease in C3-deficient mice is associated with decreased donor Th1/Th17 differentiation.

Graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation is mediated by the activation of recipient dendritic cells and subsequent proliferation of donor T cells. The complement system was recently shown to modulate adaptive immunity through an interaction of the complement system and lymphocytes. Complement proteins participate in the activation of dendritic cells, antigen presentation to T cells, and proliferation of T cells. Our studies with a murine model of bone marrow transplantation demonstrate that complement system regulates alloimmune responses in GVHD. Mice deficient in the central component of the complement system (C3(-/-)) had significantly lower GVHD-related mortality and morbidity compared with wild-type recipient mice. The numbers of donor-derived T cells, including IFN-γ(+), IL-17(+), and IL-17(+)IFN-γ(+) subsets, were decreased in secondary lymphoid organs of C3(-/-) recipients. Furthermore, the number of recipient CD8α(+)CD11c(+) cells in lymphoid organs was reduced. We conclude that C3 regulates Th1/17 differentiation in bone marrow transplantation, and define a novel function of the complement system in GVHD.

Lovastatin inhibits T-cell proliferation while preserving the cytolytic function of EBV, CMV, and MART-1-specific CTLs.

Statin treatment has been shown to reduce graft-versus-host disease while preserving graft-versus-tumor effect in allogeneic stem cell transplantation. Herein, we investigated whether lovastatin treatment affects the function of human cytolytic T lymphocytes (CTLs). Upon T-cell receptor stimulation, lovastatin significantly inhibited the proliferation of both CD4+ and CD8+ T cells from healthy donors whereas their intracellular cytokine production including interferon-γ and tumor necrosis factor-α remained the same with a slight decrease of interleukin-2. Moreover, the specific lysis of target cells by CTL lines derived from patients and normal donors specific for Epstein-Barr virus-encoded antigen latent membrane protein-2 or cytomegalovirus-encoded antigen pp65 was uncompromised in the presence of lovastatin. In addition, we evaluated the effect of lovastatin on the proliferation and effector function of the CD8+ tumor-infiltrating lymphocytes (TILs) derived from melanoma patients specific for MART-1 antigen. Lovastatin significantly reduced the expansion of antigen-specific TILs upon MART-1 stimulation. However, the effector function of TILs, including the specific lysis of target cells and secretion of cytokine interferon-γ, remained intact with lovastatin treatment. Taken together, these data demonstrated that lovastatin inhibits the proliferation of Epstein-Barr virus, cytomegalovirus, and MART-1-specific CTLs without affecting cytolytic capacity. The differential effect of lovastatin on the proliferation versus cytotoxicity of CTLs might shed some light on elucidating the possible mechanisms of graft-versus-host disease and graft-versus-tumor effect elicited by alloimmune responses.

Targeted mass spectrometric analysis of N-terminally truncated isoforms generated via alternative translation initiation.

Alternative translation initiation is a mechanism whereby functionally altered proteins are produced from a single mRNA. Internal initiation of translation generates N-terminally truncated protein isoforms, but such isoforms observed in immunoblot analysis are often overlooked or dismissed as degradation products. We identified an N-terminally truncated isoform of human Dok-1 with N-terminal acetylation as seen in the wild-type. This Dok-1 isoform exhibited distinct perinuclear localization whereas the wild-type protein was distributed throughout the cytoplasm. Targeted analysis of blocked N-terminal peptides provides rapid identification of protein isoforms and could be widely applied for the general evaluation of perplexing immunoblot bands.

14-3-3 zeta protein secreted by tumor associated monocytes/macrophages from ascites of epithelial ovarian cancer patients.

Tumor associated monocytes/macrophages (MO/MA) are known contributors to the immune-inflammatory cell environment of advanced epithelial ovarian carcinoma (EOC). The secreted proteome of ascitic MO/MA was examined as an aid to the discovery of novel proteins in EOC that are likely to have biological relevance in the inflammatory pathways of EOC. Ascitic fluid MO/MA were isolated from EOC patients, grown short-term in serum-free media. MO/MA supernatants were analyzed for secreted proteins by HPLC fractionation followed by LC-tandem mass spectrometric analysis. The 14-3-3 zeta adaptor protein was identified in supernatants of three of three EOC patients but not in supernatants of buffy coat monocytes isolated from normal donors or the established monocyte cell line THP1. Moreover, 14-3-3 zeta was identified in ascitic fluids in eight of eight chemotherapy-naïve patients by both immunoblot and mass spectrometric analysis. Immunofluorescent staining for 14-3-3 zeta demonstrated expression of the protein on ascitic and peritumoral macrophages in EOC patients. 14-3-3 zeta was also expressed on endothelial cells in the peritumoral stroma and partially on tumor cells. Uptake of 14-3-3 zeta was observed in EOC cell lines co-cultured with the recombinant protein expressed in E. coli. It is demonstrated for the first time that the important adaptor protein 14-3-3 zeta is common to the secretome of ascitic MO/MA and the ascites of advanced EOC patients.

