A large-scale effort, termed the Secreted Protein Discovery Initiative (SPDI), was undertaken to identify novel secreted and transmembrane proteins. In the first of several approaches, a biological signal sequence trap in yeast cells was utilized to identify cDNA clones encoding putative secreted proteins. A second strategy utilized various algorithms that recognize features such as the hydrophobic properties of signal sequences to identify putative proteins encoded by expressed sequence tags (ESTs) from human cDNA libraries. A third approach surveyed ESTs for protein sequence similarity to a set of known receptors and their ligands with the BLAST algorithm. Finally, both signal-sequence prediction algorithms and BLAST were used to identify single exons of potential genes from within human genomic sequence. The isolation of full-length cDNA clones for each of these candidate genes resulted in the identification of >1000 novel proteins. A total of 256 of these cDNAs are still novel, including variants and novel genes, per the most recent GenBank release version. The success of this large-scale effort was assessed by a bioinformatics analysis of the proteins through predictions of protein domains, subcellular localizations, and possible functional roles. The SPDI collection should facilitate efforts to better understand intercellular communication, may lead to new understandings of human diseases, and provides potential opportunities for the development of therapeutics.
Increased levels of B lymphocyte stimulator (BLyS) are associated with systemic autoimmunity in animal models of spontaneous autoimmune disease, and transgenic animals expressing BLyS develop typical autoimmune disease. Here, we demonstrate significant elevations of BLyS in the patients with systemic lupus erythematosus (SLE). The BLyS isolated from the sera of SLE patients had the same m.w. as the natural soluble form and was able to stimulate B cell activation in vitro. Increased BLyS in SLE patients was partially associated with higher levels of anti-dsDNA Ab of the IgG, IgM, and IgA classes, but not associated with the disease activity. Our results suggest that BLyS may be a useful marker for early activation of an autoimmune diathesis and likely plays a critical role in triggering activation of self-Ag-driven autoimmune B cells in human SLE. BLyS may provide an effective therapeutic target in systemic autoimmunity.
The tolerability, pharmacodynamic effects, and pharmacokinetics of belimumab (LymphoStat-B) were evaluated in cynomolgus monkeys. Belimumab is a fully human IgG1λ antibody directed against B-lymphocyte stimulator (BLyS) protein. BLyS is a TNF family member that supports B-lymphocyte maturation and survival and has been implicated in the pathogenesis of autoimmune diseases and B-lymphocyte malignancies. Belimumab was developed to antagonize BLyS activity in autoimmune diseases and B-lymphocyte malignancies, where undesirable effects of B-lymphocyte activity may cause or contribute to disease. Pharmacodynamic effects of belimumab were monitored by immunophenotyping of peripheral blood. Pathology end points, including tissue immunophenotyping, are described after 13 and 26 weeks of treatment and after a 34-week treatment-free (recovery) period. Belimumab was safe and well tolerated in repeat-dose toxicology studies at 5–50 mg/kg for up to 26 weeks. Monkeys exposed to belimumab had significant decreases in peripheral blood B lymphocytes by 13 weeks of exposure, continuing into the recovery period, despite total lymphocyte counts similar to the controls. There were concomitant decreases in spleen and lymph node B-lymphocyte representation after 13 or 26 weeks of treatment with belimumab. Microscopically, monkeys treated with belimumab for 13 or 26 weeks had decreases in the number and size of lymphoid follicles in the white pulp of the spleen. All findings were generally reversible within a 34-week recovery period. These data confirm the specific pharmacologic activity of belimumab in reducing B lymphocytes in the cynomolgus monkey. The favorable safety profile and lack of treatment-related infections also support continued clinical development of belimumab.
Signal and stop-transfer sequences are the known determinants involved in topogenesis of integral membrane proteins. To study the characteristics of stop-transfer sequences, artificial proteins have been created on the DNA level based on the cDNA of the asialoglycoprotein receptor H1. Its internal signal/anchor domain initiates translocation of the downstream sequence across the endoplasmic reticulum membrane. The ability of several hydrophobic sequences inserted into the translocating polypeptide to stop further transfer was analyzed by translation of the fusion proteins using the wheat germ extract and rabbit reticulocyte lysate systems with dog pancreas microsomes. We discovered that some of the sequences behave differently with respect to translocation across the membrane depending on the translation system. Expression of one of the fusion proteins in fibroblasts showed that the reticulocyte lysate system reflects more closely the in vivo situation than the wheat germ system. Our results suggest that in a homologous system the translating ribosomes interact with the translocation machinery and influence the termination of polypeptide transfer by hydrophobic sequences.
