Cell Surface Membrane Proteins Research Articles

On-cell nuclear magnetic resonance spectroscopy to probe cell surface interactions

Nuclear magnetic resonance (NMR) spectroscopy allows determination of atomic-level information about intermolecular interactions, molecular structure, and molecular dynamics in the cellular environment. This may be broadly divided into studies focused on obtaining detailed molecular information in the intracellular context ("in-cell") or those focused on characterizing molecules or events at the cell surface ("on-cell"). In this review, we outline some key NMR techniques applied for on-cell NMR studies through both solution-state and solid-state NMR and survey studies that have used these techniques to uncover key information. We particularly focus on application of on-cell NMR spectroscopy to characterize ligand interactions with cell surface membrane proteins such as G-protein coupled receptors (GPCRs), receptor tyrosine kinases, etc. These techniques allow for quantification of binding affinities, competitive binding assays, delineation of portions of ligands involved in binding, ligand bound-state conformational determination, evaluation of receptor structuring and dynamics, and inference of distance constraints characteristic of the ligand-receptor bound state. Excitingly, it is possible to avoid the barriers of production and purification of membrane proteins while obtaining directly physiologically-relevant information through on-cell NMR. We also provide a briefer survey of the applicability of on-cell NMR approaches to other classes of cell surface molecule.

Lateral Interaction Between CD147 and B7-H3 Determines the Stemness and Survival of Breast Cancer Patients

Oncogenic cell-surface membrane proteins contribute to the phenotypic and functional characteristics of cancer stem cells (CSCs). We quantitatively analyzed cell-surface membrane proteins that are in close proximity to CD147 in CSCs. Moreover, we compared CSCs to non-CSCs to identify CSC-specific cell-surface membrane proteins that are the nearest neighbors of CD147 and revealed that lateral interaction between CD147 and CD276 (B7-H3) concealed within the lipid raft microdomain in CSCs confers resistance to docetaxel. Furthermore, we found that the co-expression of CD147 and CD276 was linked to unfavorable clinical outcome in patients with HER2+ breast cancer receiving chemotherapy (disease-free survival: p = 0.04, METABRIC). Subsequent immunohistochemical analysis indicated that CD147 and CD276 were exclusively associated with an inadequate chemotherapeutic response. Altogether, our data suggest that the lateral interaction between CD147 and its proximal partners may be significantly associated with clinicopathological characteristics and is a critical phenotypic determinant of breast cancer stemness. Funding Information: This research was funded by the National Research Foundation of Korea (NRF) (grant numbers: NRF-2016R1D1A1B04931656 and NRF-2021M3E5D3016636 to K.M.K., NRF-2016R1A5A1010764 and NRF-2020M3E5D9080791 to E.C.Y., and NRF-2017-Global Ph.D. Fellowship Program to J.L.). This research was supported by 2017 Research Grant (No. 520170409) and 2019 Research Grant (PoINT) from Kangwon National University and the BK21 FOUR funded by the Ministry of Education (MOE, Korea) and NRF. Declaration of Interests: The authors declare no competing interests. Ethical Approval: The Institutional Review Board of Seoul National University Hospital approved this study protocol

Anti-BCMA Immunotoxins: Design, Production, and Preclinical Evaluation.

Multiple myeloma (MM) is a B-cell malignancy that is incurable for a majority of patients. B-cell maturation antigen (BCMA) is a lineage-restricted differentiation protein highly expressed in multiple myeloma cells but not in other normal tissues except normal plasma B cells. Due to the restricted expression and being a cell surface membrane protein, BCMA is an ideal target for immunotherapy approaches in MM. Recombinant immunotoxins (RITs) are a novel class of protein therapeutics that are composed of the Fv or Fab portion of an antibody fused to a cytotoxic agent. RITs were produced by expressing plasmids encoding the components of the anti-BCMA RITs in E. coli followed by inclusion body preparation, solubilization, renaturation, and purification by column chromatography. The cytotoxic activity of RITs was tested in vitro by WST-8 assays using BCMA expressing cell lines and on cells isolated from MM patients. The in vivo efficacy of RITs was tested in a xenograft mouse model using BCMA expressing multiple myeloma cell lines. Anti-BCMA recombinant immunotoxins are very effective in killing myeloma cell lines and cells isolated from myeloma patients expressing BCMA. Two mouse models of myeloma showed that the anti-BCMA immunotoxins can produce a long-term complete response and warrant further preclinical development.

