GPI-anchored Cell Surface Protein Research Articles

The major surface protein of malaria sporozoites is GPI-anchored to the plasma membrane

Glycosylphosphatidylinositol (GPI) anchor protein modification in Plasmodium species is well known and represents the principal form of glycosylation in these organisms. The structure and biosynthesis of GPI anchors of Plasmodium spp. has been primarily studied in the asexual blood stage of P. falciparum and is known to contain the typical conserved GPI structure of EtN-P-Man3GlcN-PI. Here, we have investigated the circumsporozoite protein (CSP) for the presence of a GPI-anchor. CSP is the major surface protein of Plasmodium sporozoites, the infective stage of the malaria parasite. While it is widely assumed that CSP is a GPI-anchored cell surface protein, compelling biochemical evidence for this supposition is absent. Here, we employed metabolic labeling and mass-spectrometry based approaches to confirm the presence of a GPI anchor in CSP. Biosynthetic radiolabeling of CSP with [3H]-palmitic acid and [3H]-ethanolamine, with the former being base-labile and therefore ester-linked, provided strong evidence for the presence of a GPI anchor on CSP, but these data alone were not definitive. To provide further evidence, immunoprecipitated CSP was analyzed for presence of myo-inositol (a characteristic component of GPI anchor) using strong acid hydrolysis and GC-MS for a highly sensitive and quantitative detection. The single ion monitoring (SIM) method for GC-MS analysis confirmed the presence of the myo-inositol component in CSP. Taken together, these data provide confidence that the long-assumed presence of a GPI anchor on this important parasite protein is correct.

Abstract LB045: Exploiting biparatopic binding to deliver novel, differentiated ALPP/ALPPL2 targeting protein drug conjugates

Abstract The placental alkaline phosphatases ALPP and ALPPL2 are attractive targets for antibody and protein-drug conjugate (ADC and PDC) approaches. These GPI-anchored cell-surface proteins, which share very high sequence identity, are over-expressed in a number of different solid tumor indications including testicular, ovarian, endometrial, gastric, pancreatic and non-small cell lung cancers but have very little expression on normal adult tissues. Consequently, there is significant interest in exploiting ALPP/ALPPL2 for the development of new targeted oncology therapeutics, and an ALPP/ALPPL2 ADC, utilizing a classical full-length IgG antibody, is currently in Phase I clinical trials. The exploitation of biparatopic binding is an attractive approach for improving the therapeutic index of ADCs. This mode of target engagement increases the effective number of binding sites on the tumor cells and can promote internalisation and trafficking, thereby delivering increased efficacy for the same payload bolus and opening up patient populations with lower target expression. This can lead to a widening of the therapeutic window, by improving the efficacy achievable for a given amount of payload administered (often defined as the “platform” toxicity). For the development of biparatopic conjugates, the use of small protein domain binders offers advantages over the use of full-length antibodies, as they can readily be reformatted into a variety of biparatopic architectures to deliver homogeneous drug conjugates in high yield. Additionally, smaller protein formats have the capacity to penetrate deeper into solid tumors than large antibodies. We have identified a series of highly selective, high affinity ALPP/ALPPL2 single domain VHH binders, which target distinct epitopes on the protein and which, importantly, show no binding to the closely related ALPI or ALPL isoforms (which have high normal tissue expression). Using protein engineering approaches, a series of homogenous site-specifically labelled biparatopic PDCs have been developed employing the clinically validated vcMMAE (vedotin) payload. In pre-clinical studies, these PDC agents demonstrate excellent anti-tumor efficacy in vivo in ALPP/ALPPL2 positive cell-line derived xenograft models of gastric, pancreatic and ovarian cancer displaying a range of target expression. In benchmarking studies, lead biparatopic PDCs show increased internalisation and target-mediated in vitro cell kill potency compared to an ALPP/ALPPL2 targeting ADC. Additionally, in multiple CDX models, lead biparatopic PDCs demonstrate improved efficacy over an ALPP/ALPPL2 ADC both on a mg/kg and on a payload equivalents basis, in both single dose and multi-dose studies, providing complete regressions. Based on this excellent efficacy profile and favourable developability characteristics, preclinical development activities have been initiated. Citation Format: Graham Cotton, Estelle G. McLean, Paul Trumper, Mark Wappett, Stephanie Gatdula, Stacey Bell, Greg Papadakos, Kieron Lucas, Stephanie Burton, Chiara Saladino, Georgiana Parau, Aidan McCann, Jennifer Thom, Aaron N. Cranston, Tim Harrison. Exploiting biparatopic binding to deliver novel, differentiated ALPP/ALPPL2 targeting protein drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB045.