Abstract The tumor microenvironment contains diverse types of myeloid cells, including tumor-associated macrophages (TAMs), tumor-associated neutrophils (TANs) and myeloid-derived suppressor cells (MDSCs). TAMs and TANs exhibit a spectrum of functional phenotypes ranging from immunosuppressive that promote tumor growth, to pro-inflammatory that unleash anti-tumor immunity. Therapies that shift the inhibitory myeloid cell phenotypes toward more pro-inflammatory function are expected to positively impact anti-tumor immune responses and convert checkpoint inhibitor (CPIs)-resistant tumors into CPI-sensitive tumors. TREM1 (triggering receptor expressed on myeloid cells-1) is a transmembrane protein enriched in myeloid cells, including intratumoral TAMs, TANs and MDSCs. In a number of tumor types, TREM1 expression negatively correlated with patient survival. Furthermore, a survey of the immune infiltrates in a variety of human solid tumor types by flow cytometry and gene expression analysis revealed a high frequency of intratumoral TREM1+ myeloid cells in all tested tumors. We developed anti-human and anti-mouse TREM1 monoclonal antibodies (mAbs), termed PY159 and PY159m, respectively, to target TREM1+ myeloid cells. These mAbs triggered signaling pathways downstream of TREM1 and induced a highly selective pro-inflammatory cytokine signature in ex vivo assays. Additionally, PY159 treatment increased the myeloid cell expression of HLA-DR and the costimulatory molecule CD40, indicating an enhanced potential for costimulation and immune activation. Consistent with ex vivo results, in vivo treatment with PY159m promoted both innate and adaptive immune pathways in syngeneic mouse tumors revealed by gene expression profiling. These findings suggest that anti-TREM1 therapy re-educates TREM1+ myeloid cells into pro-inflammatory cells. Furthermore, PY159m was sufficient to drive anti-tumor activity as a single agent in a number of syngeneic tumor models. Strikingly, PY159m also converted anti-PD-1 mAb-resistant tumors into treatment-sensitive tumors with combination therapy, demonstrating the utility of targeting TREM1+ myeloid cells as a combination approach to improve CPI therapy responses. Mice cured of their tumors by PY159m/PD-1 mAb combination therapy were resistant to tumor re-challenge, demonstrating that targeting TREM1+ myeloid cells also supports adaptive immunity and induces long-term immunological memory. Based on these preclinical findings, we are developing PY159 as a therapeutic agent for monotherapy and/or CPI combination therapy for solid tumors. Citation Format: Vladi Juric, Chris Chan, Erin Mayes, Manith Norng, Tiep Le, Subhadra Dash, Venkataraman Sriram, Erick Lu, Joshua L. Pollack, Mikhail Binnewies, Joanna Waszczuk, Xiaoyan Du, Shilpa Mankikar, Aritra Pal, Kevin P. Baker, Linda Liang. Targeting of TREM1+ myeloid cells to promote antitumor immunity [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C105. doi:10.1158/1535-7163.TARG-19-C105
The tumor microenvironment (TME) often contains high levels of suppressive myeloid cells that contribute to innate checkpoint inhibitor (CPI) resistance. Pionyr's Myeloid Tuning approach involves altering the composition and/or the function of myeloid cells in the TME. Myeloid reprogramming alters the function of immunosuppressive myeloid cells to acquire an immunostimulatory phenotype. Triggering receptor expressed on myeloid cells-1 (TREM1) is an immunoglobulin superfamily cell surface receptor enriched on tumor-associated myeloid cells. To investigate the potential of TREM1 modulation as an anti-cancer therapeutic strategy, Pionyr developed an afucosylated humanized anti-TREM1 monoclonal antibody termed PY159 and characterized it in pre-clinical and translational biomarker assays described below.
Methods
PY159 responses in human whole blood and dissociated primary tumor cells in vitro were evaluated by flow cytometry and measurement of secreted cytokines and chemokines by MSD. TREM1 expression in human tumors was validated by scRNAseq, flow cytometry, and immunohistochemistry (IHC). In vivo efficacy and pharmacodynamic studies of a surrogate anti-mouse TREM1 antibody, termed PY159m, were evaluated using syngeneic mouse tumor models, either as a single-agent or in combination with anti-PD-1. To select tumor types and patients most likely to benefit from PY159 therapy, Pionyr developed qualitative and quantitative monoplex and multiplex IHC assays that detect TREM1 expression levels in human tumor tissues.
