Development Of Novel Monoclonal Antibodies Research Articles

Development of novel monoclonal antibodies for blocking NF-κB activation induced by CD2v protein in African swine fever virus

BackgroundCD2v, a critical outer envelope glycoprotein of the African swine fever virus (ASFV), plays a central role in the hemadsorption phenomenon during ASFV infection and is recognized as an essential immunoprotective protein. Monoclonal antibodies (mAbs) targeting CD2v have demonstrated promise in both diagnosing and combating African swine fever (ASF). The objective of this study was to develop specific monoclonal antibodies against CD2v.MethodsIn this investigation, Recombinant CD2v was expressed in eukaryotic cells, and murine mAbs were generated through meticulous screening and hybridoma cloning. Various techniques, including indirect enzyme-linked immunosorbent assay (ELISA), western blotting, immunofluorescence assay (IFA), and bio-layer interferometry (BLI), were employed to characterize the mAbs. Epitope mapping was conducted using truncation mutants and epitope peptide mapping.ResultsAn optimal antibody pair for a highly sensitive sandwich ELISA was identified, and the antigenic structures recognized by the mAbs were elucidated. Two linear epitopes highly conserved in ASFV genotype II strains, particularly in Chinese endemic strains, were identified, along with a unique glycosylated epitope. Three mAbs, 2B25, 3G25, and 8G1, effectively blocked CD2v-induced NF-κB activation.ConclusionsThis study provides valuable insights into the antigenic structure of ASFV CD2v. The mAbs obtained in this study hold great potential for use in the development of ASF diagnostic strategies, and the identified epitopes may contribute to vaccine development against ASFV.

Development of Novel Monoclonal Antibodies to Wheat Alpha-Amylases Associated with Grain Quality Problems That Are Increasing with Climate Change.

Accurate, rapid testing platforms are essential for early detection and mitigation of late maturity α-amylase (LMA) and preharvest sprouting (PHS) in wheat. These conditions are characterized by elevated α-amylase levels and negatively impact flour quality, resulting in substantial economic losses. The Hagberg-Perten Falling Number (FN) method is the industry standard for measuring α-amylase activity in wheatmeal. However, FN does not directly detect α-amylase and has major limitations. Developing α-amylase immunoassays would potentially enable early, accurate detection regardless of testing environment. With this goal, we assessed an expression of α-amylase isoforms during seed development. Transcripts of three of the four isoforms were detected in developing and mature grain. These were cloned and used to develop E. coli expression lines expressing single isoforms. After assessing amino acid conservation between isoforms, we identified peptide sequences specific to a single isoform (TaAMY1) or that were conserved in all isoforms, to develop monoclonal antibodies with targeted specificities. Three monoclonal antibodies were developed, anti-TaAMY1-A, anti-TaAMY1-B, and anti-TaAMY1-C. All three detected endogenous α-amylase(s). Anti-TaAMY1-A was specific for TaAMY1, whereas anti-TaAMY1-C detected TaAMY1, 2, and 4. Thus, confirming that they possessed the intended specificities. All three antibodies were shown to be compatible for use with immuno-pulldown and immuno-assay applications.

Development of Novel Monoclonal Antibodies Against Nattokinase.

Nattokinase is a protease produced by Bacillus subtilis var. natto that exhibits various beneficial biological effects. Thus, a reliable assay to determine nattokinase levels is needed. In this study, we developed novel mouse monoclonal antibodies (mAbs) that recognize nattokinase, and created a specific and sensitive enzyme-linked immunosorbent assay (ELISA) to measure nattokinase levels. The ELISA was developed using a combination of new mouse antinattokinase mAbs used as capture antibodies coated onto 96-well plates, with a peroxidase-conjugated antibody used for detection. This ELISA enabled detection of nattokinase at 1 ng/mL. We believe that the novel mAbs developed in this study will be useful in future for elucidating nattokinase function.