The discovery of a new antibody for BRIL-fused GPCR structure determination

G-protein-coupled receptors (GPCRs)—the largest family of cell-surface membrane proteins—mediate the intracellular signal transduction of many external ligands. Thus, GPCRs have become important drug targets. X-ray crystal structures of GPCRs are very useful for structure-based drug design (SBDD). Herein, we produced a new antibody (SRP2070) targeting the thermostabilised apocytochrome b562 from Escherichia coli M7W/H102I/R106L (BRIL). We found that a fragment of this antibody (SRP2070Fab) facilitated the crystallisation of the BRIL-tagged, ligand bound GPCRs, 5HT1B and AT2R. Furthermore, the electron densities of the ligands were resolved, suggesting that SPR2070Fab is versatile and adaptable for GPCR SBDD. We anticipate that this new tool will significantly accelerate structure determination of other GPCRs and the design of small molecular drugs targeting them.

A liposome-based cancer vaccine for a rapid and high-titre anti-ErbB-2 antibody response

Vaccines are arguably the most important medical technology developed to date. However, effective treatment of diseases such as breast cancer have so far evaded standard vaccination strategies. One popular target for cancer treatment is the cell surface membrane protein, ErbB-2, also known as Her-2 or neu. It is localised to the cell surface and has raised expression in 15–30% of all breast cancers, as well as in ovarian, colon and lung cancer. Here, a liposomal system comprised of spatially separated ErbB-2 peptide, to activate B cells, and ovalbumin peptide OVA323−339, to provide non-cognate T cell support, was used to generate antibodies against the epitope of the ErbB-2 protein targeted by Pertuzumab, a monoclonal antibody licensed for the treatment of ErbB-2 expressing cancers. After just 7 days a raised (7.3-fold, p<0.01), isotype-switched, humoral immune response specific for the ErbB-2 peptide was achieved in mice with pre-existing immunity to OVA which were exposed to liposomes with external ErbB-2 and internal OVA323−339. The absence of pre-existing OVA immunity in the mice or OVA323−339 peptide in the liposomes removed the effect. The effect of this anti-ErbB-2 antibody response was characterised against an ErbB-2 overexpressing tumour cell line both in vitro and in vivo. Notably, antibody responses were demonstrated to induce cell death in vitro, resulting in 96% reduction in viable cells. This study, therefore, demonstrates the feasibility of this approach to generate a rapid, high-titre, isotype-switched, antibody response that specifically targets ErbB-2 overexpression on tumour cells and is capable of inducing cell death in vitro in the absence of complement or immune cells.

Extracellular vesicles as a platform to study cell-surface membrane proteins.

Producing intact recombinant membrane proteins for structural studies is an inherently challenging task due to their requirement for a cell-lipid environment. Most of the procedures developed involve isolating the protein by solubilization with detergent and further reconstitutions into artificial membranes. These procedures are highly time consuming and suffer from further drawbacks, including low yields and high cost. We describe here an alternative method for rapidly obtaining recombinant cell-surface membrane proteins displayed on extracellular vesicles (EVs) derived from cells in culture. Interaction between these membrane proteins and ligands can be analyzed directly on EVs. Moreover, EVs can also be used for protein structure determination or immunization purposes.

The Lysosome Pathway Degrades CD81 on the Cell Surface by Poly-ubiquitination and Clathrin-Mediated Endocytosis.

