Mason Lu

Modulation of Notch Signaling by Antibodies Specific for the Extracellular Negative Regulatory Region of NOTCH3

The Notch pathway regulates the development of many tissues and cell types and is involved in a variety of human diseases, making it an attractive potential therapeutic target. This promise has been limited by the absence of potent inhibitors or agonists that are specific for individual human Notch receptors (NOTCH1-4). Using an unbiased functional screening, we identified monoclonal antibodies that specifically inhibit or induce activating proteolytic cleavages in NOTCH3. Remarkably, the most potent inhibitory and activating antibodies bind to overlapping epitopes within a juxtamembrane negative regulatory region that protects NOTCH3 from proteolysis and activation in its resting autoinhibited state. The inhibitory antibodies revert phenotypes conveyed on 293T cells by NOTCH3 signaling, such as increased cellular proliferation, survival, and motility, whereas the activating antibody mimics some of the effects of ligand-induced Notch activation. These findings provide insights into the mechanisms of Notch autoinhibition and activation and pave the way for the further development of specific antibody-based modulators of the Notch receptors, which are likely to be of utility in a wide range of experimental and therapeutic settings.

Interaction between Antibody-Diversification Enzyme AID and Spliceosome-Associated Factor CTNNBL1

Activation-induced deaminase (AID) deaminates deoxycytidine residues in immunoglobulin genes, triggering antibody diversification. Here, by use of two-hybrid and coimmunoprecipitation assays, we identify CTNNBL1 (also known as NAP) as an AID-specific interactor. Mutants of AID that interfere with CTNNBL1 interaction yield severely diminished hypermutation and class switching. Targeted inactivation of CTNNBL1 in DT40 B cells also considerably diminishes IgV diversification. CTNNBL1 is a widely expressed nuclear protein that associates with the Prp19 complex of the spliceosome, interacting with its CDC5L component. The results, therefore, identify residues in AID involved in its in vivo targeting and suggest they might act through interaction with CTNNBL1, giving possible insight into the linkage between AID recruitment and target-gene transcription.

Mice Deficient in APOBEC2 and APOBEC3

ABSTRACT The activation-induced deaminase/apolipoprotein B-editing catalytic subunit 1 (AID/APOBEC) family comprises four groups of proteins. Both AID, a lymphoid-specific DNA deaminase that triggers antibody diversification, and APOBEC2 (function unknown) are found in all vertebrates examined. In contrast, APOBEC1, an RNA-editing enzyme in gastrointestinal cells, and APOBEC3 are restricted to mammals. The function of most APOBEC3s, of which there are seven in human but one in mouse, is unknown, although several human APOBEC3s act as host restriction factors that deaminate human immunodeficiency virus type 1 replication intermediates. A more primitive function of APOBEC3s in protecting against the transposition of endogenous retroelements has, however, been proposed. Here, we focus on mouse APOBEC2 (a muscle-specific protein for which we find no evidence of a deaminating activity on cytidine whether as a free nucleotide or in DNA) and mouse APOBEC3 (a DNA deaminase which we find widely expressed but most abundant in lymphoid tissue). Gene-targeting experiments reveal that both APOBEC2 (despite being an ancestral member of the family with no obvious redundancy in muscle) and APOBEC3 (despite its proposed role in restricting endogenous retrotransposition) are inessential for mouse development, survival, or fertility.

Generation of Monoclonal Antibody MS17-57 Targeting Secreted Alkaline Phosphatase Ectopically Expressed on the Surface of Gastrointestinal Cancer Cells

