Dependent Binding Research Articles

Abstract A93: Development of blocking antibodies to human CD47 with lower side effects on RBCs and platelets

Abstract CD47 is a major immune “checkpoint” molecule for macrophage activation. It is highly expressed on many types of cancers, including human acute myeloid leukemia (AML), non-Hodgkin’s lymphoma (NHL), and solid tumors such as small-cell lung cancer (SCLC). Binding of CD47 to its receptor, SIRPα, on macrophages leads to inhibition of macrophage activation and phagocytosis. CD47 overexpression is considered to be one of the mechanisms cancer cells employ to evade immune surveillance and correlates with poor prognosis in cancers. Several strategies have been used to block CD47 binding to SIRPα, including anti-CD47 blocking antibodies and SIRPα Fc proteins. However, since CD47 is also expressed on normal cells, a major concern is that these anti-CD47 antibodies may also induce platelet aggregation and red blood cell hemagglutination. In addition, since anti-CD47 antibodies bind to many different cell types in the peripheral circulation, the half-life for these antibody drugs could be lower. Here we report the generation and characterization of a panel of high-affinity anti-human CD47 blocking antibodies. The lead antibodies bound to human CD47 with sub-nanomolar affinities and displayed a range of activities in blocking CD47/SIRPα interaction. These antibodies showed robust activities in stimulating macrophage-mediated phagocytosis of cancer cells. Importantly, our antibodies did not induce red blood cell hemagglutination in in vitro assays. One of the lead antibodies demonstrates pH- dependent binding to human CD47. It has lower binding affinity to human CD47 at neutral pH but higher binding affinity to CD47 at more acidic pH, suggesting that this antibody may preferentially bind to CD47 in tumor microenvironment. Currently we are evaluating these anti-CD47 antibodies in preclinical efficacy and safety models. Citation Format: Richard Zhang, Haishan Lin. Development of blocking antibodies to human CD47 with lower side effects on RBCs and platelets [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A93.

Caspase-6 Is a Key Regulator of Innate Immunity, Inflammasome Activation, and Host Defense

Caspases regulate cell death, immune responses, and homeostasis. Caspase-6 is categorized as an executioner caspase but shows key differences from the other executioners. Overall, little is known about the functions of caspase-6 in biological processes apart from apoptosis. Here, we show that caspase-6 mediates innate immunity and inflammasome activation. Furthermore, we demonstrate that caspase-6 promotes the activation of programmed cell death pathways including pyroptosis, apoptosis, and necroptosis (PANoptosis) and plays an essential role in host defense against influenza A virus (IAV) infection. In addition, caspase-6 promoted the differentiation of alternatively activated macrophages (AAMs). Caspase-6 facilitated the RIP homotypic interaction motif (RHIM)-dependent binding of RIPK3 to ZBP1 via its interaction with RIPK3. Altogether, our findings reveal a vital role for caspase-6 in facilitating ZBP1-mediated inflammasome activation, cell death, and host defense during IAV infection, opening additional avenues for treatment of infectious and autoinflammatory diseases and cancer.

The LQT-associated calmodulin mutant E141G induces disturbed Ca2+-dependent binding and a flickering gating mode of the CaV1.2 channel.

Calmodulin (CaM) mutations are associated with congenital long QT (LQT) syndrome (LQTS), which may be related to the dysregulation of the cardiac-predominant Ca2+ channel isoform CaV1.2. Among various mutants, CaM-E141G was identified as a critical missense variant. However, the interaction of this CaM mutant with the CaV1.2 channel has not been determined. In this study, by utilizing a semiquantitative pull-down assay, we explored the interaction of CaM-E141G with CaM-binding peptide fragments of the CaV1.2 channel. Using the patch-clamp technique, we also investigated the electrophysiological effects of the mutant on CaV1.2 channel activity. We found that the maximum binding (Bmax) of CaM-E141G to the proximal COOH-terminal region, PreIQ-IQ, PreIQ, IQ, and NT (an NH2-terminal peptide) was decreased (by 17.71-59.26%) compared with that of wild-type CaM (CaM-WT). In particular, the Ca2+-dependent increase in Bmax became slower with the combination of CaM-E141G + PreIQ and IQ but faster in the case of NT. Functionally, CaM-WT and CaM-E141G at 500 nM Ca2+ decreased CaV1.2 channel activity to 24.88% and 55.99%, respectively, compared with 100 nM Ca2+, showing that the inhibitory effect was attenuated in CaM-E141G. The mean open time of the CaV1.2 channel was increased, and the number of blank traces with no channel opening was significantly decreased. Overall, CaM-E141G exhibits disrupted binding with the CaV1.2 channel and induces a flickering gating mode, which may result in the dysfunction of the CaV1.2 channel and, thus, the development of LQTS. The present study is the first to investigate the detailed binding properties and single-channel gating mode induced by the interaction of CaM-E141G with the CaV1.2 channel.