CD81 is a highly conserved four-transmembrane protein in mammals and widely expressed on many tissues. It belongs to the tetraspanin family and forms complexes with various cell surface membrane proteins. It also functions in cell migration and B-cell activation, which is induced by CD81 complexing with CD19, CD21 and the B-cell receptor. Thus, CD81 is thought to play a key role in regulating cell function and fate. However, little is known about the degradation mechanism of CD81. Here we found that CD81 on the plasma membrane is degraded by the lysosome pathway via endocytosis. The expression levels of CD81 in HEK293T cells treated with a proteasome inhibitor (lactacystin) and lysosome inhibitors (chloroquine and bafilomycin A1) were analyzed by flow cytometry. The expression of CD81 on the cell surface was increased by the lysosome inhibitors, but not lactacystin. A pulldown assay revealed that CD81 was conjugated with a K63- and K29-linked poly-ubiquitin chain before its degradation, and the poly-ubiquitination site was Lys8 at the N-terminal intracellular domain of CD81. Furthermore, mutant CD81, in which Lys8 was substituted with alanine (Ala), extended the CD81 half-life compared with wildtype. CD81 was mainly localized on the plasma membrane in normal cells, but also co-localized with lysosomal LAMP1 and early endosomal EEA1 in chloroquine-treated cells. Furthermore, a clathrin-mediated endocytosis inhibitor, chlorpromazine, stabilized CD81 expression on the cell surface. Hence, we demonstrated that CD81 is internalized by clathrin-mediated endocytosis and subsequently degraded via a lysosome pathway requiring the K63- and K29-linked poly-ubiquitination of CD81.

Abstract 4833: Antibody-drug conjugate targeting glypican-1 shows tumor growth inhibition in cholangiocarcinoma

Abstract Cholangiocarcinoma is one of the most highly malignant cancers. Many patients need systemic chemotherapy for tumor development and recurrence, but their prognosis is poor. Therefore new treatment options are urgently required. We confirmed that the expression of glypican-1 (GPC1) was enhanced in cholangiocarcinoma. GPC1 is a cell surface membrane protein and has been reported as a poor prognostic factor in pancreatic cancer. In this study, we aimed to develop a new therapy for cholangiocarcinoma by the antibody-drug conjugate (ADC) targeting GPC1. By immunohistochemical analysis, enhanced expression of GPC1 was observed in clinical specimens of cholangiocarcinoma. KKU-055 and KKU-100 cells, which are cell lines of cholangiocarcinoma, had high expression of GPC1. In the KKU-055 xenograft model when we administered fluorescently-labeled GPC1 antibody to mice, it accumulated in tumor tissue. We developed a new anti-GPC1 monoclonal antibody (mAb) with highly internalizing activity. The anti-GPC1 mAb was conjugated with the cytotoxic agent monomethyl auristatin F (MMAF). GPC1-ADC showed potent antitumor effect toward KKU-055 and KKU-100 cells compared with the control ADC. In the KKU-055 xenograft model, GPC1-ADC had significant and potent tumor growth inhibition in a dose-dependent manner. In summary, our newly-developed GPC1-ADC showed significant tumor growth inhibition against GPC1-positive cholangiocarcinoma cell lines. Our preclinical data demonstrated that targeting GPC1 by ADC is a promising therapy for GPC1-positive cholangiocarcinoma. Citation Format: Keiichiro Yokota, Satoshi Serada, Shigehiro Tsujii, Kosuke Hiramatsu, Tsutomu Namikawa, Ichiro Murakami, Kazuhiro Hanazaki, Tetsuji Naka. Antibody-drug conjugate targeting glypican-1 shows tumor growth inhibition in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4833.

A serine in the first transmembrane domain of the human E3 ubiquitin ligase MARCH9 is critical for down-regulation of its protein substrates

The membrane-associated RING-CH (MARCH) family of membrane-bound E3 ubiquitin ligases regulates the levels of cell-surface membrane proteins, many of which are involved in immune responses. Although their role in ubiquitin-dependent endocytosis and degradation of cell-surface proteins is extensively documented, the features of MARCH proteins and their substrates that drive the molecular recognition events leading to ubiquitin transfer remain poorly defined. In this study, we sought to determine the features of human MARCH9 that are required for regulating the surface levels of its substrate proteins. Consistent with previous studies of other MARCH proteins, we found that susceptibility to MARCH9 activity is encoded in the transmembrane (TM) domains of its substrates. Accordingly, substitutions at specific residues and motifs within MARCH9's TM domains resulted in varying degrees of functional impairment. Most notably, a single serine-to-alanine substitution in the first of its two TM domains rendered MARCH9 completely unable to alter the surface levels of two different substrates: the major histocompatibility class I molecule HLA-A2 and the T-cell co-receptor CD4. Solution NMR analysis of a MARCH9 fragment encompassing the two TM domains and extracellular connecting loop revealed that the residues contributing most to MARCH9 activity are located in the α-helical portions of TM1 and TM2 that are closest to the extracellular face of the lipid bilayer. This observation defines a key region required for substrate regulation. In summary, our biochemical and structural findings demonstrate that specific sequences in the α-helical MARCH9 TM domains make crucial contributions to its ability to down-regulate its protein substrates.