Results
PY159 treatment in vitro induced signaling, upregulated monocyte activation markers, and induced proinflammatory cytokines. In human tumors, TREM1 was detected on tumor-associated neutrophils, tumor-associated macrophages, and monocytic myeloid-derived suppressive cells. The surrogate PY159m anti-mouse TREM1 antibody exhibited anti-tumor efficacy in several syngeneic mouse tumor models, both as single-agent and in combination with anti-PD-1. Screening for TREM1 expression in tumor tissues demonstrated that TREM1+ tumor associated myeloid cells were highly enriched in the TME of multiple solid tumor indications. The monoplex and multiplex IHC assays offered insights into the localization of TREM1+ myeloid cells and their spatial relationship with other immune cells present in the TME to determine what immune composition will be more favorable for response to PY159 therapy.
Conclusions
Collectively, the available nonclinical data support PY159 as a TREM1 agonist that reprograms myeloid cells and unleashes anti-tumor immunity. PY159 safety and efficacy are currently being evaluated in first-in-human clinical trial (NCT04682431) involving select advanced solid tumors patients resistant and refractory to standard of care therapies alone and in combination with a CPI. The TREM1 IHC assay is successfully being used on FFPE archival tumor tissues from enrolled patients to determine TREM1 expression levels.
Abstract An increasing body of evidence has implicated FGF2 as one of the drivers of resistance to various inhibitors of VEGF-mediated angiogenesis. This resistance may play a role as a key limitation to the efficacy of therapies targeted at VEGF and its receptors. We investigated the potential for FP-1039/GSK3052230, a ligand trap that sequesters FGFs and inhibits their signaling, to enhance the activity of VEGF antagonist therapies in certain preclinical models of renal cell (RCC) and hepatocellular (HCC) carcinomas. First, we examined whether FP-1039/GSK3052230 has single agent efficacy against human RCC and HCC xenografts that express relatively high levels of FGF2, a profile that would mimic FGF2-driven resistance to VEGF therapy. We determined that this expression profile represents 34% of clear cell RCC (ccRCC) and 31% of HCC patients, based on the cancer genome atlas (TCGA) data. Human ccRCC xenografts with high FGF2 expression and low VEGFA expression demonstrated a significant inhibition in tumor growth when treated with FP-1039/GSK3052230 alone (TGI: 39-81%). In addition, we show that the high FGF2 expression profile is similarly predictive for the anti-tumor response of a human HCC model to single-agent FP-1039/GSK3052230 (TGI: 31-55%). In contrast, RCC models with low FGF2 expression, representing 66% of all ccRCC in the TCGA, are relatively insensitive to FP-1039/GSK3052230 as a single-agent. However, combination therapy of FP-1039/GSK3052230 with pazopanib in these tumors is significantly more effective than either agent alone. FP-1039/GSK3052230 not only slows tumor growth, but can induce ∼25% tumor regression when administered to mice bearing ccRCC xenografts that have become resistant to pazopanib. Together, our data demonstrate that FP-1039/GSK3052230 may be an effective therapy against RCC and HCC, both as a single agent in disease driven by FGF2 and in combination with VEGF antagonist therapies that represent the current standards of care for advanced disease. Citation Format: David I. Bellovin, Servando Palencia, Kevin Hestir, Ernestine Lee, M. Phillip DeYoung, Thomas Brennan, Gerrit Los, Kevin Baker. FP-1039/GSK3052230, an FGF ligand trap, enhances VEGF antagonist therapy in preclinical models of RCC and HCC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5449. doi:10.1158/1538-7445.AM2014-5449
To identify and characterize a fully human antibody directed against B lymphocyte stimulator (BLyS), a tumor necrosis factor-related cytokine that plays a critical role in the regulation of B cell maturation and development. Elevated levels of BLyS have been implicated in the pathogenesis of autoimmune diseases.A human phage display library was screened for antibodies against human BLyS. A human monoclonal antibody, LymphoStat-B, specific for human BLyS was obtained from the library screening and subsequent affinity optimization mutagenesis. The antibody was tested for inhibition of human BLyS in vitro and in an in vivo murine model. Additionally, the consequences of BLyS inhibition were tested in vivo by administration of LymphoStat-B to cynomolgus monkeys.LymphoStat-B bound with high affinity to human BLyS and inhibited the binding of BLyS to its 3 receptors, TACI, BCMA, and BLyS receptor 3/BAFF-R. LymphoStat-B potently inhibited BLyS-induced proliferation of B cells in vitro, and administration of LymphoStat-B to mice prevented human BLyS-induced increases in splenic B cell numbers and IgA titers. In cynomolgus monkeys, administration of LymphoStat-B resulted in decreased B cell representation in both spleen and mesenteric lymph nodes.A fully human monoclonal antibody has been isolated that binds to BLyS with high affinity and neutralizes human BLyS bioactivity in vitro and in vivo. Administration of this antibody to cynomolgus monkeys resulted in B cell depletion in spleen and lymph node. This antibody may prove therapeutically useful in the treatment of autoimmune diseases in humans.