APOL1 C-Terminal Variants May Trigger Kidney Disease through Interference with APOL3 Control of Actomyosin

SummaryThe C-terminal variants G1 and G2 of apolipoprotein L1 (APOL1) confer human resistance to the sleeping sickness parasite Trypanosoma rhodesiense, but they also increase the risk of kidney disease. APOL1 and APOL3 are death-promoting proteins that are partially associated with the endoplasmic reticulum and Golgi membranes. We report that in podocytes, either APOL1 C-terminal helix truncation (APOL1Δ) or APOL3 deletion (APOL3KO) induces similar actomyosin reorganization linked to the inhibition of phosphatidylinositol-4-phosphate [PI(4)P] synthesis by the Golgi PI(4)-kinase IIIB (PI4KB). Both APOL1 and APOL3 can form K+ channels, but only APOL3 exhibits Ca2+-dependent binding of high affinity to neuronal calcium sensor-1 (NCS-1), promoting NCS-1-PI4KB interaction and stimulating PI4KB activity. Alteration of the APOL1 C-terminal helix triggers APOL1 unfolding and increased binding to APOL3, affecting APOL3-NCS-1 interaction. Since the podocytes of G1 and G2 patients exhibit an APOL1Δ or APOL3KO-like phenotype, APOL1 C-terminal variants may induce kidney disease by preventing APOL3 from activating PI4KB, with consecutive actomyosin reorganization of podocytes.

Using Magnetic Nanoparticles and Protein–Protein Interactions to Measure pH at the Nanoscale

Dynamic magnetization of magnetic nanoparticles provides ready access to passive sensing in nanometer scale environments. Here, we use commercially available streptavidin labeled 100 nm magnetic nanoparticles to develop a nanoscale pH sensor. The sensor works based on pH dependent binding of streptavidin to rat tail type-I collagen. The major protein-protein interactions in this case are well rationalized from zeta potential/mobility measurements, which yield a good measure of the relative pIs of the proteins. The pI is the pH at which the protein has zero overall surface charge. Nanoparticle streptavidin has a stable negative charge over the entire range of the measurements from pH 6 to 7.5 (pI ~2), whereas collagen has a steadily increasing positive charge as the pH drops below 7.5 (pI 7.8). Interactions between protein1 and NP-protein2 based on an initial pI difference allow sophisticated nanoscale sensors to be constructed that respond to small changes in pH via corresponding systematic changes in protein-protein interactions. More generally, any type of protein-protein interaction can in principle be measured using magnetic nanoparticles.

EEF1A2 mutations in epileptic encephalopathy/intellectual disability: Understanding the potential mechanism of phenotypic variation

EEF1A2 encodes protein elongation factor 1-alpha 2, which is involved in Guanosine triphosphate (GTP)-dependent binding of aminoacyl-transfer RNA (tRNA) to the A-site of ribosomes during protein biosynthesis and is highly expressed in the central nervous system. De novo mutations in EEF1A2 have been identified in patients with extensive neurological deficits, including intractable epilepsy, globe developmental delay, and severe intellectual disability. However, the mechanism underlying phenotype variation is unknown. Using next-generation sequencing, we identified a novel and a recurrent de novo mutation, c.294C>A; p.(Phe98Leu) and c.208G>A; p.(Gly70Ser), in patients with Lennox–Gastaut syndrome. The further systematic analysis revealed that all EEF1A2 mutations were associated with epilepsy and intellectual disability, suggesting its critical role in neurodevelopment. Missense mutations with severe molecular alteration in the t-RNA binding sites or GTP hydrolysis domain were associated with early-onset severe epilepsy, indicating that the clinical expression was potentially determined by the location of mutations and alteration of molecular effects. This study highlights the potential genotype–phenotype relationship in EEF1A2 and facilitates the evaluation of the pathogenicity of EEF1A2 mutations in clinical practice.