Abstract 3202: Radiation increases invasive activity of breast cancer cells via altering lysosome exocytosis

Abstract Radiotherapy is a standard treatment for many localized solid cancers. However, previous studies have shown that radiation may increase the invasive activity of cancer cells and potentially distant metastasis. Recently, lysosome exocytosis has been linked to cancer cell invasiveness and progression. In this study, we evaluate the role of lysosome exocytosis on invasive activity of breast cancer cells upon radiation.We used both human and murine breast cancer cell lines (MDA-MB-231 and 4T1). The cells were treated with a single radiation dose of 4 Gy. Cell invasive activity was measured by matrigel chemoinvasion assay. Lysosome exocytosis was quantified by fluorescein isothiocyanate conjugated dextran (FITC-dextran) intake assay, cell-surface lysosomal associated membrane protein 1 (LAMP1) expression and cellular lysosome distribution assay. To validate the lysosome function, lysosome inhibitors, bafilomycin A1 and chloroquine were used. Short hairpin RNA (shRNA) was used to knockdown ARL8B, which is a small GTPase protein that regulates lysosome distribution and exocytosis. The invasive activity and lysosome exocytosis of tested breast cancer cell lines were increased after radiation treatment. Treatment with lysosome inhibitor bafilomycin A1 or chloroquine decreased the invasive activity of cancer cells, with or without radiation treatment. The protein level of ARL8B increased in the lysosome fraction upon radiation. Down-regulation of ARL8B with shRNA led to a decrease in lysosome exocytosis with a concomitant inhibition radiation induced invasive activity of the breast cancer cells without affecting the basal invasiveness. In addition, overexpression of ARL8B increased the invasive activity of breast cancer cells, which was similar to the result obtained after radiation.In summary, radiation enhances lysosome exocytosis in breast cancer cells that can lead to their increased invasive activity. Our findings provide a novel mechanism to understand cancer invasion after radiotherapy and suggest novel approaches to counteract this undesirable effect of radiotherapy in the future. Citation Format: Ping-Hsiu Wu, Yasuhito Onodera, Amato J. Giaccia, Quynh-Thu Le, Hiroki Shirato, Jin-Min Nam. Radiation increases invasive activity of breast cancer cells via altering lysosome exocytosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3202.

Therapeutic Monoclonal Antibodies to Complex Membrane Protein Targets: Antigen Generation and Antibody Discovery Strategies.

Cell surface membrane proteins comprise a wide array of structurally and functionally diverse proteins involved in a variety of important physiological and homeostatic processes. Complex integral membrane proteins, which are embedded in the lipid bilayer by multiple transmembrane-spanning helices, are represented by families of proteins that are important target classes for drug discovery. Such protein families include G-protein-coupled receptors, ion channels and transporters. Although these targets have typically been the domain of small-molecule drugs, the exquisite specificity of monoclonal antibodies offers a significant opportunity to selectively modulate these target proteins. Nevertheless, the isolation of antibodies with desired pharmacological functions has proved difficult because of technical challenges in preparing membrane protein antigens for antibody drug discovery. In this review, we describe recent progress in defining strategies for the generation of membrane protein antigens. We also describe antibody-isolation strategies that identify antibodies that bind the membrane protein and modulate protein function.

Fluorogenic Probing of Membrane Protein Trafficking.