Development of novel monoclonal antibodies against nsp12 of SARS-CoV-2.

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a global pandemic of coronavirus disease 19. Coronaviruses, including SARS-CoV-2, use RNA-dependent RNA polymerase (RdRP) for viral replication and transcription. Since RdRP is a promising therapeutic target for infection of SARS-CoV-2, it would be beneficial to develop new experimental tools for analysis of the RdRP reaction of SARS-CoV-2. Here, we succeeded to develop novel mouse monoclonal antibodies (mAbs) that recognize SARS-CoV-2 nsp12, catalytic subunit of the RdRP. These anti-nsp12 mAbs, RdMab-2, -13, and -20, specifically recognize SARS-CoV-2 nsp12 by western blotting analysis, while they exhibit less or no cross-reactivity to SARS-CoV nsp12. In addition, SARS-CoV-2 nsp12 was successfully immunoprecipitated using RdMab-2 from lysates of cells overexpressing SARS-CoV-2 nsp12. RdMab-2 was able to detect SARS-CoV-2 nsp12 transiently expressed in established culture cells such as HEK293T cells by indirect immunofluorescence technique. These novel mAbs against SARS-CoV-2 nsp12 are useful to elucidate the RdRP reaction of SARS-CoV-2 and biological cell response against it.

Development of novel monoclonal antibodies and immunoassays for sensitive and specific detection of SULF1 endosulfatase

BackgroundCell-surface heparan sulfate proteoglycans (HSPGs) function as receptors or co-receptors for ligand binding and mediate the transmission of critical extracellular signals into cells. The complex and dynamic modifications of heparan sulfates on the core proteins are highly regulated to achieve precise signaling transduction. Extracellular endosulfatase Sulf1 catalyzes the removal of 6-O sulfation from HSPGs and thus regulates signaling mediated by 6-O sulfation on HSPGs. The expression of Sulf1 is altered in many cancers. Further studies are needed to clarify Sulf1 role in tumorigenesis, and new tools that can expand our knowledge in this field are required. MethodsWe have developed and validated novel SULF1 monoclonal antibodies (mAbs). The isotype and subclass for each of these antibodies were determined. These antibodies provide invaluable reagents to assess SULF1- tissue and blood levels by immunohistochemistry and ELISA assays, respectively. ResultsThis study reports novel mAbs and immunoassays developed for sensitive and specific human Sulf1 protein detection. Using these SULF1 mAbs, we developed an ELISA assay to investigate whether blood-derived SULF1 may be a useful biomarker for detecting cancer early. Furthermore, we have demonstrated the utility of these antibodies for Sulf1 protein detection, localization, and quantification in biospecimens using various immunoassays. ConclusionsThis study describes novel Sulf1 mAbs suitable for various immunoassays, including Western blot analysis, ELISA, and immunohistochemistry, which can help understand Sulf1 pathophysiological role. General significanceNew tools to assess and clarify SULF1 role in tumorigenesis are needed. Our novel Sulf1 mAbs and immunoassays assay may have utility for such application.

Development of novel monoclonal antibodies with specific binding affinity for denatured human CD26 in formalin-fixed paraffin-embedded and decalcified specimens.