Neutrophil Functionalities in Patients with Paroxysmal Nocturnal Haemoglobinuria Are Not Affected By Complement C5- Inhibition

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare, acquired haematological stem cell disorder characterized by haemolysis, thrombophilia, and cytopenia. PNH is caused by a somatic mutation in the phosphatidylinositol glycan class A (PIG-A) gene at the level of pluripotent, hematopoietic stem cells leading to a lack of GPI anchored cell surface proteins, such as complement-inactivating proteins, (e.g., CD55, CD59) on stem cells, polymorphonuclear neutrophil granulocytes (PMN), erythrocytes, and platelets. Subsequently, uncontrolled complement overactivity leads to the systemic complications. Phenotypic mosaicism is a distinct characteristic of PNH. For diagnostic purposes, the absence of the GPI-anchor on granulocytes and monocytes in the flow cytometry is considered the gold standard. However, the exact consequences of GPI deficiency in PMNs and monocytes have not been studied sufficiently yet. Since the implementation of complement inhibitors, the primary cause of morbidity and mortality in PNH has been significantly reduced. Despite this, the tendency towards thrombosis, as well as the consequences of the deficiency of GPI-linked proteins on PMNs and monocytes, remain largely unclear. Therefore, the main objective of this project is to gain a deeper understanding of the impact of PNH-PMNs and monocytes on thrombo-inflammation in PNH. Specifically, the impact of complement inhibitors on these factors has been compared. Blood from PNH patients (n=14) with a median clone size of 74 % were compared with an age- and sex-matched control group (n =20). Various cellular markers indicating a state of activation (CD11b, TREM1, CD64) or showing platelet adherence (CD42), were measured using flow cytometry. Unmutated PMNs and monocytes from PNH patients (PMN) served as intrinsic control, GPI positive PMNs (WT-PMN (PNH)) and monocytes from a control group served as extrinsic control. We compared unstimulated PNH and WT cells for their basal phenotype, and then functional changes upon stimulation (phorbol 12-myristate 13-acetate (PMA)). Our data present a contrary perspective to our initial assumptions, indicating that PNH-PMNs have a direct impact on unmutated PMNs, resulting in the generation of a thrombogenic state and increased levels of activation. The unmutated GPI-positive PMNs and monocytes exhibit a significantly stronger direct aggregation with platelets compared to GPI-deficient clones. It is hypothesized that the PNH clones influence the activity and aggregation propensity of the remaining unmutated granulocytes through additional cellular mechanisms. In a subgroup analysis, the impact of complement factor 5 inhibitors (C5i) on the aforementioned factors was investigated detecting no significant differences in terms of PMN activation or platelet aggregation. Thus, it can be hypothesized that thrombosis and hyper-inflammation are not solely driven by the PNH clones directly and modulated C5 inhibition. Additional cellular mechanisms and signaling pathways appear to play an important role, and these aspects have not been adequately addressed by the current therapy.

In vivoimmune inhibitory role of Ly-6A protein

Abstract Ly-6A is a GPI-anchored cell surface protein with reported cell adhesion and cell signaling role in CD4 +T cells. Ly-6A shows low expression on naïve T cells; however, its expression is elevated upon T cell activation. Additionally, IFN-γ and IL-27 cytokines increase Ly-6A expression, a feature observed in immune checkpoint inhibitory (ICI) proteins, such as PD-1 and CTLA-4. Many reports using cell lines and primary cells show, as for ICI proteins, Ly-6A expression inhibiting clonal expansion of antigen receptor-stimulated CD4 +T cells. While the published in vitro and ex vivo studies provide strong evidence for Ly-6A as T cell inhibitory protein, in vivo role of Ly-6A is unknown. Using a tumor transplantation mouse model, we have found that Ly-6A-deficient mice support slower or no growth of B16-F10 melanoma and MC38 adenocarcinoma tumors compared to the wildtype C57BL/6 control mice. We report that Ly-6A-deficient mice injected with B16-F10 tumor cells have elevated number of activated T cells in the draining lymph nodes (DLN) on day-3 post-injection. Furthermore, significantly elevated proliferation of DLN cells from B16-F10 injected Ly-6A-deficient mice was observed on day-7, as assessed by ex vivo proliferation assays. Transcriptome analyses of the tumor tissues showed, among others, significantly elevated expression of IFN-γ inducible and chemokine genes. Additionally, CD73 expression, as assessed by confocal microscopy, was lower in B16-F10 tumor tissues from Ly-6A-deficient mice than the wildtype tumors. Our results reveal a novel immune inhibitory role of Ly-6A protein and potential ICI target for cancer immunotherapy, either as a standalone or combinatorial ICI blockade strategy. Supported by funds from Department of Biology, Villanova University (SRG) & Sigma Xi

Identification of Allosteric Inhibitors of CD73 Using Three‐Phrase Computational Protocol for Drug Discovery