Comparative analysis of peritoneum and tumor eicosanoids and pathways in advanced ovarian cancer.

PURPOSE: To describe the eicosanoid profile and differentially expressed eicosanoid and arachidonic acid pathway genes in tissues from patients with advanced epithelial ovarian cancer (EOC). EXPERIMENTAL DESIGN: We first employed electrospray tandem mass spectrometry to determine tissue-specific concentrations of the eicosanoids prostaglandin E2 (PGE2), the hydroxyeicosatetraenoic acids (12-HETE and 5-HETE), and leukotriene (LTB4), selected for tumor growth potential, and two other bioactive lipids (15-HETE and 13-HODE) with tumor cell proliferation interference potential. The cellular location of eicosanoid activity was identified by immunofluorescence antibody costaining and confocal microscopy. Differential analysis of eicosanoid and arachidonic pathway genes was done using a previously validated cDNA microarray platform. Tissues used included EOC tumor, tumor-free malignant peritoneum (MP), and benign peritoneum (BP) from patients with benign pelvic disease. RESULTS: (a) Eicosanoid products were detected in tumor, MP, and BP specimens. PGE2 levels were significantly elevated in tumors in an overall comparison with MP or BP (P < 0.001). Combined levels of PGE2, 12-HETE, 5-HETE, and LTB4 increased progressively from low to high concentrations in BP, MP, and tumors (P = 0.012). Neither 15-HETE nor 13-HODE showed a significant opposite trend toward levels found in BP. (b) Tissue specimens representing common EOC histotypes showed strong coexpressions of cyclooxygenases (COX-1) and prostaglandin E synthases (PGES-1) on tumor cells, whereas intratumoral or peritumoral MO/MA coexpressed COX-1 and COX-2 and PGES-1 and PGES-2, respectively. (c) cDNA microarray analysis of MP, BP, and tumor showed that a number of eicosanoid and arachidonic acid pathway genes were differentially expressed in MP and BP compared with tumor, except for CYP2J2, which was increased in tumors. CONCLUSIONS: Elevated levels of eicosanoid metabolites in tumors and differential expression of eicosanoid and arachidonic acid pathway genes in the peritoneum support the involvement of bioactive lipids in the inflammatory tumor environment of EOC.

Expression of CD40 and growth-inhibitory activity of CD40 ligand in ovarian cancer cell lines.

OBJECTIVE: Soluble recombinant human CD40 ligand trimer (rhuCD40Lt) has shown antitumor activity in preclinical and clinical studies. We evaluated the effect of rhuCD40Lt on epithelial ovarian carcinoma (EOC) cell lines. METHODS: Expression of the receptor, CD40, was determined by reverse transcriptase-polymerase chain reaction and flow cytometry, and antiproliferative effects of rhuCD40Lt, either alone or in combination with recombinant interferon-gamma (rIFN-gamma), were examined in 8 EOC lines. RESULTS: Expression of CD40 was elevated in 5 out of 8 EOC cell lines examined by flow cytometry, and the presence of CD40 transcripts was detected by RT-PCR in all 8 cell lines. CD40 expression was increased by rIFN-gamma, but treatment with rhuCD40Lt decreased CD40 expression in 4 of the 5 lines that had shown elevated CD40 expression. rhuCD40Lt had a growth-inhibitory effect on 2774 cells, which also exhibited the highest level of CD40 expression. Growth-inhibitory effect of rhuCD40Lt was additive with rIFN-gamma on 2774, NMP-1, a cisplatin-resistant subline of OVCAR3, and HEY cell lines. The number of apoptotic tumor cells was increased following treatment with rhuCD40Lt. CONCLUSIONS: CD40 is expressed on EOC cell lines, and expression was found at the transcript level in all of the EOC lines examined. rIFN-gamma enhances CD40 expression, though a decrease in CD40 expression was observed following treatment with rhuCD40Lt. Growth-inhibitory activity of rhuCD40Lt on EOC lines that express CD40 could be enhanced when rhuCD40Lt treatment was combined with rIFN-gamma. These results suggest that future studies of the combination of rhuCD40Lt and rIFN-gamma might warrant consideration.

Monocyte/macrophage and T-cell infiltrates in peritoneum of patients with ovarian cancer or benign pelvic disease.