A 110-kDa type II transmembrane glycoprotein with dipeptidyl peptidase IV (DPPIV) activity in its extracellular region, CD26 has a multitude of biological functions and plays an important role in the regulation of inflammatory responses and tumor biology. Our work has focused on CD26 as a novel therapeutic target for various tumors and immune disorders, and we have recently developed a humanized anti-CD26 monoclonal antibody (mAb), YS110, which has promising safety profile and clinical activity in patients with malignant pleural mesothelioma. The development of an anti-human CD26 mAb that can clearly and reliably detect the denatured CD26 molecule in formalin-fixed paraffin-embedded (FFPE) tissues in the clinical setting is therefore of the utmost importance. To develop novel anti-CD26 mAbs capable of binding to denatured CD26, we immunized mice with urea-treated CD26 protein. Hybridoma supernatants were screened for specific reactivity with human CD26 by immunostaining through the use of a set of FFPE human CD26-positive or negative tumor cell lines. This screening method enables us to develop novel anti-human CD26 mAbs suitable for immunohistochemical staining of CD26 in FFPE non-tumor and tumor tissue sections with reliable clarity and intensity. Specifically, these mAbs display strong binding affinity to denatured human CD26 rather than undenatured human CD26, and are capable of detecting denatured human CD26 in decalcified specimens. These novel anti-CD26 mAbs are potentially useful for the analysis of CD26 expression in cancer patients with bony metastasis, and may help decide the appropriateness of YS110 therapy for future cancer patients.

Novel monoclonal antibody-based treatment strategies in adults with acute lymphoblastic leukemia.

Adult acute lymphoblastic leukemia (ALL) has a poor overall survival compared with pediatric ALL where cure rates are observed in more than 90% of patients. The recent development of novel monoclonal antibodies targeting CD20, CD19, and CD22 has changed the long-term outcome of this disease, both in the frontline setting (e.g. rituximab) and for patients with relapsed/refractory disease (e.g. inotuzumab ozogamicin and blinatumomab). The CD3-CD19 bispecific T-cell-engaging antibody blinatumomab is also the first drug approved in ALL for patients with persistent or recurrent measurable residual disease, providing a new treatment paradigm for these patients. Several new agents are also in development that use novel constructs or target alternative surface epitopes such as CD123, CD25, and CD38. Herein, we review the role of monoclonal antibodies in adult ALL and summarize the current and future approaches in ALL, including novel combination therapies and the possibility of early incorporation of these agents into treatment regimens.

Development of novel monoclonal antibodies against CD109 overexpressed in human pancreatic cancer

Pancreatic cancer is one of the most aggressive and lethal types of cancer, and more effective therapeutic agents are urgently needed. Overexpressed cell surface antigens are ideal targets for therapy with monoclonal antibody (mAb)-based drugs, but none have been approved for the treatment of pancreatic cancer. Here, we report development of two novel mouse mAbs, KU42.33C and KU43.13A, against the human pancreatic cancer cell line BxPC-3. Using ELISA, flow cytometry, competitive assay and immunoprecipitation followed by mass spectrometry, we discovered that these two mAbs target two distinct epitopes on the external domain of CD109 that are overexpressed by varying amounts in human pancreatic cancer cell lines. Treatment with these two naked antibodies alone did not affect tumour cell growth or migration in vitro. Of the two mAbs, only KU42.33C was useful in determining the expression of CD109 in tumour cells by Western blot and immunohistochemistry. Interestingly, immunohistochemistry of human pancreatic carcinoma tissue arrays with mAb KU42.33C showed that 94% of the 65 human pancreatic adenocarcinoma cases were CD109 positive, with no expression in normal pancreatic tissues. Our results suggest that these two novel mAbs are excellent tools for determining the expression level of CD109 in the tumour specimens and sera of patients with a wide range of cancers, in particular pancreatic cancer, and for investigating its diagnostic, prognostic and predictive value. Further research is warranted and should aim to unravel the therapeutic potential of the humanised forms or conjugated versions of such antibodies in patients whose tumours overexpress CD109 antigen.

Development of novel monoclonal antibodies to dog leukocyte antigen DR displaying direct and immune-mediated cytotoxicity toward canine lymphoma cell lines.