A three-phase computational protocol for drug discovery has been developed that includes 1) 3D shape characterization of binding sites of a protein to predict optimum performance with selected molecular docking programs; 2) Implementation of predictive algorithms for pharmacokinetic properties and ADME profiling for targeted searches in the ZINC15 database; 3) Flexible molecular docking/dynamics protocols of small molecule/protein complexes for drug discovery. Focusing on cancer immunotherapy to implement this computational approach, the allosteric site of ecto-5’-nucleotidase (CD73), a regulator of the extracellular concentration of adenosine, is targeted for large scale small molecule searches. CD73 which hydrolyzes AMP to adenosine, is a GPI-anchored cell surface protein cleaved and activated in the extracellular environment as a homodimer. CD73 exhibits higher expression on lymphocytes, endothelial, and epithelial cells during a stress response and his been reported activated for various types of cancer. CD73 participates in blocking antitumor responses and enhances tumor growth and metastasis by producing high levels of adenosine in the extracellular environment. Protein coordinates of CD73 were downloaded for these from Rutgers Consortium of Structural Biology (RCSB) Protein Data Bank (PDB) and the potential inhibitory molecules from the ZINC15 Database. AutoDock Vina was selected as the ideal docking program for the target based on comparing predicting binding constants with actual Ki's and RMSD analysis of the co-crystallized ligand with the re-docked ligand. Once molecules with high affinity predicted scores were identified from docking, molecular dynamics studies were performed on the highest scoring protein/small molecule complexes. Studies were carried out using GROMACS, implemented with AMBER force field. Dissociation constants (Ki's) for the protein-ligand complex were calculated throughout MD runs. Conformational changes in the binding sites were observed visually and quantitified with PCA analysis. Novel hydrogen bonding networks and hydrophobic networks of residues in the 3D structures were identified to connect the active sites to the allosteric binding site using a MD protocol.

Role of Prostatic Stem Cell Antigen (PSCA) and Snail in Different Prostatic Lesions) (An immunohistochemical study

Background: Prostatic carcinoma (PCa) represents the second most common cancer, and the fifth leading cause of cancer death among males worldwide. PSCA is a GPI-anchored cell surface protein. It belongs to the Thy-1/Ly-6 family which shows a functional diversity ranging from T-cell activation to apoptosis regulation. Snail is one of zinc finger proteins which are transcriptional repressors of E-cadherin. Aim: To study PSCA and Snail expression in different prostatic lesions to evaluate their roles in PCa. Material and Methods: This retrospective study was done upon 80 different prostatic lesions; 17 cases of BPH, 13 cases of HGPIN, and 50 cases of PCa. PSCA and Snail immunostaining was done and assessed for each case. Results: There was a highly significant statistical correlation between both PSCA and Snail expressions and histopathological type (P-value<0.01). PSCA expression showed a highly significant statistical correlation with Gleason score, tumor grade and stage (P-value<0.01), and a significant correlation with PSA, and peri-neural invasion (P-value<0.05). Snail expression showed a highly significant statistical correlation with Gleason score and tumor grade (P-value<0.01), and a significant correlation with lymph node metastasis and tumor stage (P-value<0.05). There was a highly significant statistical correlation between PSCA and Snail immune-expression (P-value<0.01). Conclusion: PSCA and Snail expressions correlate with the most important prognostic clinicopathological variables in PCa, thus they may represent a useful predictor of prognosis.

Semaphorin 7A: A novel marker of disease activity in Gaucher disease.

Gaucher disease (GD) is a recessively inherited lysosomal storage disorder in which sphingolipids accumulates in the macrophages that transform into Gaucher cells. A growing body of evidence indicates that red blood cells (RBCs) represent important actors in GD pathophysiology. We previously demonstrated that altered RBC properties including increased Lyso-GL1 levels, dyserythropoiesis, and iron metabolism defect in GD patients contribute to anemia and hyperferritinemia. Since RBC defects also correlated well with markers of GD severity and were normalized under enzyme replacement therapy (ERT), the identification of molecules that are deregulated in GD RBCs represents an important issue in the search of pertinent markers of the disease. Here, we found a decreased expression of the GPI-anchored cell surface protein Semaphorin 7A (Sema7A) in RBCs from untreated GD (GD UT) patients, in parallel with increased levels of the soluble form in the plasma. Sema7A plays a role in neural guidance, atherosclerosis, and inflammatory diseases and represents a promigratory cue in physiological and pathological conditions. We showed that the decreased expression of Sema7A in RBCs correlated with their abnormal properties and with markers of GD activity. Interestingly, ERT restored the level of Sema7A to normal values both in RBCs and in plasma from GD patients. We then proposed that SemaA7A represents a simple and pertinent marker of inflammation in GD. Finally, because Sema7A is known to regulate the activity of immune cells, the increased level of soluble Sema7A in GD patients could propagate inflammation in several tissues.

Abstract B057: BMS-986148, an anti-mesothelin antibody-drug conjugate (ADC), alone or in combination with nivolumab demonstrates clinical activity in patients with select advanced solid tumors