Methods to differentially label cell-surface and intracellular membrane proteins are indispensable for understanding their function and the regulation of their trafficking. We present an efficient strategy for the rapid and selective fluorescent labeling of membrane proteins based on the chemical-genetic fluorescent marker FAST (fluorescence-activating and absorption-shifting tag). Cell-surface FAST-tagged proteins could be selectively and rapidly labeled using fluorogenic membrane-impermeant 4-hydroxybenzylidene rhodanine (HBR) analogs. This approach allows the study of protein trafficking at the plasma membrane with various fluorometric techniques, and opens exciting prospects for the high-throughput screening of small molecules able to restore disease-related trafficking defects.

The major facilitator transporter Str3 is required for low-affinity heme acquisition in Schizosaccharomyces pombe

In the fission yeast Schizosaccharomyces pombe, acquisition of exogenous heme is largely mediated by the cell membrane-associated Shu1. Here, we report that Str3, a member of the major facilitator superfamily of transporters, promotes cellular heme import. Using a strain that cannot synthesize heme de novo (hem1Δ) and lacks Shu1, we found that the heme-dependent growth deficit of this strain is rescued by hemin supplementation in the presence of Str3. Microscopic analyses of a hem1Δ shu1Δ str3Δ mutant strain in the presence of the heme analog zinc mesoporphyrin IX (ZnMP) revealed that ZnMP fails to accumulate within the mutant cells. In contrast, Str3-expressing hem1Δ shu1Δ cells could take up ZnMP at a 10-μm concentration. The yeast Saccharomyces cerevisiae cannot efficiently transport exogenously supplied hemin. However, heterologous expression of Str3 from S. pombe in S. cerevisiae resulted in ZnMP accumulation within S. cerevisiae cells. Moreover, hemin-agarose pulldown assays revealed that Str3 binds hemin. In contrast, an Str3 mutant in which Tyr and Ser residues of two putative heme-binding motifs (530YX3Y534 and 552SX4Y557) had been replaced with alanines exhibited a loss of affinity for hemin. Furthermore, this Str3 mutant failed to rescue the heme-dependent growth deficit of a hem1Δ shu1Δ str3Δ strain. Further analysis by absorbance spectroscopy disclosed that a predicted extracellular loop region in Str3 containing the two putative heme-binding motifs interacts with hemin, with a KD of 6.6 μm Taken together, these results indicate that Str3 is a second cell-surface membrane protein for acquisition of exogenous heme in S. pombe.

Evidence of positive selection towards Zebuine haplotypes in the BoLA region of Brangus cattle

The Brangus breed was developed to combine the superior characteristics of both of its founder breeds, Angus and Brahman. It combines the high adaptability to tropical and subtropical environments, disease resistance, and overall hardiness of Zebu cattle with the reproductive potential and carcass quality of Angus. It is known that the major histocompatibility complex (MHC, also known as bovine leucocyte antigen: BoLA), located on chromosome 23, encodes several genes involved in the adaptive immune response and may be responsible for adaptation to harsh environments. The objective of this work was to evaluate whether the local breed ancestry percentages in the BoLA locus of a Brangus population diverged from the estimated genome-wide proportions and to identify signatures of positive selection in this genomic region. For this, 167 animals (100 Brangus, 45 Angus and 22 Brahman) were genotyped using a high-density single nucleotide polymorphism array. The local ancestry analysis showed that more than half of the haplotypes (55.0%) shared a Brahman origin. This value was significantly different from the global genome-wide proportion estimated by cluster analysis (34.7% Brahman), and the proportion expected by pedigree (37.5% Brahman). The analysis of selection signatures by genetic differentiation (Fst) and extended haplotype homozygosity-based methods (iHS and Rsb) revealed 10 and seven candidate regions, respectively. The analysis of the genes located within these candidate regions showed mainly genes involved in immune response-related pathway, while other genes and pathways were also observed (cell surface signalling pathways, membrane proteins and ion-binding proteins). Our results suggest that the BoLA region of Brangus cattle may have been enriched with Brahman haplotypes as a consequence of selection processes to promote adaptation to subtropical environments.

Calcium influx mediates the chemoattractant-induced translocation of the arrestin-related protein AdcC in Dictyostelium.