Background Therapeutic antibody development is one of the fastest growing areas of the pharmaceutical industry. Generating high-quality monoclonal antibodies against a given therapeutic target is crucial for successful drug development. However, due to immune tolerance, making it difficult to generate antibodies using conventional approaches. Methodology/Findings Mixed four human gastric cancer (GC) cell lines were used as the immunogen in A/J mice; sixteen highly positive hybridoma colonies were selected via fluorescence-activated cell sorting-high throughput screening (FACS-HTS) using a total of 20,000 colonies in sixty-seven 96-well plates against live cells (mixed human GC cells versus human PBMC controls). MS17-57 and control commercial Alkaline Phosphatase (ALP) mAbs were used to confirm the target antigens (Ags), which were identified as ALPs expressed on the GC cell surface through a combination of western blot, immunoprecipitation and mass spectrometry (MS). MS identified the Ags recognized by MS17-57 to be two variants of a secreted ALP, PALP and IALP (Placental and intestinal ALP). These proteins belong to a hydrolase enzyme family responsible for removing phosphate groups from many types of molecules. Immunofluorescence staining using MS17-57 demonstrated higher staining of gastrointestinal (GI) cancer tissues compared to normal GI tissues (P<0.03), and confirmed binding of MS17-57 to be restricted to a functional epitope expressed on the cancer cell surface. Proliferation assays using the PALP/IALP-expressing GC cell lines demonstrated that MS17-57 inhibited cell growth by 32±8%. Transwell cell migration assays documented that MS17-57 can inhibit PALP/IALP-expressing GI cancer cell migration by 25±5%. MS17-57 mAb inhibited tumor growth in nude mice. Conclusions Our findings indicate that PALP and IALP can be ectopically expressed on extracellular matrix of GI cancers, and that MS17-57 directed against PALP/IALP can inhibit GI cancer cells growth and migration in vitro and in vivo. This investigation provides an example of identification of cancer biomarkers representing promising therapeutic targets using mAb generated through a novel HTS technology.

Melanoma Expression Genes Identified through Genome-Wide Association Study of Breslow Tumor Thickness

rs12203592 falls within an enhancer region of IRF4; the T allele impairs transcription factor binding, leading to reduced expression of IRF4 and tyrosinase (Praetorius et al., 2013). In a recent meta-analysis, the association between rs12203592 polymorphism and SCC was significant in dominant, recessive, and codominant models (Wu et al., 2016). The additive genetic model was selected a priori for our study, but exploration of the codominant model confirmed the association between SCC risk and the TT versusCCgenotype (HR1⁄4 2.24, 95% confidence interval 1⁄4 1.22e4.09, P 1⁄4 0.009). Previous studies investigating genetic risks for cSCC in OTRs have been limited by small sample sizes and candidate gene approach; the recent GWAS for cSCC has informed our candidate gene selection in this cohort. Our study design enabled investigation of these genes in the context of Fitzpatrick skin type, an important risk factor. Our data show that genotype data can improve risk stratification for posttransplantation cSCC beyond the clinical pigmentation phenotype. Strengths of this study include a wellcharacterized cohort with Fitzpatrick type and histopathologic confirmation of cSCC outcomes. The genotyping array is the same as that used in Asgari et al. (2016), allowing direct validation of SNPs reported in that publication. The primary limitation is small sample size, but this pilot data supports development of cohort studies to generate

Suppression of stromal-derived Dickkopf-3 (DKK3) inhibits tumor progression and prolongs survival in pancreatic ductal adenocarcinoma

DKK3 is produced by the stroma in pancreatic cancer, promotes tumor progression and resistance to therapy, and is a therapeutic target. Killing tumors by targeting their neighbors Pancreatic cancer is infamous for its bad prognosis, as well as for its dense stroma. Most therapies target the tumor cells themselves rather than the stroma, but now, Zhou et al. identified a therapeutic target called DKK3, which is produced by pancreatic stellate cells. The authors showed that this protein was present in most of the human pancreatic tumors in their study sample. They also demonstrated the effectiveness of ablating DKK3, either by genetic means or with a monoclonal antibody. The antibody treatment reduced tumor growth and extended survival in mouse models, especially when combined with an immune checkpoint inhibitor, demonstrating its therapeutic potential. Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, and it is unclear whether its stromal infiltrate contributes to its aggressiveness. Here, we demonstrate that Dickkopf-3 (DKK3) is produced by pancreatic stellate cells and is present in most human PDAC. DKK3 stimulates PDAC growth, metastasis, and resistance to chemotherapy with both paracrine and autocrine mechanisms through NF-κB activation. Genetic ablation of DKK3 in an autochthonous model of PDAC inhibited tumor growth, induced a peritumoral infiltration of CD8+ T cells, and more than doubled survival. Treatment with a DKK3-blocking monoclonal antibody inhibited PDAC progression and chemoresistance and prolonged survival. The combination of DKK3 inhibition with immune checkpoint inhibition was more effective in reducing tumor growth than either treatment alone and resulted in a durable improvement in survival, suggesting that DKK3 neutralization may be effective as a single targeted agent or in combination with chemotherapy or immunotherapy for PDAC.