Islet Complexins are Complex

While Ca2+ activity is required for islet hormone secretion, proper glucose stimulated glucagon, insulin and somatostatin secretion requires a synergistic combination of electrical, paracrine, juxtacrine and cell intrinsic signaling pathways. This complexity can be unraveled by reconciling islet cell electrical activity, second messenger response and secretory vesicle mobilization in the presence and absence of paracrine regulatory factors. One molecule of interest is Complexin 2, a Protein Kinase A (PKA) dependent SNARE binding protein known to be expressed in alpha, beta and delta cells. Previous alpha cell studies showed that paracrine signaling by insulin and somatostatin combine to reduce glucagon secretion by lowering cAMP and PKA activity independent of Ca2+ activity. In contrast, beta cell insulin secretion is increased by cAMP/PKA and Ca2+ activity. Here, we test the hypothesis that Complexin 2 regulates insulin and glucagon secretion at the secretory vesicle level via phosphorylation by Protein Kinase A (PKA) which is in turn regulated by somatostatin paracrine signaling. Using islets from mice that are null for Complexin 2 gene (Cplxn2 KO), we measured insulin, glucagon and somatostatin secretion in response to glucose. We found increases in both glucagon and insulin output despite a concomitant increase in somatostatin secretion. In order to image Ca2+ signaling in Cplxn2 KO mice we used 2D confocal and 3D dual inverted selective plane illumination microscopy (diSPIM), which performs at high speed using light sheet illumination and camera detection. Live islet imaging experiments with GCaMP6 expressed in Cplxn 2 KO alpha cells showed that somatostatin, which normally inhibits Ca2+ oscillations, was unable to attenuate Ca2+ oscillations in Cplxn2 KO alpha cells during direct alpha cell cAMP/PKA activation.

Observing Single-Molecule Protein-DNA Interactions and DNA Transcription In Vitro using Transcriptomic Tethered Particle Motion

The regulation of genes acquired through horizontal gene-transfer plays an important role in the spread of antibiotic resistance in bacteria. However, transcriptional regulation of horizontally acquired genes by nucleoid-associated proteins (NAPs) like H-NS and Lsr2 remain little understood. We investigate the molecular mechanism of transcription and gene silencing by DNA associated proteins in-vitro at a single molecule level. Utilizing 3rd generation click-chemistry, we have established a tethered particle motion (TPM) platform that is remarkably stable (many days), easily prepared, adjustable, self-contained, inherently anti-fouling and highly parallelized. With this platform, we study the sequence dependent binding of H-NS and Lsr2 at various protein concentrations and buffer conditions. Well over 100 particles can be observed simultaneously, allowing for statistically efficient experimentation with single molecule resolution. Each particle is anchored to the substrate using a short ∼1000 bp DNA tether. The NAPs interact with and bind to the DNA. Changes in particle motion reveal the interactions between the proteins and the DNA, quantified in terms of the equilibrium dissociation constant, on- and off-rates, molecule-to-molecule variation and protein induced DNA persistence length changes. These experiments are performed for DNA sequences incorporating a variety of known and hypothesized NAP binding nucleation sites. To directly connect the biomechanical effects of NAPs to gene transcription, we employ an extension of TPM we have coined transcriptomic TPM. In our transcription system we attach DNA strands containing the gene of interest to a tethered particle. T7 RNA Polymerase is attached to the substrate and repeatedly transcribes the genetic sequence attached to the particles. We determine the transcriptional dynamics from the motion of the reporter particle. We report on our progress and discuss the future investigative avenues opened up by this system.

Study on the role of calmodulin in sperm function through the enrichment and identification of calmodulin-binding proteins in bovine ejaculated spermatozoa.