Abstract Background: Mesothelin, a GPI-anchored cell surface protein, is highly expressed in several tumor types and can be targeted for antibody (ab)-based cancer therapy. BMS-986148 is a fully human IgG1 anti-mesothelin monoclonal ab conjugated to tubulysin to promote selective cytotoxic delivery to tumor cells. In preclinical models, combination of anti–mesothelin-tubulysin with anti–PD-1 promoted a synergistic antitumor response and influx of tumor-infiltrating lymphocytes. Here, we present initial data for BMS-986148 ± nivolumab (NIVO; anti–PD-1) from a phase 1/2a trial in a biomarker-defined population of patients (pts) with select advanced solid tumors (NCT02341625). Methods: During dose escalation (ESC), pts received BMS-986148 0.1 to 1.6 mg/kg IV Q3W, BMS-986148 0.4 or 0.8 mg/kg IV Q1W, or BMS-986148 0.8 mg/kg + NIVO 360 mg IV Q3W. During monotherapy (mono) dose expansion (EXP), pts with mesothelin-selected mesothelioma (ie, H score ≥ 100 for tumor mesothelin expression), non-small cell lung cancer (NSCLC), or ovarian cancer received BMS-986148 1.2 mg/kg mono IV Q3W, and during combination (combo) dose EXP, pts with mesothelin-selected mesothelioma or pancreatic cancer received BMS-986148 0.8 mg/kg + NIVO 360 mg combo IV Q3W. The primary endpoint was safety. Secondary endpoints included pharmacokinetics (PK) and antitumor activity. Results: As of April 1, 2019, 126 pts have been treated in this trial; 96 pts received BMS-986148 mono (Q3W, n = 84; Q1W, n = 12), and 30 pts received BMS-986148 + NIVO combo. PK analysis in pts treated during ESC demonstrated that total antibody and active ADC exposures increased in a dose-proportional manner. Unconjugated tubulysin concentrations were low but sustained over the dosing interval. The maximum tolerated dose (MTD) for mono was 1.2 mg/kg Q3W, and the 0.8 mg/kg dose was determined to be tolerable for the combo. Across all cohorts, any-grade and grade 3/4 treatment-related AEs (TRAEs) were reported in 87% and 44% of all pts, respectively. Overall, the most common TRAEs (any grade; grade 3-4) reported in ≥ 15% of pts were AST increased (43%; 17%), ALT increased (41%; 16%), fatigue (37%; 5%), nausea (29%; 0%), decreased appetite (22%; 1%), and blood alkaline phosphatase increased (18%; 4%). The frequency of TRAEs and dose-limiting toxicities related to liver function tests increased with increasing dose of BMS-986148. Most TRAEs resolved with dose interruption, dose reduction, or treatment discontinuation. Serious TRAEs were reported in 18% of pts with mono and 23% of pts with combo. Fifteen percent of pts discontinued due to TRAEs, and 1 pt died due to a TRAE (pneumonitis; 1.2 mg/kg Q3W mono). The ORR was 6% (0 CR, 3 PR) with mono in EXP and 20% (0 CR, 6 PR) with the combo in ESC and EXP. Among pts with mesothelioma, the ORR was 4% with mono in EXP and 31% with combo in ESC and EXP. Among pts with ovarian carcinoma, the ORR was 9% with mono in EXP. Of note, durable responses lasting up to ≈ 20 mo in pts with ovarian cancer with mono and ≈ 9 mo in pts with mesothelioma with mono and combo were observed. Conclusions: BMS-986148 ± NIVO was tolerable and demonstrated a clinically manageable safety profile. Preliminary clinical activity was observed with BMS-986148 ± NIVO in select patient populations, including pts with mesothelioma. Citation Format: Jeffrey Clarke, Siu-Chung Chu, Lillian L Siu, Jean-Pascal Machiels, Benjamin Markman, Kimberley Heinhuis, Michael Millward, Martijn Lolkema, Sandip Pravin Patel, Paul de Souza, Giuseppe Curigliano, Armando Santoro, Michelle Brown, Ronald Fleming, Heather Vezina, Chunsheng He, Sylvie Rottey. BMS-986148, an anti-mesothelin antibody-drug conjugate (ADC), alone or in combination with nivolumab demonstrates clinical activity in patients with select advanced solid tumors [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 B057. doi:10.1158/1535-7163.TARG-19-B057

CD109 acts as a gatekeeper of the epithelial trait by suppressing epithelial to mesenchymal transition in squamous cell carcinoma cells in vitro

There is increasing evidence that the expression of CD109, a GPI-anchored cell surface protein is dysregulated in squamous cell carcinoma (SCC). However, the functional role of CD109 in SCC progression is poorly understood. In current study, we demonstrate that CD109 is a critical regulator of epithelial phenotype in SSC cells. CD109 levels inversely correlate with TGF-β signaling, EMT, migration, and invasion in cultured SCC cells. CRISPR/Cas9-mediated knockout CD109 (CD109 KO) in SCC cells represses epithelial traits and promotes the mesenchymal phenotype, as evidenced by elevated expression of mesenchymal proteins and markers of epithelial to mesenchymal transition. Treatment with recombinant CD109 protein causes CD109 KO cells to regain their epithelial traits. CD109 loss results in pronounced alterations of gene expression as detected by microarray analysis and in dysregulation of 15 important signalling pathways as shown by KEGG pathway cluster analysis. Validation using 52 human oral SCC tumor samples show that CD109 levels inversely correlate with tumor grade and the activation state of one such pathway, the TGF-β signaling pathway. Taken together, our findings highlight a novel role for CD109 as a gatekeeper of the epithelial phenotype by regulating TGF-β pathway in SCC cells.