Regulatory effect of anti-gp130 functional mAb on IL-6 mediated RANKL and Wnt5a expression through JAK-STAT3 signaling pathway in FLS

We investigated the effect on rheumatoid arthritis (RA) of an anti-gp130 monoclonal antibody (mAb) and its mechanism using RA fibroblast-like synoviocytes (FLS) and a collagen antibody–induced arthritis (CAIA) mouse model. We determined the interleukin 6 (IL-6), IL-6 receptor α (IL-6Rα), gp130, receptor activator of nuclear factor κB ligand (RANKL), matrix metalloproteinase 3 (MMP3), TIMP metallopeptidase inhibitor 1 (TIMP1), and Bcl-2 levels in RA and osteoarthritis (OA) serum and synovial fluid. RA FLS were cultured with or without IL-6/IL-6Rα; WNT5A and RANKL levels were detected. We generated an anti-gp130 mAb (M10) with higher affinity and specificity, blocked IL-6 signaling with it, and assessed its effects on the CAIA model, WNT5A and RANKL expression, and signal transducer and activator of transcription 3 (STAT3) phosphorylation. The IL-6 signaling system in patients with RA was increased; RANKL, MMP3, TIMP1, and Bcl-2 in RA bone were elevated. IL-6/IL-6Rα increased RA FLS WNT5A and RANKL expression. M10 ameliorated arthritis in the CAIA model, and inhibited RANKL, WNT5A, and Bcl-2 expression in RA FLS by blocking IL-6 signaling, likely via Janus kinase–STAT3 pathway downregulation. The IL-6–soluble IL-6Rα–gp130 complex is hyperactive in RA and OA. M10 may be the basis for a novel RA treatment drug.

Abstract 599: MS17-38 mAb targeting of PODXL-v2 inhibits gastric cancer growth and metastasis

Background: Gastric cancer is the primary cause of cancer-specific mortality worldwide. PODXL (podocalyxin-like protein, or podocalyxin) is a cell-surface glycoprotein that belongs to CD34 family of sialomucins. PODXL-v1 is widely expressed on vascular endothelium, mesothelial cells, and platelets; in contrast, PODXL-v2, a truncated short form of PODXL is specifically expressed or up-regulated in numerous cancer cell types as well as in small blood vessels in tumor tissues. There remains considerable interest in generating monoclonal antibodies (mAbs) directed against specific tumor targets for antigen (Ag) discovery, diagnosis and therapy. Methods: Mixed live cells from four human GC cell lines were used as the immunogen in A/J mice. MS17-38 mAb was generated by hybridoma technology and from high throughput screening (HTS), with GC cell lines versus normal human PBMC live cells. Purified MS17-38 mAb was used to confirm the target Ag through a combination of western blot (WB), immunoprecipitation (IP) and mass spectrometry (MS). Peptide microarrays of the identified Ag were layered on chips and double screening was performed to achieve epitope mapping of binding of the mAb its target. Dose-dependent treatment of GC cells with MS17-38 mAb and target siRNA-was followed by staining with CCK-8 and CyQuant GR dyes and read at 450nm and 480/520nm for cell proliferation and transwell migration assays. GC MKN-45 cells were injected subcutaneously (s.c.) into the flank of nu/nu mice treated with MS17-38 mAb or control mAb. In addition, tumor tissues from 42 human GC patients were stained by immunohistochemistry (IHC) and evaluated for PODXL expression using MS17-38, and the survival of the GC patients compared to target antigen expression levels. Results: MS17-38 mAb was generated by live cells HTS and was demonstrated to bind to a specific conformational epitope of PODXL-v2 on GC cells. PODXL-v2 expression inhibition by PODXL-v2 siRNA or the PODXL-v2-neutralizing antibody MS17-38 resulted in inhibition of GC cell growth and prevention of GC cell migration in vitro. Furthermore, in vivo studies demonstrated that MS17-38 mAb can potently inhibit tumor growth and prevent MKN-45 metastasis to the lungs in nu/nu mouse models. Finally, high expression of PODXL-v2 was associated with advanced GC stage and short survival time (P Conclusions: Our findings indicate that PODXL-v2 is specifically expressed in the extracellular matrix of GC, and MS17-38 mAb directed against a conformational epitope of PODXL-v2 identified through HTS can functionally inhibit GC cancer cell growth and migration/metastasis both in vitro and in vivo. MS17-38 is a promising functional antibody directed against GC that could potentially be developed into a therapeutic mAb for clinical application. Citation Format: Runhua Feng, Ming Li, Yuling Wang, Dongqing Zhang, Xiaolian Gao, Xuehua Chen, Joe X. Zhou, Victor G. Prieto, Jian H. Song, Shenying Fang, Binya Liu, Zhenggang Zhu, Xiangcang Ye, Lee M. Ellis, Mason Lu, Jeffery E. Lee. MS17-38 mAb targeting of PODXL-v2 inhibits gastric cancer growth and metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 599.