Spontaneous canine lymphoma (CL) has become a promising, nonrodent model for advancing the therapeutic strategies of human hematological malignancies. As new resources for veterinary and comparative studies on CL-associated antigens, we developed 2 novel mouse monoclonal antibodies, denoted B5 and E11, that recognized the canine major histocompatibility Class II DR antigens (dog leukocyte antigen DR). Using flow cytometry and solid phase immunoenzymatic assays, we showed that the antigens recognized by B5 and E11 were strongly expressed in several CL cell lines and the ex vivo canine neoplastic cells of B and mixed B/T immunophenotypes. Additionally, we evaluated a minimal cross-reactivity of B5 and E11 with the human B-cell line, Raji. By the ectopic expression of the hybrid murine/canine I-E/DR dimers in the HEK293 cells, we demonstrated that the epitope of B5 was localized to the invariant DRα chain, whereas the epitope of E11 was collectively formed by the DRα and DRβ chains. Both epitopes were conformational and conserved in all the tested unrelated individuals of different dog breeds. In vitro treatment of 2 CL B-cell lines (CLBL1 and CLB70) with B5 and E11 rapidly induced a direct apoptotic cell death. Similarily, both mouse monoclonal antibodies efficiently killed the above cell lines through the mechanisms of complement-dependent and antibody-mediated cellular phagocytosis. Collectively, our data support the further development of B5 and E11 as novel tools for dog leukocyte antigen DR-targeted, preclinical trials involving CL.

Development of novel monoclonal antibodies against starch and ulvan - implications for antibody production against polysaccharides with limited immunogenicity

Monoclonal antibodies (mAbs) are widely used and powerful research tools, but the generation of mAbs against glycan epitopes is generally more problematic than against proteins. This is especially significant for research on polysaccharide-rich land plants and algae (Viridiplantae). Most antibody production is based on using single antigens, however, there are significant gaps in the current repertoire of mAbs against some glycan targets with low immunogenicity. We approached mAb production in a different way and immunised with a complex mixture of polysaccharides. The multiplexed screening capability of carbohydrate microarrays was then exploited to deconvolute the specificities of individual mAbs. Using this strategy, we generated a set of novel mAbs, including one against starch (INCh1) and one against ulvan (INCh2). These polysaccharides are important storage and structural polymers respectively, but both are generally considered as having limited immunogenicity. INCh1 and INCh2 therefore represent important new molecular probes for Viridiplantae research. Moreover, since the α-(1-4)-glucan epitope recognised by INCh1 is also a component of glycogen, this mAb can also be used in mammalian systems. We describe the detailed characterisation of INCh1 and INCh2, and discuss the potential of a non-directed mass-screening approach for mAb production against some glycan targets.

Development of Novel Monoclonal Antibodies for Evaluation of Transmembrane Prostate Androgen-Induced Protein 1 (TMEPAI) Expression Patterns in Gastric Cancer.

Transmembrane prostate androgen-induced protein 1 (TMEPAI) is a single-span membrane protein, functionally involved in transforming growth factor beta signaling pathway. The particular protein presented in cells in three isoforms, which differs in the length of the soluble N-terminal extracellular domain, making it challenging for the immunochemical recognition. By using quantitative real-time polymerase chain reaction, we identified significant upregulation of PMEPA1 gene expression in malignant tissues of patients with gastric adenocarcinoma. The main part of commercially available anti-TMEPAI antibodies are having polyclonal nature or not suitable for immunocytochemical localization of target protein in tissue specimens. Hence, we decide to generate a set of novel rat monoclonal antibodies (mAb) directed against conservative C-terminal cytoplasmic epitope. Immunoblotting analysis showed that monoclonal antibodies, 2E1, 6C6, and 10A7 were able to recognize specifically target protein in transiently transfected HEK293T and CHO-K1 cells. Especially established mAb, named 10A7, showed the excellent binding ability to target protein in immunohistochemistry. By using developed antibodies, we observed pronounced expression of TMEPAI in normal gastric epithelial cells while tumor cells from gastric adenomas, and adenocarcinoma samples were mostly negative for target protein expression. Also, we found that gastric epithelium cells lose the TMEPAI expression concurrently with severe dysplasia progression, which probably caused by a mechanism involving specific microRNA.