Abstract 2316: Systemic Control of CAR T Cell Activity Using a Secret Passageway Fusion Receptor

Abstract Chimeric antigen receptor (CAR) T cells are modified T cells that have been genetically engineered to recognize and kill any cancer cell expressing a specific cell surface tumor antigen. Despite the remarkable success of CAR T cell technologies to date, adoption of the technologies has been limited by severe side-effects such as a potentially fatal cytokine release syndromes (CRS), neurologic toxicities, and on-target off-tumor toxicities, etc. Moreover, chronic exposure of CAR T cells to tumor antigen can lead to CAR T cell exhaustion, which results in inactivation of the CAR T cell therapy. Therefore, an ability to sensitively control CAR T cell fate in vivo, either to suppress its activity when confronted with a cytokine storm, or to rejuvenate its activity when encountering CAR T cell exhaustion, would be highly desirable. We have developed a CAR T cell regulatory platform that enables delivery of any desired modulator selectively into an engineered CAR T cell without simultaneously permitting any modulator uptake by other cells. For this purpose, a fusion receptor that contains two subdomains, a ligand binding module and a membrane-anchoring/internalizing module, is co-expressed with the classical CAR construct in the desired T cell. In the embodiment described here, the ligand binding domain is comprised of human FK506 binding protein (FKBP), while the membrane-anchoring/internalizing domain is comprised of sequences from human folate receptor alpha (FRα). Because the fusion construct is human, it is not expected to be highly immunogenic. Moreover, because FRα is a GPI-anchored cell surface protein that constitutively internalizes, the fusion construct is predicted to constantly enter cells by endocytosis throughout the lifespan of the CAR T cell. We show here that this fusion construct can be co-expressed with an anti-CD19 CAR in the same human T cell with a T2A linker in between in one lentiviral vector. We then demonstrate that any CAR T cell modulator can be selectively targeted to CAR T cells in vivo by linking the desired modulator to FK506; i.e. the ligand that binds FKBP. Upon binding FKBP, the FK506-modulator conjugate is shown to rapidly internalize into the targeted CAR T cell via standard FRα endocytosis. Furthermore, when the linker connecting FK506 to the desired modulator is designed to be cleaved within early endosomes, the modulator is demonstrated to be released following entry into the CAR T cell, where it becomes immediately available for modulation of CAR T cell properties. In the panels shown in this poster, examples of the use of this universal CAR T cell regulatory strategy to control the activities of CAR T cells following their infusion into animal models are provided. Citation Format: Boning Zhang, John V Napoleon, Philip Low. Systemic Control of CAR T Cell Activity Using a Secret Passageway Fusion Receptor [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 2316.

Abstract A090: Exploring folate receptor α immunotherapy of breast carcinomas: Human monocytic cell-mediated killing triggered by IgG1 and IgE antibodies

Abstract Background: Folate receptor α is a GPI-anchored cell surface protein that mediates the intracellular transport of folates. It is over-expressed in a number of malignancies, including ovarian carcinoma and mesothelioma and has limited normal tissue expression. More recently it has been found to be over-expressed in breast cancer, and particularly a less favourable prognostic form, known as ‘triple-negative’ breast cancer (TNBC). We examined a therapeutic antibody candidate recognising FRα; which we are preparing for Phase 1 clinical trials for its potential to target human immune effector cells against breast cancer cells. As part of this effort, we endeavoured to identify a breast cancer cell line model in which the ability for this antibody to induce Antibody-dependent Cell-mediated breast tumour cell death could be evaluated in vitro. Methods: Twenty-two breast cancer cell lines were evaluated by flow cytometry for surface expression of FRα using the anti-FRα antibody compared with IGROV1, a well characterised ovarian cancer cell line used as a positive control. We confirmed these findings with mRNA levels for cell lines available from the publically accessible Cancer Cell Line Encyclopaedia (CCLE). The two highest expressing breast cancer cell lines, T47D and CAL51 were then studied in a functional flow cytometric assay which can simultaneously measure antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis (ADCP). The human monocytic U937 cells which express Fc receptors for IgG and IgE antibodies, served as effectors. We compared the capacity of the the anti-FRα antibodies (IgG1 and IgE isotypes) to activate monocytic cells against the FRα-expressing ovarian carcinoma IGROV1 cells, the breast carcinoma T47D and CAL51 cells and the FRα non-expressing breast cancer cell line, HCC1428. Results: There was wide variety in the levels of cell surface expression of FRα; by breast cancer cell lines, expressed as Mean Fluorescent Intensity (&amp;gt;than an isotype control). The highest expressing breast cancer cell lines, T47D and CAL51 had values of 1273 (SEM 13.6) and 1638 (SEM 24.7) respectively in comparison with the non-expressing cell line, HCC1428 with a value of 8 (SEM 0.54). Expression levels in the ovarian cancer cell line, IGROV1 were 16580 (SEM 2534). These values correlated well with mRNA levels from the CCLE database (Spearman's rank, r=0.6011, p=0.0065) which varied from 1x104-1x1011 amongst breast cancer cell lines but were still below those detected with IGROV1 ovarian cancer cells with an mRNA level of 7x1012. We detected no clear evidence of expressing/non-expressing cell lines clustering amongst any known molecular subtype of TNBC. In vitro functional assay assessments of breast carcinoma cell killing in the non-FRα expressing cell line, HCC1428, revealed that neither IgG nor IgE induced breast cancer cell cytotoxicity above that induced by a non-targeting isotype antibody control. In contrast anti-FRα IgG1 and IgE antibodies could induce 20-30% more ADCP and ADCC, respectively, of the FRα+ IGROV1 tumour cells than the isotype control. The breast carcinoma T47D cells, likewise, were killed by ADCP when exposed to anti-FRα IgG, while the anti-FRα IgE antibody induced high levels of ADCC of ~40% above isotype control. Furthermore, treatment of breast carcinoma cells with anti-FRα antibodies induced direct signalling effects that may lead to reduced growth and survival cascades. Conclusions: We have identified two breast cancer cell line models which could be candidates for further in vitro and in vivo assessment of the efficacy and mechanisms of the novel anti-FRα; IgE against breast carcinomas. We have demonstrated similar levels of human immune effector cell-mediated breast cancer cell killing in one of these cell lines, T47D, to a well characterised ovarian cancer cell line, IGROV1, not seen in the FRα; non-expressing cell line, HCC1428. This tumour antigen warrants further characterisation as a potential new immune therapy in TNBC, where immune infiltration is already known to be favourably prognostic. Citation Format: Matthew W. Fittall, Anthony Cheung, Gyula M. Petranyi, Diana Rodriguez-Dominguez, Heather J. Bax, Panagiotis Karagiannis, Kristina M. Ilieva, Andrew Tutt, Sophia N. Karagiannis. Exploring folate receptor α immunotherapy of breast carcinomas: Human monocytic cell-mediated killing triggered by IgG1 and IgE antibodies. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A090.

