Mammalian Expression System Research Articles

1C22 内耳タンパク質モーターprestinの哺乳類細胞発現系の構築

1C22 内耳タンパク質モーターprestinの哺乳類細胞発現系の構築

Fundamentals of Baculovirus Expression and Applications.

In 1982 E. coli produced human insulin, the world's first recombinant DNA drug, was approved by the FDA. Since this historical event, remarkable progress has been made in developing bacterial, yeast, mammalian and insect cell protein expression systems that are used to produce recombinant proteins for both research and clinical applications. Of the available approaches, the insect cell based baculovirus expression vector system (BEVS) has proven to be a particularly adaptable system for producing a diverse collection of proteins. Along with E. coli, the system has been valuable for the production of proteins for structural studies, including adequate quantities of difficult to produce G protein-coupled receptors. BEVS has also been used for production of the human papilloma virus vaccine, Cervarix, the first FDA approved insect cell produced product and FluBlok, a vaccine based on the influenza virus hemagglutinin protein. Baculoviruses, modified to contain mammalian promoters (BacMam viruses), have proven to be efficient gene delivery vectors for mammalian cells and provide an alternative transient mammalian cell based protein expression approach to that of plasmid DNA based transfection methodologies. Here we provide an update on recent advances in baculovirus vector development with a focus on the numerous applications of these viruses in basic research and biotechnology.

Abstract B018: Developmental and functional characterization of immunoglobulin kappa chain-bridged single chain bi-specific antibody for targeted antitumor therapy

Abstract The immune system has routinely been the focus of cancer treatments. One of the strategies that targets the immune system is by use of monoclonal antibodies to specifically target tumor antigens leading to immune activation and tumor destruction. Therefore, many biopharmaceutical companies are interested/invested in antibody development, including the development of next-generation antibodies such as bispecific antibodies. The Development Center for Biotechnology (DCB) in Taiwan has developed a new platform for the generation of immunoglobulin kappa chain-bridged single chain bispecific antibody. Using this platform, DCB constructed a kappa domain-bridged HER2/CD3 bispecific single chain antibody known as the LBscbHER2-CD3. Utilizing a mammalian expression system, the antibody can be easily purified by affinity chromatography via kappa fragment capture. Binding affinity studies have revealed that the purified LBscbHER2-CD3 antibody is able to independently as well as simultaneously bind to HER2+ tumor cells and CD3+ T cells at low nanomolar concentrations. In co-culture experiments, the addition of purified LBscbHER2-CD3 antibody resulted in a significant increase in IL-2 production which is a strong indicator of human T cell activation. In addition, the co-engagement of either IL-2 primed or un-primed human T cells and HER2+ tumor cells by the LBscbHER2-CD3 antibody facilitated the killing of tumor cells from breast, colon and pancreatic cancers. Lastly, when administered in vivo, LBscbHER2-CD3 showed strong tumor growth inhibition in SCID mice xenograft models with implanted HER2+ SW480 cancer cells, HT29 human colon carcinoma cells, and Capan-1 human pancreatic cancer cells. In conclusion, the LBscbHER2-CD3 bispecific antibody has strong affinity for both the cancer cell-expressing receptor and the T cell antigen which leads to cancer cell elimination in vitro and in vivo. These results suggest that LBscbHER2-CD3 may have therapeutic potential for drug development in the treatment of human cancers. Citation Format: Yu Hsun Lo, Jei-Hwa Yu, Yu-Hsen Hsui, Ta-Tung Yuan. Developmental and functional characterization of immunoglobulin kappa chain-bridged single chain bi-specific antibody for targeted antitumor therapy. [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 B018.

Small-Scale Screening to Large-Scale Over-Expression of Human Membrane Proteins for Structural Studies.

Membrane protein structural studies are frequently hampered by poor expression. The low natural abundance of these proteins implies a need for utilizing different heterologous expression systems. E. coli and yeast are commonly used expression systems due to rapid cell growth at high cell density, economical production, and ease of manipulation. Here we report a simplified, systematically developed robust strategy from small-scale screening to large-scale over-expression of human integral membrane proteins in the mammalian expression system for structural studies. This methodology streamlines small-scale screening of several different constructs utilizing fluorescence size-exclusion chromatography (FSEC) towards optimization of buffer, additives, and detergents for achieving stability and homogeneity. This is followed by the generation of stable clonal cell lines expressing desired constructs, and lastly large-scale expression for crystallization. These techniques are designed to rapidly advance the structural studies of eukaryotic integral membrane proteins including that of human membrane proteins.

Plant expression systems for early stage discovery and development of lead therapeutic antibodies.

Antibodies for human clinical applications are predominantly produced in mammalian expression systems, with Chinese hamster ovary (CHO) cells being the gold standard. CHO cells are ideal for the manufacturing of the IgG class of antibodies, but not for the production of complex antibodies like secretory IgAs (SIgAs) and IgMs, which remain unavailable for clinical use. In contrast, plant seeds and leaves hold the promise to produce SIgAs, IgMs and similar complex antibody formats to scalable amounts. Using transient transformation of Nicotiana benthamiana leaves, complex antibody formats can be produced up to milligram amounts in less than a month. Based on these merits, we propose a model for early-phase exploration and designing of innovative antibody formats for therapeutic application. Further in this essay, we elaborate how the model was followed during the selection of novel antibodies for seed-based production. This exploratory model led to the engineering of novel light-chain devoid porcinized-llama antibodies (VHH-Fc) of the IgG (VHH-IgG) and IgA (VHH-IgA) isotypes and also tetravalent dimeric and SIgAs. The proposed strategy may lead to plant-based rapid engineering of innovative antibodies more apt and efficacious for therapy, and in the coarse also add to the understanding of their mode of action.