Development of novel monoclonal antibodies directed against catechins for investigation of antibacterial mechanism of catechins

Catechins are major polyphenolic compounds of green tea. To investigate mechanism for antibacterial action of catechins, 11 monoclonal antibodies (MAbs) were raised against a 3-succinyl-epicatechin (EC)-keyhole limpet hemocyanin (KLH) conjugate. Amino acid sequences of variable regions determined for MAbs b-1058, b-1565, and b-2106 confirmed their innovative character. MAb b-1058 strongly interacted with its target substances in the following order of magnitude: theaflavin-3,3′-di-O-gallate (TFDG)>theaflavin-3-O-gallate (TF3G)≥theaflavin-3′-O-gallate (TF3’G)>gallocatechin gallate (GCg)>penta-O-galloyl-β-d-glucose (PGG)>epigallocatechin gallate (EGCg), as determined using surface plasmon resonance (SPR) on MAb-immobilized sensor chips. The affinity profiles of MAbs b-1058 and b-2106 to the various polyphenols tested suggested that flavan skeletons with both carbonyl oxygen and hydroxyl groups are important for this interaction to take place. S. aureus cells treated with EGCg showed green fluorescence around the cells after incubation with FITC-labeled MAb b-1058. The fluorescence intensity increased with increasing concentrations of EGCg. These MAbs are effective to investigate antibacterial mechanism of catechins and theaflavins.

Droplet-Digital PCR Provides a Rapid, Accurate and Cost-Effective Method for Identification of Biomarker FcγRIIIa-F158V Genotypes

Development of novel monoclonal antibodies, vaccines and oncolytic virus therapies have relied on analysis of biomarkers as potential predictors of success. One well studied biomarker is the CD16/ FcγRIIIa receptor residue 158 F/V. Identifying variants through genotyping of the FcγRIIIa locus is widely practiced and highly varied with commonly used methods including: Sanger sequencing, flow-cytometry, PCR/RFLP, Goldengate (replaced by Infinium) and TaqMAN analysis. While each of these methods have considerable backing in publications related to CD16 FcγRIIIa 158 F/V, the majority present significant short comings in identifying both homozygotes (wild-type and mutant) and heterozygotes in a time and cost-efficient manner. Utilization of droplet-digital PCR with FcγRIIIa-F158V specific probes results in the accurate genotyping using direct recognition of sequence in genomic samples at a lower average cost and faster turnaround. Here we demonstrate the use of ddPCR to accurately identify FcγRIIIa-F158V genotypes with confirmation by Illumina sequencing in 128 patient samples.

ATPS-82PROBING THE TUMORIGENIC PROPERTIES OF TWO PORE POTASSIUM CHANNELS USING INHIBITORY KCNK9 mAbs

Ion channels govern portals that facilitate ionic passage across membranes in all organisms. KCNK9, a member of the family of two-pore domain potassium K+ channels, mediates leak currents critical to maintaining cell resting membrane potentials. Since its identification, KCNK9 has been implicated in various human disorders including cancer. KCNK9 overexpression has been observed in melanoma, colorectal, breast and lung cancers, and KCNK9 has been found to promote neoplastic cell survival and growth. However, KCNK9's underlying contributions to malignancy remain elusive due to the absence of specific modulators. Here, we describe the development of novel monoclonal antibodies directed against the KCNK9 extracellular domain and their functional effects. We show that one antibody (Y4) with the highest affinity binding, inhibits KCNK9 activity by inducing internalization of cell-surface channels. When tested on KCNK9-expressing cells, Y4 reduces cell viability; increases cell death and triggers complement-dependent cytotoxicity. Systemic administration of Y4 effectively inhibits growth of human lung cancer xenografts and breast cancer metastasis in a syngeneic murine model (P < 0.05). Since KCNK9 expression has been reported in human glioma and linked to regulation of glioblastoma cell survival, we extended our study to glioblastoma and examined KCNK9 expression in a panel of glioblastoma cell lines as well as primary cultures from patient-derived tumors. We found abundant KCNK9 expression in some aggressive glioblastoma models (e.g. Mayo39). We are currently investigating the functional effects of antibodies in these models. In conclusion, we have generated a high affinity KCNK9-specific monoclonal antibody that inhibits tumor cell survival, primary tumor formation and metastasis. Antibody-based KCNK9 targeting can shed light on KCNK9's significance in glioblastoma development and become promising therapeutic strategy in KCNK9-expressing brain malignancy.