Abstract 2836: Development of a companion diagnostic IHC assay for the biomarker-driven selection of C4.4a positive patients

Abstract C4.4a (LYPD3), a GPI-anchored cell surface protein, has been identified previously as a cancer- and metastasis-associated cell surface protein. It is expressed in a variety of cancer indications and, particularly, in the squamous cell subtype of non-small cell lung cancer (NSCLC) and head and neck cancer. Targeting C4.4a with a specific antibody-drug conjugate showed a potent and selective antitumor activity in various human xenograft models and in patient-derived NSCLC tumor models (Willuda J et al., AACR 2014). Since it has been previously shown in vivo that the expression of C4.4a is required for the response to anti-C4.4a treatment, we initiated the development of an IHC assay that might be used to detect C4.4a expression in patient tumor samples. For the development of this companion diagnostic IHC assay, a novel monoclonal rabbit anti-C4.4a antibody was generated. Since C4.4a is post-transcriptionally modified by shedding, we aimed to identify an antibody that binds to a similar epitope as the therapeutic C4.4a antibody. The immunizations of rabbits with specific peptide sequences of C4.4a and with the full length C4.4a recombinant protein produced several antibody candidates. Those antibody candidates were characterized in regard to their staining characteristics, domain binding, and specificity for C4.4a. Furthermore, the staining pattern of the antibody candidates and the therapeutic C4.4a antibody were compared side by side to ensure that stainings using the newly generated antibodies are predictive for anti-C4.4a treatment. The most promising antibodies were selected to set up and optimize an IHC assay on the Ventana staining platform. In summary, we developed a companion diagnostic IHC assay that might be used to detect C4.4a expression in patient tumor samples. Citation Format: Claudia Schneider, Joseph Couto, Yifei Zhu, Zhiming Liao, Robert Pytela, Alton Hiscox, Sabine Wittemer-Rump, Ulf Forssmann, Lars Linden, Joerg Willuda, Daniel Forler, Matthew Nelson, Ricarda Finnern, Thomas Krahn, Khusru Asadullah. Development of a companion diagnostic IHC assay for the biomarker-driven selection of C4.4a positive patients. [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 2836. doi:10.1158/1538-7445.AM2014-2836

Regulation of PSCA expression by m‐Numb in the stomach (904.2)

Prostate stem cell antigen (PSCA) is a GPI‐anchored cell surface protein expressed in the normal epithelium of several tissues including the isthmus region of the gastric mucosa. Recently, a genome‐wide association study revealed several SNPs in the PSCA‐gene to be associated with gastric cancer and duodenal ulcer risk. However, the underlying regulatory mechanisms of PSCA are still unclear.Methods: Msi1‐KO mice with lowered expression of m‐Numb in the stomach and wild‐type ICR mice were used for in vivo studies. In vitro studies were carried out with the mouse gastric epithelial cell line MGE507. Overexpression and suppression of m‐Numb in MGE507 cells was achieved by transfection with lentiviral particles carrying an m‐Numb expression vector and shRNA expression vector, respectively. Gene expression was analysed with qRT‐PCR, and western blotting was used to measure protein levels.Results: In Msi1‐KO mice, a significantly reduced expression‐ and protein level of PSCA was found when comparing the gastric mucosa to wild‐type ICR mice. Expectedly, the protein level of m‐Numb was also verified as lower in the KO‐line. Silencing m‐Numb expression in MGE507 cells resulted in a reduction in protein level of PSCA, and overexpression of m‐Numb led to an increased PSCA‐level.Conclusion: PSCA gene expression and protein level was regulated by m‐Numb in the mouse stomach and in mouse gastric epithelial cells.