Calmodulin is a small, highly conserved acidic protein present at high levels in spermatozoa that mediates numerous intracellular Ca2+ -dependent events. Sperm motility and fertilizing ability results from an array of biochemical pathways under Ca2+ control, in which the importance of calmodulin is not fully understood. The role of calmodulin in sperm function has been mostly assessed using antagonists. Nevertheless, few known calmodulin-regulated enzymes have been described in spermatozoa regarding their involvement in sperm function. To further understand the role of this important Ca2+ mediator in spermatozoa, different studies were also undertaken to investigate and to identify sperm calmodulin-binding proteins and determine their localization and subcellular distribution as an attempt to elucidate the role of this important Ca2+ mediator. In the present study, sperm calmodulin-binding proteins were identified by mass spectrometry after Ca2+ -dependent biotinylated-calmodulin binding on sperm head proteins subjected to 2D electrophoresis and transferred on a polyvinylidene difluoride membrane. Calmodulin binding protein identification was also done on detergent extracted whole sperm proteins pulled down in a Ca2+ -dependent manner by calmodulin-conjugated sepharose beads. In this latter group, 300 proteins were identified in at least two experiments out of three, and those identified in the three independent experiments were analyzed for overrepresented biological processes using the Bos taurus Gene Ontology database. Proteins with known function in reproductive processes, fertilization, sperm-egg recognition, sperm binding to the zona pellucida, regulation of sperm capacitation, and sperm motility were identified and further emphasize the importance of calmodulin in sperm function.

Selectin Binding Sites Are Involved in Cell Adhesive Properties of Head and Neck Squamous Cell Carcinoma.

The formation of distant metastases often determines the fate of patients with head and neck squamous cell carcinoma (HNSCC). The expression of cell adhesion molecules (CAMs) and their ligands of the leukocyte adhesion cascade has been associated with metastatic competence in several malignant entities. In this study, human HNSCC cell lines were analyzed in vitro and in a spontaneous metastatic xenograft model. Immunohistochemical analyses of several CAMs were performed on xenograft tumors and tissue microarrays (TMA) from 453 patients with head and neck squamous cell carcinomas with full histo-pathological and clinical follow-up data. UTSCC 24A and 24B cells bind to E-selectin in vitro, show E-selectin dependent binding to human umbilical vein endothelial cells (HUVECs), and express sLeX. All HNSCC cells engrafted into severe combined immunodeficient (SCID) mice, and UTSCC 24A cells formed sporadically spontaneous lung metastases. The expression of CAMs varied between the cell lines, but a correlation between tumor growth and metastatic potential did not exist. None of the CAMS or their ligands could be identified to be of prognostic relevance in the TMA study. The in vitro results indicate that E-selectin and sLeX are involved in the adhesion of HNSCC cells to endothelium. However, specific prognostic markers chosen from the leukocyte adhesion cascade for HNSCC were not identified.

Deficiency of nuclear receptor interaction protein leads to cardiomyopathy by disrupting sarcomere structure and mitochondrial respiration

BackgroundCardiomyopathy is a common and lethal complication in patients with limb-girdle muscular dystrophy (LGMD), one of the most prevalent forms of muscular dystrophy. The pathogenesis underlying LGMD-related cardiomyopathy remains unclear. NRIP (gene name DCAF6), a Ca2+-dependent calmodulin binding protein, was reduced in dystrophic muscles from LGMD patients. Mice lacking NRIP exhibit a myopathic phenotype resembling that in LGMD patients, making NRIP deficiency a potential culprit leading to cardiomyopathy. This study aimed to determine if NRIP deficiency leads to cardiomyopathy and to explore the underlying molecular mechanisms. Methods and resultsNRIP expression was reduced in both human and mouse failing hearts. Muscle-specific NRIP knockout (MCK-Cre::Dcaf6flox/flox) mouse heart and isolated cardiomyocytes exhibited markedly reduced contractility. Transmission electron microscopy revealed abnormal sarcomere structures and mitochondrial morphology in MCK-Cre::Dcaf6flox/flox hearts. Protein co-immunoprecipitation and confocal imaging revealed that NRIP interacts with α-actinin 2 (ACTN2) at the Z-disc. We found that NRIP facilitated ACTN2-mediated F-actin bundling, and that NRIP deficiency resulted in reduced binding between Z-disc proteins ACTN2 and Cap-Z. In addition, NRIP-deficiency led to increased mitochondrial ROS and impaired mitochondrial respiration/ATP production owing to elevated cellular NADH/NAD+ ratios. Treatment with mitochondria-directed antioxidant mitoTEMPO or NAD+ precursor nicotinic acid restored mitochondrial function and cardiac contractility in MCK-Cre::Dcaf6flox/flox mice. ConclusionsNRIP is essential to maintain sarcomere structure and mitochondrial/contractile function in cardiomyocytes. Our results revealed a novel role for NRIP deficiency in the pathogenesis of LGMD and heart failure. Targeting NRIP, therefore, could be a powerful new approach to treat myocardial dysfunction in LGMD and heart failure patients.