Abstract P211: Normo-aldosteronemic Aldosterone-producing Adenoma: Immunochemical Characterization and Diagnostic Implications

Background: Primary Aldosteronism (PA), the most common form of endocrine hypertension, is usually identified by a high aldosterone-renin ratio (ARR) alongside elevated plasma aldosterone concentration (PAC) values. Normo-aldosteronemic PA is also being diagnosed when only the high ARR was driven by low renin because PAC was normal. However, whether this entity truly exists remains contentious since most such cases did not undergo surgical confirmation and even when adrenalectomy was performed no demonstration of an aldosterone-producing adenoma (APA) could be obtained at immunohistochemistry. Case Description: In 2003 a young lady presented with severe hypertension, low plasma renin activity (PRA), but consistently normal PAC values. She undertook a magnetic resonance that revealed a small adenoma on the right side, but adrenal vein sampling (AVS) showed left lateralization of aldosterone secretion. Therefore, she underwent left laparoscopic adrenal surgery, which determined complete normalization of blood pressure and PRA long term follow up (12 years). In 2014 the development of novel monoclonal antibodies for the human aldosterone synthase CYP11B2 and 11β-hydroxylase CYP11B1, allowed us to immunochemically characterize the resected adrenal gland. Double immunostaining for CYP11B2 and CYP11B1 showed a small CYP11B2-positive adenoma, thus unequivocally proving the presence of an APA. Conclusions: This case provides compelling evidence for the existence of normo-aldosteronemic APA and suggests that many cases that we dismiss as “low renin-essential hypertension” might instead have an undetected surgically curable APA.

Abstract 2733: Development of novel monoclonal antibodies for a cyclic DNA adduct derived from oxidation of ω-6 polyunsaturated fatty acids

Abstract Dietary polyunsaturated fatty acids (ω-3 and ω-6 PUFAs) play a role in certain human cancers. The oxidation of PUFAs produces reactive α,β-unsaturated aldehydes (enals) that can modify DNA producing mutagenic lesions. Of particular interest, 7-(1′,2′-dihydroxyheptyl)-1,N6- ethenodeoxyadenosine (DHHϵdA) is a novel DNA adduct in vitro and in vivo derived from lipid peroxidation of ω-6 PUFAs. Despite its recent identification in vivo, the role of DHHϵdA in tumorigenesis is not yet known. Previously, the endogenously formed DHHϵdA was detected by liquid chromatography tandem mass spectrometry (LC-MS/MS). Due to its low levels in vivo (1-30 adducts/109 DNA bases), a relatively high quantity of DNA is needed for its detection and quantification. In this study we developed a monoclonal antibody against DHHϵdA in order to detect and quantify this adduct in cells and tissues. Two antigens were synthesized by conjugating activated hapten: 5′-carboxy-7-(1′,2′-dihydroxyheptyl) adenosine (5′-carboxy DHHϵA) to BSA and KLH carrier proteins. KLH-conjugated hapten was used for mice immunization, whereas BSA-based hapten was used to test for an immune response. After immunization and several screenings, 6 monoclonal cell lines were chosen for further tests. Normal and competitive ELISA was performed to characterize the antibodies from all cell lines. Competitive ELISA revealed no cross-reactivity towards normal nucleosides and α- and γ-OHPdG, the cyclic 1,N2-propano adduct derived from acrolein, and 8-oxo-dG for all of the tested antibodies. However, we observed a weak (&amp;lt; 1:100) specificity for HNE-dG, an adduct derived from (E)-4-hydroxy-2-nonenal produced from oxidized ω-6 PUFAs. Because of the relatively low level of endogenous HNE-dG compared to DHHϵdA, this weak cross-reactivity should not affect the specificity of the developed antibody to detect DHHϵdA. Two cell lines were discarded after antibodies from them showed moderate competitive effects against 1,N6-etheno-2′-deoxyadenosine (ϵdA). From the remaining 4 cell lines the best one was chosen to produce purified antibody. We believe that this antibody will be a useful tool in the studies of the role of DHHϵdA as an endogenous DNA lesion in cancer development associated with ω-6 PUFAs. This work was supported by the NCI grant CA134892. Citation Format: Marcin Dyba, Brandon Da Silva, Jishen Pan, Fung-Lung Chung. Development of novel monoclonal antibodies for a cyclic DNA adduct derived from oxidation of ω-6 polyunsaturated fatty acids. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2733. doi:10.1158/1538-7445.AM2015-2733