Abstract LB-206: Target validation of C4.4a (LYPD3) as a target for antibody-drug conjugates for the treatment of squamous cell carcinoma of different origin.

Abstract C4.4a (LYPD3) is a GPI-anchored cell surface protein with unknown physiological function, although it has been speculated that it might play a role in tumor invasion and metastasis formation. In addition, over-expression of C4.4a correlates with a malignant phenotype and poor prognosis in several cancer indications. As a result of comprehensive gene expression profiling, we report that the C4.4a transcript is being significantly up-regulated in human non-small cell lung cancer (NSCLC) as well as in other tumors compared to the respective normal tissue. This finding was confirmed in IHC studies showing strong C4.4a protein expression on the surface of tumor cells particularly in lung squamous cell carcinoma. Interestingly, a high incidence of C4.4a protein expression was found in a number of additional cancers such as stomach, bladder, colorectal, ovarian, prostate, hepatocellular as well as SCC of the head and neck. In marked contrast to tumors, C4.4a expression in normal tissues is restricted to esophagus, cervix and the outer epithelial layer of skin (stratum spinosum and stratum granulosum). Employing FACS and IHC analyses, several model systems were identified expressing C4.4a in vitro and in vivo. Subsequently, Mab-Zap assays were used to demonstrate efficient internalization of C4.4a in endogenously expressing cells as well as in cells transfected to over-express C4.4a. In summary, the observed strong over-expression of C4.4a in tumors, its very limited expression in normal tissue as well as its ability to rapidly internalize make it a promising target for the generation of antibody-drug conjugates for the treatment of several cancer indications, in particular lung squamous cell carcinoma. Citation Format: Gabriele Leder, Yong-Jiang Cao, Lars Linden, Sven Golfier, Oliver von Ahsen, Claudia Schneider, Joerg Willuda, Kirk McLean, Hans-Dieter Pohlenz, Bertolt Kreft, Karl Ziegelbauer. Target validation of C4.4a (LYPD3) as a target for antibody-drug conjugates for the treatment of squamous cell carcinoma of different origin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-206. doi:10.1158/1538-7445.AM2013-LB-206

CD73‐generated adenosine promotes osteoblast differentiation

CD731 is a GPI-anchored cell surface protein with ecto-5'-nucleotidase enzyme activity that plays a crucial role in adenosine production. While the roles of adenosine receptors (AR) on osteoblasts and osteoclasts have been unveiled to some extent, the roles of CD73 and CD73-generated adenosine in bone tissue are largely unknown. To address this issue, we first analyzed the bone phenotype of CD73-deficient (cd73(-/-)) mice. The mutant male mice showed osteopenia, with significant decreases of osteoblastic markers. Levels of osteoclastic markers were, however, comparable to those of wild-type mice. A series of in vitro studies revealed that CD73 deficiency resulted in impairment in osteoblast differentiation but not in the number of osteoblast progenitors. In addition, over expression of CD73 on MC3T3-E1 cells resulted in enhanced osteoblastic differentiation. Moreover, MC3T3-E1 cells expressed adenosine A(2A) receptors (A(2A)AR) and A(2B) receptors (A(2B)AR) and expression of these receptors increased with osteoblastic differentiation. Enhanced expression of osteocalcin (OC) and bone sialoprotein (BSP) observed in MC3T3-E1 cells over expressing CD73 were suppressed by treatment with an A(2B)AR antagonist but not with an A(2A) AR antagonist. Collectively, our results indicate that CD73 generated adenosine positively regulates osteoblast differentiation via A(2B)AR signaling.

Stem Cell Antigen-1 (Sca-1) Regulates Mammary Tumor Development and Cell Migration

BackgroundStem cell antigen-1 (Sca-1 or Ly6A) is a glycosyl phostidylinositol (GPI)-anchored cell surface protein associated with both stem and progenitor activity, as well as tumor initiating-potential. However, at present the functional role for Sca-1 is poorly defined.Methodology/Principal FindingsTo investigate the role of Sca-1 in mammary tumorigenesis, we used a mammary cell line derived from a MMTV-Wnt1 mouse mammary tumor that expresses high levels of endogenous Sca-1. Using shRNA knockdown, we demonstrate that Sca-1 expression controls cell proliferation during early tumor progression in mice. Functional limiting dilution transplantations into recipient mice demonstrate that repression of Sca-1 increases the population of tumor propagating cells. In scratch monolayer assays, Sca-1 enhances cell migration. In addition, knockdown of Sca-1 was shown to affect cell adhesion to a number of different extracellular matrix components. Microarray analysis indicates that repression of Sca-1 leads to changes in expression of genes involved in proliferation, cell migration, immune response and cell organization.Conclusions/SignificanceSca-1 exerts marked effects on cellular activity and tumorgenicity both in vitro and in vivo. A better understanding of Sca-1 function may provide insight into the broader role of GPI-anchored cell surface proteins in cancer.