Arrestins are key adaptor proteins that control the fate of cell-surface membrane proteins and modulate downstream signaling cascades. The Dictyostelium discoideum genome encodes six arrestin-related proteins, harboring additional modules besides the arrestin domain. Here, we studied AdcB and AdcC, two homologs that contain C2 and SAM domains. We showed that AdcC - in contrast to AdcB - responds to various stimuli (such as the chemoattractants cAMP and folate) known to induce an increase in cytosolic calcium by transiently translocating to the plasma membrane, and that calcium is a direct regulator of AdcC localization. This response requires the calcium-dependent membrane-targeting C2 domain and the double SAM domain involved in AdcC oligomerization, revealing a mode of membrane targeting and regulation unique among members of the arrestin clan. AdcB shares several biochemical properties with AdcC, including in vitro binding to anionic lipids in a calcium-dependent manner and auto-assembly as large homo-oligomers. AdcB can interact with AdcC; however, its intracellular localization is insensitive to calcium. Therefore, despite their high degree of homology and common characteristics, AdcB and AdcC are likely to fulfill distinct functions in amoebae.

Paired Expression Analysis of Tumor Cell Surface Antigens.

Adoptive immunotherapy with antibody-based therapy or with T cells transduced to express chimeric antigen receptors (CARs) is useful to the extent that the cell surface membrane protein being targeted is not expressed on normal tissues. The most successful CAR-based (anti-CD19) or antibody-based therapy (anti-CD20) in hematologic malignancies has the side effect of eliminating the normal B cell compartment. Targeting solid tumors may not provide a similar expendable marker. Beyond antibody to Her2/NEU and EGFR, very few antibody-based and no CAR-based therapies have seen broad clinical application for solid tumors. To expand the way in which the surfaceome of solid tumors can be analyzed, we created an algorithm that defines the pairwise relative overexpression of surface antigens. This enables the development of specific immunotherapies that require the expression of two discrete antigens on the surface of the tumor target. This dyad analysis was facilitated by employing the Hotelling’s T-squared test (Hotelling–Lawley multivariate analysis of variance) for two independent variables in comparison to a third constant entity (i.e., gene expression levels in normal tissues). We also present a unique consensus scoring mechanism for identifying transcripts that encode cell surface proteins. The unique application of our bioinformatics processing pipeline and statistical tools allowed us to compare the expression of two membrane protein targets as a pair, and to propose a new strategy based on implementing immunotherapies that require both antigens to be expressed on the tumor cell surface to trigger therapeutic effector mechanisms. Specifically, we found that, for MYCN amplified neuroblastoma, pairwise expression of ACVR2B or anaplastic lymphoma kinase (ALK) with GFRA3, GFRA2, Cadherin 24, or with one another provided the strongest hits. For MYCN, non-amplified stage 4 neuroblastoma, neurotrophic tyrosine kinase 1, or ALK paired with GFRA2, GFRA3, SSK1, GPR173, or with one another provided the most promising paired-hits. We propose that targeting these markers together would increase the specificity and thereby the safety of CAR-based therapy for neuroblastoma.

Dissecting Multivalent Lectin-Carbohydrate Recognition Using Polyvalent Multifunctional Glycan-Quantum Dots.

Multivalent protein–carbohydrate interactions initiate the first contacts between virus/bacteria and target cells, which ultimately lead to infection. Understanding the structures and binding modes involved is vital to the design of specific, potent multivalent inhibitors. However, the lack of structural information on such flexible, complex, and multimeric cell surface membrane proteins has often hampered such endeavors. Herein, we report that quantum dots (QDs) displayed with a dense array of mono-/disaccharides are powerful probes for multivalent protein–glycan interactions. Using a pair of closely related tetrameric lectins, DC-SIGN and DC-SIGNR, which bind to the HIV and Ebola virus glycoproteins (EBOV-GP) to augment viral entry and infect target cells, we show that such QDs efficiently dissect the different DC-SIGN/R-glycan binding modes (tetra-/di-/monovalent) through a combination of multimodal readouts: Förster resonance energy transfer (FRET), hydrodynamic size measurement, and transmission electron microscopy imaging. We also report a new QD-FRET method for quantifying QD-DC-SIGN/R binding affinity, revealing that DC-SIGN binds to the QD >100-fold tighter than does DC-SIGNR. This result is consistent with DC-SIGN’s higher trans-infection efficiency of some HIV strains over DC-SIGNR. Finally, we show that the QDs potently inhibit DC-SIGN-mediated enhancement of EBOV-GP-driven transduction of target cells with IC50 values down to 0.7 nM, matching well to their DC-SIGN binding constant (apparent Kd = 0.6 nM) measured by FRET. These results suggest that the glycan-QDs are powerful multifunctional probes for dissecting multivalent protein–ligand recognition and predicting glyconanoparticle inhibition of virus infection at the cellular level.