Abstract 1857: Intracellular protein GP128 is a novel tumor angiogenesis marker on gastric cancer cell lines and GI tissues: Ectopic expression on the cell surface defined by MS38-2.1 mAb

Our aim is to generate monoclonal antibodies (mAbs) against novel markers on the cancer cell surface using an innovated high throughput screening (HTS) strategy. Mixed live cells from four gastric cancer cell lines (MKN45, SGC7901, BGC823 and MKN28) were used as the immunogen for A/J mice: spleen cells were then fused with mouse SP2/0 myeloma cells to generate monoclonal antibody hybridomas. The 16 highly positive hybridoma colonies were selected from 20,000 colonies in sixty-seven 96-well plates through screening using live cell (gastric cancer cells versus normal PBMC) staining and FACS-HTS technology. Protein-A affinity purified mAbs were used to define the antigens, which were confirmed as novel biomarkers on the cancer cell surface using microarray, mass and proteomic analyses. Protein microarray analysis with the MS38-2.1 mAb identified an intracellular protein GP128 (e.g., SPRR3) as the most likely a novel cancer biomarker. The immunohistochemical profiles of MS38-2.1 mAb in GI (gastric and colorectal) cancer tissues were found to be significantly higher (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1857. doi:1538-7445.AM2012-1857

Phase II trial of irinotecan and cisplatin combination as first-line chemotherapy in patients with advanced esophageal squamous-cell carcinoma: An interim report.

137 Background: Although the irinotecan and cisplatin combination has been used in esophageal cancer treatment, we tested the combination specifically in unresectable or metastatic esophageal squamous cell carcinoma with different doses to decrease the toxicity and keep the efficacy. METHODS Patients were eligible if they had histologic proof of unresectable or metastatic squamous cell carcinoma of the esophagus, between 18-75 years of age with a Karnofsky performance status ≥ 80. No prior chemotherapy was allowed. Patients were treated with irinotecan 130 mg/m2 and cisplatin 60 mg/m2 repeated 3 weeks. Response was evaluated every two cycles of treatment by using RECIST criteria. The sample size was calculated using Simons 2-stage design. The primary end point of the study was objective response rate. The second end points are PFS and toxicity. Accrual was planned to a total of 46 patients with the targeted response rate of 50%. The first stage requires at least 7 or more out of 16 patients to have a confirmed partial or complete response before proceeding to the second stage. RESULTS Twenty one patients have been enrolled so far, of which 16 patients were assessable for response. The overall response rate was 43.8%(7/16), including one complete response (6.3%) and six partial response (37.5%). Among 13 patients who presented with dysphagia at baseline, 10 (62.5%) had the symptom either completely resolved or significantly improved. The most common toxicities were neutropenia, diarrhea and alopecia. Seven patients (33.3%) developed grade 3 neutropenia and one had (4.8%) with febrile neutropenia. One patient (4.8%) had grade 3 diarrhea. There were no treatment-related deaths. CONCLUSIONS The combination of irinotecan plus cisplatin with lower doses showed equivalent efficacy and less toxicity in patients with unresectable or metastatic squamous-cell carcinoma of the esophagus. The study is ongoing. No significant financial relationships to disclose.