A novel fragment of antigen binding (Fab) surface display platform using glycoengineered Pichia pastoris

A fragment of antigen binding (Fab) surface display system was developed using a glycoengineered Pichia pastoris host strain genetically modified to secrete glycoproteins with mammalian mannose-type Man5GlcNAc2 N-linked glycans. The surface display method described here takes advantage of a pair of coiled-coil peptides as the linker while using the Saccharomyces cerevisiae Sed1p GPI-anchored cell surface protein as an anchoring domain. Several Fabs were successfully displayed on the cell surface using this system and the expression level of the displayed Fabs was correlated to that of secreted Fabs from the same glycoengineered host in the absence of the cell wall anchor. Strains displaying different model Fabs were mixed and, through cell sorting, the strain displaying more expressed Fab molecule or the strain displaying the Fab with higher affinity for an antigen was effectively enriched by FACS. This novel yeast surface display system provides a general platform for the display of Fab libraries for affinity and/or expression maturation using glycoengineered Pichia.

Receptor-like Protein-tyrosine Phosphatase α Enhances Cell Surface Expression of Neural Adhesion Molecule NB-3

Neural adhesion molecule NB-3 plays an important role in the apical dendrite development of layer V pyramidal neurons in the visual cortex, and receptor-like protein-tyrosine phosphatase α (PTPα) mediates NB-3 signaling in this process. Here we investigated the role of PTPα in regulating cell surface expression of NB-3. We found that cortical neurons from PTPα knock-out mice exhibited a lower level of NB-3 at the cell surface. When expressed in COS1 cells, NB-3 was enriched in the Golgi apparatus with a low level of cell surface expression. However, co-expression of PTPα increased the cell surface distribution of NB-3. Further analysis showed that PTPα facilitated Golgi exit of NB-3 and stabilized NB-3 protein at the cell surface by preventing its release from the plasma membrane. The extracellular region of PTPα but not its catalytic activity is necessary for its effect on NB-3 expression. Thus, the PTPα-mediated increase of NB-3 level at the cell surface represents a novel function of PTPα in NB-3 signaling in neural development.

The in vivo pig‐a gene mutation assay, a potential tool for regulatory safety assessment

The Pig-a (phosphatidylinositol glycan, Class A) gene codes for a catalytic subunit of the N-acetylglucosamine transferase complex involved in an early step of glycosylphosphatidyl inositol (GPI) cell surface anchor synthesis. Pig-a is the only gene involved in GPI anchor synthesis that is on the X-chromosome, and research into the origins of an acquired genetic disease involving GPI anchor deficiency (paroxysmal nocturnal hemoglobinuria) indicates that cells lacking GPI anchors, or GPI-anchored cell surface proteins, almost always have mutations in the Pig-a gene. These properties of the Pig-a gene and the GPI anchor system have been exploited in a series of assays for measuring in vivo gene mutation in blood cells from humans, rats, mice, and monkeys. In rats, flow cytometric measurement of Pig-a mutation in red blood cells requires microliter volumes of blood and data can be generated in hours. Spontaneous mutant frequencies are relatively low (<5 × 10(-6)) and rats treated with multiple doses of the potent mutagen, N-ethyl-N-nitrosourea, display Pig-a mutant frequencies that are close to the sum of the frequencies produced by the individual exposures. A general observation is that induced mutant frequencies are manifested earlier in reticulocytes (about 2 weeks after treatment) than in total red blood cells (about 2 months after exposure). Based on data from a limited number of test agents, the assay shows promise for regulatory applications, including integration of gene mutation measurement into repeat-dose toxicology studies.

Microdeletions within the hydrophobic core region of cellular prion protein alter its topology and metabolism

The cellular prion protein (PrP C) is a GPI-anchored cell-surface protein. A small subset of PrP C molecules, however, can be integrated into the ER-membrane via a transmembrane domain (TM), which also harbors the most highly conserved regions of PrP C, termed the hydrophobic core (HC). A mutation in HC is associated with prion disease resulting in an enhanced formation of a transmembrane form ( CtmPrP), which has thus been postulated to be a neurotoxic molecule besides PrP Sc. To elucidate a possible physiological function of the transmembrane domain, we created a set of mutants carrying microdeletions of 2–8 aminoacids within HC between position 114 and 121. Here, we show that these mutations display reduced propensity for transmembrane topology. In addition, the mutants exhibited alterations in the formation of the C1 proteolytic fragment, which is generated by α-cleavage during normal PrP C metabolism, indicating that HC might function as recognition site for the protease(s) responsible for PrP C α-cleavage. Interestingly, the mutant G113V, corresponding to a hereditary form of prion disease in humans, displayed increased CtmPrP topology and decreased α-cleavage in our in vitro assay. In conclusion, HC represents an essential determinant for transmembrane PrP topology, whereas the high evolutionary conservation of this region is rather based upon preservation of PrP C α-cleavage, thus highlighting the biological importance of this cleavage.