Molecular Recognition of Rhodopsin Kinase GRK1 and Recoverin Is Tuned by Switching Intra- and Intermolecular Electrostatic Interactions.

G protein-coupled receptor kinase 1 (GRK1) or rhodopsin kinase is under specific control of the neuronal Ca2+-sensor protein recoverin, which is a critical feedback mechanism responsible for the modulation of the shape and sensitivity of the rod cell photoresponse. This process requires the precise matching of interacting protein surfaces and the dynamic changes in protein conformations. Here we study the molecular recognition process of recoverin and GRK1 by testing the hypothesis of a cation-π interaction pair in the recoverin-GRK1 complex. The critical role of residue K192 in recoverin was investigated by site-directed mutagenesis and subsequent structural and functional analysis. The following methods were used: isothermal titration calorimetry, fluorescence and circular dichroism spectroscopy, Ca2+-dependent membrane binding, and protein-protein interaction analysis by back scattering interferometry and surface plasmon resonance. While neutralizing the charge at K in the mutant K192L did not prevent binding of recoverin to GRK1, reversing the charge from K to E led to more distortions in the interaction process, but both mutations increased the stability of the protein conformation. Molecular dynamics simulations provided an explanation for these findings as they let us suggest that residue 192 per se is not a major stabilizer of the interaction between recoverin and its target but rather that the native K is involved in a network of switching electrostatic interactions in wild-type recoverin.

Heparanase-2 protects from LPS-mediated endothelial injury by inhibiting TLR4 signalling

The endothelial glycocalyx and its regulated shedding are important to vascular health. Endo-β-D-glucuronidase heparanase-1 (HPSE1) is the only enzyme that can shed heparan sulfate. However, the mechanisms are not well understood. We show that HPSE1 activity aggravated Toll-like receptor 4 (TLR4)-mediated response of endothelial cells to LPS. On the contrary, overexpression of its endogenous inhibitor, heparanase-2 (HPSE2) was protective. The microfluidic chip flow model confirmed that HPSE2 prevented heparan sulfate shedding by HPSE1. Furthermore, heparan sulfate did not interfere with cluster of differentiation-14 (CD14)-dependent LPS binding, but instead reduced the presentation of the LPS to TLR4. HPSE2 reduced LPS-mediated TLR4 activation, subsequent cell signalling, and cytokine expression. HPSE2-overexpressing endothelial cells remained protected against LPS-mediated loss of cell-cell contacts. In vivo, expression of HPSE2 in plasma and kidney medullary capillaries was decreased in mouse sepsis model. We next applied purified HPSE2 in mice and observed decreases in TNFα and IL-6 plasma concentrations after intravenous LPS injections. Our data demonstrate the important role of heparan sulfate and the glycocalyx in endothelial cell activation and suggest a protective role of HPSE2 in microvascular inflammation. HPSE2 offers new options for protection against HPSE1-mediated endothelial damage and preventing microvascular disease.

Human SP-D Acts as an Innate Immune Surveillance Molecule Against Androgen-Responsive and Androgen-Resistant Prostate Cancer Cells.

Surfactant Protein D (SP-D), a pattern recognition innate immune molecule, has been implicated in the immune surveillance against cancer. A recent report showed an association of decreased SP-D expression in human prostate adenocarcinoma with an increased Gleason score and severity. In the present study, the SP-D expression was evaluated in primary prostate epithelial cells (PrEC) and prostate cancer cell lines. LNCaP, an androgen dependent prostate cancer cell line, exhibited significantly lower mRNA and protein levels of SP-D than PrEC and the androgen independent cell lines (PC3 and DU145). A recombinant fragment of human SP-D, rfhSP-D, showed a dose and time dependent binding to prostate cancer cells via its carbohydrate recognition domain. This study, for the first time, provides evidence of significant and specific cell death of tumor cells in rfhSP-D treated explants as well as primary tumor cells isolated from tissue biopsies of metatstatic prostate cancer patients. Viability of PrEC was not altered by rfhSP-D. Treated LNCaP (p53+/+) and PC3 (p53 −/−) cells exhibited reduced cell viability in a dose and time dependent manner and were arrested in G2/M and G1/G0 phase of the cell cycle, respectively. rfhSP-D treated LNCaP cells showed a significant upregulation of p53 whereas a significant downregulation of pAkt was observed in both PC3 and LNCaP cell lines. The rfhSP-D-induced apoptosis signaling cascade involved upregulation of Bax:Bcl2 ratio, cytochrome c and cleaved products of caspase 7. The study concludes that rfhSP-D induces apoptosis in prostate tumor explants as well as in androgen dependent and independent prostate cancer cells via p53 and pAkt pathways.