Abstract 640: Probing the tumorigenic properties of two pore potassium channels using inhibitory anti-KCNK9 mAbs

Abstract Ion channels are important portals that facilitate ionic passage across membranes in all organisms. KCNK9, a member of the family of two-pore domain potassium K+ channels, mediates “leak” currents critical to maintaining cell resting membrane potentials. Since its identification, KCNK9 has been implicated in various human disorders including cancer. KCNK9 overexpression has been observed in melanoma, colorectal, breast and lung tumors and KCNK9 has been found to promote tumor survival and growth, by increasing resistance to hypoxia and apoptosis. However, KCNK9's underlying pathophysiologic mechanisms have been elusive due to the absence of specific inhibitors. Here, we describe the development of novel monoclonal antibodies directed against the KCNK9 extracellular domain and their molecular/cellular effects on KCNK9-expressing cells. Murine monoclonal IgG1 antibodies were raised against recombinant KCNK9 extracellular domains expressed in a mammalian system. 40 monoclonal antibodies were generated and demonstrated channel subtype-specific recognition of human KCNK9. Among them, 1 antibody designated Y4-mAb displayed sub-nanomolar binding affinity and inhibited KCNK9 channel conductance by up to 30% when tested in a fluorescent ion-influx assay. Through confocal microscopic analysis, channel inhibition was found to result from antibody-induced internalization and endocytosis of cell-surface channels. Y4-mAb induced up to 30∼40% reduction in cell viability and increased cell death in BEN non-small cell lung carcinoma cells and BT-549 and 410.4 breast cancer cells. In addition to “direct cell killing”, this antibody activated complement, resulting in complement-dependent cancer cell cytotoxicity in vitro. Initial experiments in vivo showed that systemic Y4-mAb inhibited the capacity of BEN non-small cell lung carcinoma cells to propagate subcutaneous tumor xenografts in nude mice (p&amp;lt;0.05). In conclusion, we have generated a high affinity KCNK9-specific monoclonal antibody that perturbs KCNK9 function and inhibits tumor cell survival and xenograft formation. Anti-KCNK9 antibodies have the potential to be valuable research tools and may lead to novel therapeutics. Moreover, our strategies should be generally applicable to studying the functions of other ion channels in cancer. Citation Format: Han Sun, Liqun Luo, Bachchu Lal, John Laterra, Min Li. Probing the tumorigenic properties of two pore potassium channels using inhibitory anti-KCNK9 mAbs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 640. doi:10.1158/1538-7445.AM2015-640

Successful development of novel monoclonal antibodies against HTLV-1 bZIP factor and their applications in studying the pathogenesis of HAM/TSP

Methods We have generated rat and human monoclonal antibodies against HBZ by hybridoma methodology and a phage display system, and developed the ELISA system for the detection of HBZ protein and anti-HBZ antibodies. Using these reagents, we analyzed the expression of HBZ protein in plasma and peripheral blood mononuclear cells (PBMCs) of HTLV-1 infected individuals as well as HTLV-1 infected cell lines. Then the results were compared with the real time PCR data.