BACKGROUND: We previously showed that tumor-free peritoneum of patients with epithelial ovarian cancer (EOC) exhibited enhanced expression of several inflammatory response genes compared to peritoneum of benign disease. Here, we examined peritoneal inflammatory cell patterns to determine their concordance with selected enhanced genes. METHODS: Expression patterns of selected inflammatory genes were mined from our previously published data base. Bilateral pelvic peritoneal and subjacent stromal specimens were obtained from 20 women with EOC and 7 women with benign pelvic conditions. Sections were first stained by indirect immunoperoxidase and numbers of monocytes/macrophages (MO/MA), T cells, B cells, and NK cells counted. Proportions of CD68+ cells and CD3+ cells that coexpressed MO/MA differentiation factors (CD163, CCR1, CXCR8, VCAM1, and phosphorylated cytosolic phospholipase A2 [pcPLA2]), which had demonstrated expression in EOC peritoneal samples, were determined by multicolor immunofluorescence. RESULTS: MO/MA were present on both sides of the pelvic peritoneum in EOC patients, with infiltration of the subjacent stroma and mesothelium. CD68+ MO/MA, the most commonly represented population, and CD3+ T cells were present more often in EOC than in benign pelvic tumors. NK cells, B cells, and granulocytes were rare. CXCL8 (IL-8) and the chemokine receptor CCR1 were coexpressed more frequently on MO/MA than on CD3+ cells contrasting with CD68+/CD163+ cells that coexpressed CXCL8 less often. An important activated enzyme in the eicosanoid pathway, pcPLA2, was highly expressed on both CD68+ and CD163+ cells. The adherence molecule Vascular Cell Adhesion Molecule-1 (VCAM1) was expressed on CD31+ endothelial cells and on a proportion of CD68+ MO/MA but rarely on CD3+ cells. CONCLUSION: The pelvic peritoneum in EOC exhibits a general pattern of chronic inflammation, represented primarily by differentiated MO/MA, and distinct from that in benign conditions concordant with previous profiling results.

Cytokines, GM-CSF and IFNgamma administered by priming and post-chemotherapy cycling in recurrent ovarian cancer patients receiving carboplatin.

BACKGROUND: Monocyte/macrophages (MO/MA), a polymorphic population of innate immune cells, have the potential to mediate antitumor effects, and may also contribute to protumor effects. A priming and post-chemotherapy schedule of the myeloid cell mobilizing and immune stimulatory growth factor, granulocyte monocyte stimulating factor (GM-CSF, Leukine) and the MO/MA activating cytokine recombinant interferon gamma 1b (rIFN-gamma1b, Actimmune) has been developed. The pre- and post-chemotherapy design is based upon known in vivo kinetics and immune modulatory effects of these molecules. Carboplatin (Paraplatin) was selected as the cornerstone of treatment of epithelial ovarian cancer (EOC). METHODS: We studied hematopoietic and immunologic effects of GM-CSF and rIFN-gamma1b before and after carboplatin in patients with recurrent EOC. Potentially chemotherapy-sensitive patients with recurrent measurable tumors received subcutaneous GM-CSF (starting at 400 mug/day) for 7 days plus subcutaneous rIFN-gamma1b (100 mug) on days 5 and 7, before and after intravenous carboplatin (area under the curve of 5). We performed standard hematologic assessment and monitored monocyte (MO), dendritic cell, major cell subset counts, and antibody-dependent cell-mediated cytotoxicity (ADCC) against a Her2neu+ tumor cell line, as well as selected plasma inflammatory cytokine, chemokine and growth factor levels. RESULTS: Our analysis comprised only the first 3 months of treatment in the initial 25 patients. Relative to pretreatment baseline values, white blood cell, neutrophil, MO, and eosinophil counts increased (P

rIFN-gamma-mediated growth suppression of platinum-sensitive and -resistant ovarian tumor cell lines not dependent upon arginase inhibition.

BACKGROUND: Arginine metabolism in tumor cell lines can be influenced by various cytokines, including recombinant human interferon-gamma (rIFN-gamma), a cytokine that shows promising clinical activity in epithelial ovarian cancer (EOC). METHODS: We examined EOC cell lines for the expression of arginase in an enzymatic assay and for transcripts of arginase I and II, inducible nitric oxide synthase (iNOS), and indoleamine 2,3-dioxygenase (IDO) by reverse transcription-polymerase chain reaction. The effects of rIFN-gamma on arginase activity and on tumor cell growth inhibition were determined by measuring [3H]thymidine uptake. RESULTS: Elevated arginase activity was detected in 5 of 8 tumor cell lines, and analysis at the transcriptional level showed that arginase II was involved but arginase I was not. rIFN-gamma reduced arginase activity in 3 EOC cell lines but increased activity in the 2008 cell line and its platinum-resistant subline, 2008.C13. iNOS transcripts were not detected in rIFN-gamma-treated or untreated cell lines. In contrast, IDO activity was induced or increased by rIFN-gamma. Suppression of arginase activity by rIFN-gamma in certain cell lines suggested that such inhibition might contribute to its antiproliferative effects. However, supplementation of the medium with polyamine pathway products did not interfere with the growth-inhibitory effects of rIFN-gamma EOC cells. CONCLUSIONS: Increased arginase activity, specifically identified with arginase II, is present in most of the tested EOC cell lines. rIFN-gamma inhibits or stimulates arginase activity in certain EOC cell lines, though the decrease in arginase activity does not appear to be associated with the in vitro antiproliferative activity of rIFN-gamma. Since cells within the stroma of EOC tissues could also contribute to arginine metabolism following treatment with rIFN-gamma or rIFN-gamma-inducers, it would be helpful to examine these effects in vivo.