Calcium-dependent titin-thin filament interactions in muscle: observations and theory.

Gaps in our understanding of muscle mechanics demonstrate that the current model is incomplete. Increasingly, it appears that a role for titin in active muscle contraction might help to fill these gaps. While such a role for titin is increasingly accepted, the underlying molecular mechanisms remain unclear. The goals of this paper are to review recent studies demonstrating Ca2+-dependent interactions between N2A titin and actin in vitro, to explore theoretical predictions of muscle behavior based on this interaction, and to review experimental data related to the predictions. In a recent study, we demonstrated that Ca2+ increases the association constant between N2A titin and F-actin; that Ca2+ increases rupture forces between N2A titin and F-actin; and that Ca2+ and N2A titin reduce sliding velocity of F-actin and reconstituted thin filaments in motility assays. Preliminary data support a role for Ig83, but other Ig domains in the N2A region may also be involved. Two mechanical consequences are inescapable if N2A titin binds to thin filaments in active muscle sarcomeres: (1) the length of titin's freely extensible I-band should decrease upon muscle activation; and (2) binding between N2A titin and thin filaments should increase titin stiffness in active muscle. Experimental observations demonstrate that these properties characterize wild type muscles, but not muscles from mdm mice with a small deletion in N2A titin, including part of Ig83. Given the new in vitro evidence for Ca2+-dependent binding between N2A titin and actin, it is time for skepticism to give way to further investigation.

Abstract 63: Breast and colon cancer cells express L-selectin ligands that interact with L-selectin on white blood cells under flow conditions

Abstract Evidence suggests circulating tumor cells (CTCs) can interact with white blood cells (WBCs) and promote metastasis under certain circumstances. However, the interaction between the L-selectin ligands expressed on CTCs and the L-selectin expressed on WBCs is less understood. We hypothesize that the L-selectin of WBCs will bind to the L-selectin ligands of cancer cells under hemodynamic flow conditions. BT-20, Hs-578T, MDA-MB-231 and SK-BR-3 breast cancer cells, and LS174T colon cancer cells were tested for expression of L-selectin ligands using L-selectin hFc (R&D Systems) in flow cytometry. Flow cytometry analysis revealed all five cancer cell lines expressed L-selectin ligands that were detected by L-selectin hFc. The L-selectin ligands on the surface of cancer cells were shown to be protease sensitive, as when the cells were treated with 0.1% bromelain (a broadly active protease), L-selectin hFc binding to all cancer lines was significantly reduced and exhibited binding levels similar to the negative isotype control (hIgG). The parallel plate flow chamber adhesion assay was used to examine the interaction between cancer cell lines (BT-20, Hs-578T, and LS174T) and WBCs isolated from normal healthy human donors in an IRB approved protocol. Two populations of WBCs, PMNs and PBMCs, were separated from whole blood and separately perfused at a wall shear stress of 0.7 dyn/cm2 over a cancer cell monolayer. Interacting cells, defined as PMNs or PBMCs tethered to or rolling on the monolayer, were visualized in real time using video microscopy. Both populations of WBCs interacted with BT-20, Hs-578T, and LS174T cells. Afterward, 5 mM EDTA was perfused across the monolayer, causing the release of WBCs consistent with Ca++ dependent binding. When the WBCs were incubated with an L-selectin antibody (anti-human CD62L, BD Pharmingen) for 30 minutes on ice before perfusion, total interactions were reduced compared to the negative isotype control (mIgG, BD Pharmingen) treated WBCs. These flow chamber results indicate that the L-selectin of PMNs and PBMCs specifically bind to the L-selectin ligands of the breast and colon cancer cell lines. Altogether, the five cell lines that were tested express L-selectin ligands that bind to L-selectin hFc. BT-20, Hs-578T, and LS174T cells interact with WBCs under flow conditions in an L-selectin and divalent cation dependent manner. Ultimately, elucidating the molecular mechanisms of adhesion between WBCs and cancer cells in vasculature may provide a rational basis for developing strategies against metastasis. Citation Format: Nicholas J. Cellars, Ariel L. Lanier, Monica M. Burdick. Breast and colon cancer cells express L-selectin ligands that interact with L-selectin on white blood cells under flow conditions [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 63.