Genetic dissection of early endosomal recycling highlights a TORC1-independent role for Rag GTPases.

Endocytosed cell surface membrane proteins rely on recycling pathways for their return to the plasma membrane. Although endosome-to-plasma membrane recycling is critical for many cellular processes, much of the required machinery is unknown. We discovered that yeast has a recycling route from endosomes to the cell surface that functions efficiently after inactivation of the sec7-1 allele of Sec7, which controls transit through the Golgi. A genetic screen based on an engineered synthetic reporter that exclusively follows this pathway revealed that recycling was subject to metabolic control through the Rag GTPases Gtr1 and Gtr2, which work downstream of the exchange factor Vam6. Gtr1 and Gtr2 control the recycling pathway independently of TORC1 regulation through the Gtr1 interactor Ltv1. We further show that the early-endosome recycling route and its control though the Vam6>Gtr1/Gtr2>Ltv1 pathway plays a physiological role in regulating the abundance of amino acid transporters at the cell surface.

Abstract 4410: LSR promotes ovarian cancer cell growth following the activation of β-oxidation and its antibody inhibits lipid catabolism

Abstract Ovarian cancer is the most lethal gynecologic malignancy; thus developing new treatment options is urgently required. Molecular targeted therapies for cancers, which are generally more tolerable than widely used cytotoxic agents, have shown highly specific inhibition of target molecules. In this study, we aimed to identify a new ovarian cancer antigen and to develop a novel monoclonal antibody (mAb). Furthermore we evaluated its preclinical efficacy and analyzed the function of its antigen in ovarian cancer.To identify a new ovarian cancer antigen, cell surface membrane proteins of normal ovarian epithelial and ovarian cancer cell lines were analyzed by iTRAQ-based proteomic technology. We identified lipolysis-stimulated lipoprotein receptor (LSR) as the new therapeutic target for ovarian cancer. By the immunohistochemical analysis, significant poor prognosis was observed in high-LSR expression patients with ovarian cancer compared to patients with low-LSR expression by survival assay (p &amp;lt; 0.05). Our newly developed anti-LSR mAb showed significant inhibition of tumor growth in vivo against xenograft model of LSR-positive ovarian cancer cell line and patient derived LSR-positive ovarian cancer tissue (p &amp;lt; 0.05). In LSR-positive ovarian cancer cells, high number and large lipid droplets were observed compared to LSR-negative cells and anti-LSR mAb decreased these droplets. Moreover addition of VLDL to LSR-positive ovarian cancer cells significantly promoted the cell proliferation (p &amp;lt; 0.05) and anti-LSR mAb inhibited that in vitro (p &amp;lt; 0.05). Supporting these data, addition of VLDL to LSR-positive ovarian cancer cells significantly promoted β-oxidation-mediated lipid catabolism (p &amp;lt; 0.05) and anti-LSR mAb also inhibited that (p &amp;lt; 0.05). In addition, this anti-LSR mAb which cross-reacted with mouse LSR did not show any cytotoxicity on normal organs and lipid metabolism in mice. In summary, high expression of LSR in ovarian cancer was the poor prognostic factor. Our newly developed anti-LSR mAb showed significant tumor growth inhibition against not only LSR-positive ovarian cancer cell line but also patient derived LSR-positive ovarian cancer tissue. In LSR-positive ovarian cancer cells, high number and large lipid droplets were observed and LSR promoted cell proliferation following β-oxidation-mediated lipid catabolism. Anti-LSR mAb inhibited these processes. Our preclinical data demonstrated that targeting LSR by mAb is a promising therapy for patients with LSR-positive ovarian cancer. Citation Format: Kosuke Hiramatsu, Satoshi Serada, Takayuki Enomoto, Takuhei Yokoyama, Yusuke Takahashi, Minoru Fujimoto, Kiyoshi Yoshino, Tadashi Kimura, Tetsuji Naka. LSR promotes ovarian cancer cell growth following the activation of β-oxidation and its antibody inhibits lipid catabolism [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 4410. doi:10.1158/1538-7445.AM2017-4410