Development of novel monoclonal antibodies that define differentiation stages of human stromal (mesenchymal) stem cells.

Human mesenchymal stem cells (hMSC) are currently being introduced for cell therapy, yet, antibodies specific for native and differentiated MSCs are required for their identification prior to clinical use. Herein, high quality antibodies against MSC surface proteins were developed by immunizing mice with hMSC, and by using a panel of subsequent screening methods. Flow cytometry analysis revealed that 83.5, 1.1, and 8.5% of primary cultures of hMSC were double positive for STRO-1 and either of DJ 3, 9, and 18, respectively. However, none of the three DJ antibodies allowed enrichment of clonogenic hMSC from BMMNCs as single reagents. Using mass-spectrometric analysis, we identified the antigen recognised by DJ3 as CD44, whereas DJ9 and DJ18 recognized HLA-DRB1 and Collagen VI, respectively. The identified proteins were highly expressed throughout in vitro osteogenic- and adipogenic differentiation. Interestingly, undifferentiated cells revealed a sole cytoplasmic distribution pattern of Collagen VI, which however changed to an extracellular matrix appearance upon osteogenic- and adipogenic differentiation. In relation to this, we found that STRO-1(+/-)/Collagen VI(-) sorted hMSC contained fewer differentiated alkaline phosphatase(+) cells compared to STRO-1(+/-)/Collagen VI(+) hMSC, suggesting that Collagen VI on the cell membrane exclusively defines differentiated MSCs. In conclusion, we have generated a panel of high quality antibodies to be used for characterization of MSCs, and in addition our results may suggest that the DJ18 generated antibody against Collagen VI can be used for negative selection of cultured undifferentiated MSCs.

Melanoma: Stem cells, sun exposure and hallmarks for carcinogenesis, molecular concepts and future clinical implications

Background:The classification and prognostic assessment of melanoma is currently based on morphologic and histopathologic biomarkers. Availability of an increasing number of molecular biomarkers provides the potential for redefining diagnostic and prognostic categories and utilizing pharmacogenomics for the treatment of patients. The aim of the present review is to provide a basis that will allow the construction–or reconstruction–of future melanoma research.Methods:We critically review the common medical databases (PubMed, EMBASE, Scopus and Cochrane CENTRAL) for studies reporting on molecular biomarkers for melanoma. Results are discussed along the hallmarks proposed for malignant transformation by Hanahan and Weinberg. We further discuss the genetic basis of melanoma with regard to the possible stem cell origin of melanoma cells and the role of sunlight in melanoma carcinogenesis.Results:Melanocyte precursors undergo several genome changes –UV-induced or not– which could be either mutations or epigenetic. These changes provide stem cells with abilities to self-invoke growth signals, to suppress antigrowth signals, to avoid apoptosis, to replicate without limit, to invade, proliferate and sustain angiogenesis. Melanocyte stem cells are able to progressively collect these changes in their genome. These new potential functions, drive melanocyte precursors to the epidermis were they proliferate and might cause benign nevi. In the epidermis, they are still capable of acquiring new traits via changes to their genome. With time, such changes could add up to transform a melanocyte precursor to a malignant melanoma stem cell.Conclusions:Melanoma cannot be considered a “black box” for researchers anymore. Current trends in the diagnosis and prognosis of melanoma are to individualize treatment based on molecular biomarkers. Pharmacogenomics constitute a promising field with regard to melanoma patients' treatment. Finally, development of novel monoclonal antibodies is expected to complement melanoma patient care while a number of investigational vaccines could find their way into everyday oncology practice.