Abstract 2395: A bispecific T- cell engager antibody (BiTE) against TAG-72/ CD3 for targeted cancer immunotherapy

Abstract Background: Immunotherapies involving cytotoxic T lymphocytes (CTL’s) have been central to cancer immunotherapy. Bi-specific T cell engager antibody (BiTE) therapy has emerged as an effective immunotherapy by redirecting cytotoxic T cells against cell surface protein on tumor cells. Our BiTE antibody targets human tumor associated glycoprotein 72 (TAG-72). TAG-72 is a pan-carcinoma marker expressed on the surfaces of many cancer tissues including breast, prostate, ovary, endometrium, stomach, esophagus, and pancreas. It is not expressed in normal tissues making it an ideal target therapy for cancer. Our BiTE antibody can bind CD3 to activate the T-cell’s while simultaneously binding TAG-72 on the surface of the cancer cell leading to perforin induced cancer cell death. The in vitro and in vivo effects of TAG-72/ CD3 BiTE antibody on different cancer cells are presented here. Methods: We studied in vitro and in vivo effects of TAG-72/ CD3 BiTE antibody on different cancer cells; breast (DOX resistant MCF-7, SKBR3, MDA-MB-175 VII), ovarian (OVCAR-3), endometrium (KLE) and colon (Ls174T). In vitro cytotoxicity assay was performed by flow cytometry and immunofluorescence staining using live/ dead cell labelling fluorescent dyes. The cells were co-cultured with naïve human CD8 T cells with/ without BiTE antibody (1 µg, 5 µg and 10 µg) for different time intervals and assayed. In vivo study was carried out in NSG mice implanted with MCF-7 breast cancer cells + naïve human CD8 T cells and treated with a total of 4 doses of BiTE antibody (200µg/ dose). Results: Different tumor cells showed varying (%) of TAG-72 expression determined by flow cytometry, viz; DOX (55%), SKBR3 (16%), MDA 175 VII (8%), OVCAR-3 (27%), KLE (27%), Ls174T (30%). The BiTE antibody also showed dose dependent binding to these cells. Flow cytometry analysis showed that addition of the BiTE antibody to naïve CD8 T-cells and incubation for 4 days significantly increases tumor cell death as indicated by the Annexin V/ PI+ve signal from DOX (52.4% vs. 41.4% CD8 alone), SKBR-3 (47.9% vs. 30% CD8 alone), MDA 175 VII (23% vs 11% CD8 alone), OVCAR-3 (31.1% vs. 23.8% CD8 alone), KLE (27% vs 13% CD8 alone) and Ls174T (54% vs 29% CD8 alone) respectively (p<0.05). Further, a significant level of IFN-γ secreted by activated T-cells was only detected in the bispecific T-cell engager Ab + CD8 conditions. In vivo data showed good tumor regression at day 45 with tumors measuring (8 mm2) in group treated with BiTE antibody as compared to untreated group (31 mm2) (p<0.01). Conclusion: Our in vitro and in vivo data indicates that the TAG-72/ CD3 BiTE antibody can effectively redirect cytotoxic T cells against different cancer cells expressing TAG-72 glycoprotein and may be effective in the treatment of breast, ovarian, endometrium and colon cancers. Citation Format: Fatema Khambati, Hatem Soliman. A bispecific T- cell engager antibody (BiTE) against TAG-72/ CD3 for targeted cancer immunotherapy [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 2395.

The cytoplasmic domain of CTLA-4 control autoimmunity via inducing regulatory T cells

Abstract Regulatory T cells (Tregs) have critical roles for maintaining immune tolerance and homeostasis. Recently, it is revealed that CTLA-4 is an essential membrane protein for the suppressive function of Treg. However, the molecular mechanism of cytoplasmic domain of CTLA-4 (ctCTLA-4) and its role in Treg function and differentiation is not clearly understood. In this study, we utilized ctCTLA-4 peptide and its various mutant forms conjugated with dNP2, a cell-permeable peptide, to investigate the mechanism of ctCTLA-4 in Treg function and differentiation. We found that the ctCTLA-4 convert naïve T cells to Foxp3-positive cells in T helper 17 cell differentiation condition in vitro, while the lysine-rich motif mutant form could not. The dNP2-ctCTLA-4 also significantly inhibits experimental autoimmune encephalomyelitis (EAE) in vivo, by reducing CNS-infiltrating Th17 cells with significantly increased Treg proportion. As similar with in vitroresults, lysine-rich motif mutant form could not increase Treg proportion and inhibit EAE. To elucidate its molecular mechanisms, we conducted HuProt protein microarray to identify lysine-rich motif dependent binding partner of ctCTLA-4 related with increasing Tregs. With the results, we found that ctCTLA-4 decreased a phosphorylation of MAPK in T cell stimulation conditions resulting enhanced nuclear localization of Smad2/3, a key transcription factor for Foxp3 expression. These results suggest that ctCTLA-4 has a critical molecular mechanism of inducing Tregs to maintain peripheral immune tolerance to control autoimmunity.