Abstract 2651: A novel CD19 targeting antibody-drug conjugate, huB4-DGN462, shows promising in vitro and in vivo activity in CD19-positive lymphoma models

Abstract Background. CD19 is a cell surface membrane protein expressed in most mature and immature B cell neoplasms, making it a promising target for antibody-drug conjugate (ADC) therapy for B cell malignancies. Here we describe the preclinical activity of a novel CD19-targeting ADC, based on the potent indolinobenzodiazepine DNA-alkylating payload DGN462. Methods. The humanized anti-CD19 antibody, huB4, was conjugated to DGN462 via a cleavable disulfide linker, sulfo-SPDB. In vitro activity of the huB4-DGN462 ADC or the unconjugated DGN462 toxin was evaluated in 54 lymphoma cell lines [27 diffuse large B cell lymphomas (DLBCL); 10 mantle cell lymphomas; 6 marginal zone lymphomas; 5 anaplastic large T-cell lymphomas; 6 others]. Cell proliferation/viability after 72h of exposure was measured using a MTT assay. Apoptosis activation was defined by at least 1.5-fold increase in caspase 3/7 signal activation in respect to controls using the Promega ApoTox-Glo Triplex Assay. Gene expression profiling (GEP) was performed with the Illumina HumanHT-12 Expression BeadChips on untreated cell lines followed by GSEA (NES &amp;gt; |2|, P&amp;lt;0.05, FDR&amp;lt;0.25) and limma t-test (FC&amp;gt; |1.2|; P&amp;lt; 0.05; top 200 up and top 200 down). In vivo efficacy of huB4-DGN462 was evaluated in CD19-expressing xenograft tumor models. Results. huB4-DGN462 was cytotoxic against a broad panel of 48 B cell lymphoma cell lines (median IC50 100 pM; 95%CI, 38-214). The cytotoxic activity was not limited by P53, BCL2, MYC or CDKN2A status, or associated with DLBCL cell of origin. Consistent with overall lower CD19 expression, huB4-DGN462 was significantly (p-value=0.007) less active in eight T cell-derived lymphomas (median IC50 of 1.75 nM (95%CI, 0.5-5.75)) than in B cell lymphomas. huB4-DGN462 induced caspase 3/7 activation in 48/54 cell lines (89%) consistent with an apoptotic mechanism of action. huB4-DGN462 demonstrated compelling anti-tumor activity after a single intravenous dose in two diffuse large B-cell lymphoma cell line xenograft models: DoHH2 (a subcutaneous model) and Farage (a disseminated model). In the DoHH2 model, huB4-DGN462 resulted in a significant, dose-dependent tumor growth delay and survival benefit at 1.7 mg Ab/kg compared to a non-targeted control DGN462 ADC. In the disseminated Farage model, a significant dose-dependent increase in survival was observed in mice treated with as low as 0.17 mg Ab/kg of huB4-DGN462. At 1.7 mg Ab/kg, the life span was increased &amp;gt;400% compared to untreated mice. Conclusions. The novel ADC huB4-DGN462 presented strong preclinical anti-lymphoma activity, which provides evidence for further study. Citation Format: Eugenio Gaudio, Chiara Tarantelli, Alberto J. Arribas, Roberta Pittau Bordone, Andrea Rinaldi, Georg Stussi, Emanuele Zucca, Davide Rossi, Anastasios Stathis, Min Li, Alan Wilhem, Kate Lai, Qifeng Qiu, Stuart Hicks, Callum Sloss, Francesco Bertoni. A novel CD19 targeting antibody-drug conjugate, huB4-DGN462, shows promising in vitro and in vivo activity in CD19-positive lymphoma models [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 2651. doi:10.1158/1538-7445.AM2017-2651