SAT-191 Distribution of Annexin A1 (ANXA1) in the Anterior Pituitary Tissue and the Effect on LH Secretion

We previously showed the stimulatory effect of GnRH on annexin A5 (ANXA5) mRNA expression in the pituitary gonadotropes and the stimulating effect of ANXA5 on gonadotropin secretion (1). Annexin is a family of structurally related 12 proteins in mammals. Although molecular characteristics of annexins, for example calcium dependent phospholipid binding, are shown, their physiological role is still obscure. Among twelve annexins, we have recently reported that the expression of annexin A1 (ANXA1) is also augmented by GnRH in LβT2 gonadotrope cells like as ANXA5 (2). The extent of ANXA1 increase by GnRH, however, was much higher than that of ANXA5. Pituitary ANXA1 was already shown to be involved in cortisol inhibition of ACTH secretion and the expression in the folliculo-stellate cell was demonstrated. In the present study, we examined the distribution of ANXA1 in the pituitary tissues and the effect of ovariectomy. Further, the effect of ANXA1 on LH release in LβT2 gonadotrope was examined. ANXA1 was shown to distribute to majority of anterior pituitary cells and the intensity was highest in folliculo-stellate cell like irregular shaped cells of adult female Wistar Imamichi rats. Pituitary tissues were subjected to immunohistochemistry for ANXA1 after ovariectomy. Large round cells, activated gonadotropes, were appeared two week after the ovariectomy and ANXA1 was distributed to around these cells and folliculo-stellate cell like cells. Nuclear ANXA1 was also prominent. The administration of recombinant human ANXA1 to LβT2 gonadotrope cells increased LH release during 24 h incubation. These results indicate ANXA1 involvement in the regulation of gonadotropin secretion. Although it is not clear why only ANXA1 and A5 out of 12 members are affected by GnRH, these two annexins would be closely related in the gonadotrope. 1) Kawaminami M, Etoh S, Miyaoka H, Sakai M, Nishida M, Kurusu S, Hashimoto I., Annexin 5 messenger ribonucleic acid expression in pituitary gonadotropes is induced by gonadotropin-releasing hormone (GnRH) and modulates GnRH stimulation of gonadotropin release. Neuroendocrinology. 2002 Jan;75(1):2-11. 2) Fungbun N, Tungmahasuk D, Terashima R, Kurusu S, Kawaminami M. Annexin A1 is a novel target gene of gonadotropin-releasing hormone in LβT2 gonadotrope cells. J Vet Med Sci. 2018 Jan 27;80(1):116-124.

Ca2+-dependent regulation of sodium channels NaV1.4 and NaV1.5 is controlled by the post-IQ motif

Skeletal muscle voltage-gated Na+ channel (NaV1.4) activity is subject to calmodulin (CaM) mediated Ca2+-dependent inactivation; no such inactivation is observed in the cardiac Na+ channel (NaV1.5). Taken together, the crystal structures of the NaV1.4 C-terminal domain relevant complexes and thermodynamic binding data presented here provide a rationale for this isoform difference. A Ca2+-dependent CaM N-lobe binding site previously identified in NaV1.5 is not present in NaV1.4 allowing the N-lobe to signal other regions of the NaV1.4 channel. Consistent with this mechanism, removing this binding site in NaV1.5 unveils robust Ca2+-dependent inactivation in the previously insensitive isoform. These findings suggest that Ca2+-dependent inactivation is effected by CaM’s N-lobe binding outside the NaV C-terminal while CaM’s C-lobe remains bound to the NaV C-terminal. As the N-lobe binding motif of NaV1.5 is a mutational hotspot for inherited arrhythmias, the contributions of mutation-induced changes in CDI to arrhythmia generation is an intriguing possibility.