Automated high-throughput dense matrix protein folding screen using a liquid handling robot combined with microfluidic capillary electrophoresis

Modern molecular genetics technology has made it possible to swiftly sequence, clone and mass-produce recombinant DNA for the purpose of expressing heterologous genes of interest; however, recombinant protein production systems have struggled to keep pace. Mammalian expression systems are typically favored for their ability to produce and secrete proteins in their native state, but bacterial systems benefit from rapid cell line development and robust growth. The primary drawback to prokaryotic expression systems are that recombinant proteins are generally not secreted at high levels or correctly folded, and are often insoluble, necessitating post-expression protein folding to obtain the active product. In order to harness the advantages of prokaryotic expression, high-throughput methods for executing protein folding screens and the subsequent analytics to identify lead conditions are required. Both of these tasks can be accomplished using a Biomek 3000 liquid handling robot to prepare the folding screen and to subsequently prepare the reactions for assessment using Caliper microfluidic capillary electrophoresis. By augmenting a protein folding screen with automation, the primary disadvantage of Escherichia coli expression has been mitigated, namely the labor intensive identification of the required protein folding conditions. Furthermore, a rigorous, quantitative method for identifying optimal protein folding buffer aids in the rapid development of an optimal production process.

Development and characterization of a eukaryotic expression system for human type II procollagen.

BackgroundTriple helical collagens are the most abundant structural protein in vertebrates and are widely used as biomaterials for a variety of applications including drug delivery and cellular and tissue engineering. In these applications, the mechanics of this hierarchically structured protein play a key role, as does its chemical composition. To facilitate investigation into how gene mutations of collagen lead to disease as well as the rational development of tunable mechanical and chemical properties of this full-length protein, production of recombinant expressed protein is required.ResultsHere, we present a human type II procollagen expression system that produces full-length procollagen utilizing a previously characterized human fibrosarcoma cell line for production. The system exploits a non-covalently linked fluorescence readout for gene expression to facilitate screening of cell lines. Biochemical and biophysical characterization of the secreted, purified protein are used to demonstrate the proper formation and function of the protein. Assays to demonstrate fidelity include proteolytic digestion, mass spectrometric sequence and posttranslational composition analysis, circular dichroism spectroscopy, single-molecule stretching with optical tweezers, atomic-force microscopy imaging of fibril assembly, and transmission electron microscopy imaging of self-assembled fibrils.ConclusionsUsing a mammalian expression system, we produced full-length recombinant human type II procollagen. The integrity of the collagen preparation was verified by various structural and degradation assays. This system provides a platform from which to explore new directions in collagen manipulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0228-7) contains supplementary material, which is available to authorized users.

Utilization of Site-Specific Recombination in Biopharmaceutical Production.

Mammalian expression systems, due to their capacity in post-translational modification, are preferred systems for biopharmaceutical protein production. Several recombinant protein systems have been introduced to the market, most of which are under clinical development. In spite of significant improvements such as cell line engineering, introducing novel expression methods, gene silencing and process development, expression level is unpredictable and unstable because of the random location of integration in the genome. Site-specific recombination techniques are capable of producing stable and high producer clonal cells; therefore, they are gaining more importance in the biopharmaceutical production. Site-specific recombination methods increase the recombinant protein production by specifically inserting a vector at a locus with specific expression trait. The present review focused on the latest developments in site-specific recombination techniques, their specific features and comparisons.

Recombinant IgA production for mucosal passive immunization, advancing beyond the hurdles.

Vaccination is a successful strategy to proactively develop immunity to a certain pathogen, but most vaccines fail to trigger a specific immune response at the mucosal surfaces, which are the first port of entry for infectious agents. At the mucosal surfaces, the predominant immunoglobulin is secretory IgA (SIgA) that specifically neutralizes viruses and prevents bacterial colonization. Mucosal passive immunization, i.e. the application of pathogen-specific SIgAs at the mucosae, can be an effective alternative to achieve mucosal protection. However, this approach is not straightforward, mainly because SIgAs are difficult to obtain from convalescent sources, while recombinant SIgA production is challenging due to its complex structure. This review provides an overview of manufacturing difficulties presented by the unique structural diversity of SIgAs, and the innovative solutions being explored for SIgA production in mammalian and plant expression systems.

A Novel Dual Expression Platform for High Throughput Functional Screening of Phage Libraries in Product like Format.

High throughput screenings of single chain Fv (scFv) antibody phage display libraries are currently done as soluble scFvs produced in E.coli. Due to endotoxin contaminations from bacterial cells these preparations cannot be reliably used in mammalian cell based assays. The monovalent nature and lack of Fc in soluble scFvs prevent functional assays that are dependent on target cross linking and/or Fc functions. A convenient approach is to convert scFvs into scFv.Fc fusion proteins and express them in mammalian cell lines for screening. This approach is low throughput and is only taken after primary screening of monovalent scFvs that are expressed in bacteria. There is no platform at present that combines the benefits of both bacterial and mammalian expression system for screening phage library output. We have, therefore, developed a novel dual expression vector, called pSplice, which can be used to express scFv.Fc fusion proteins both in E.coli and mammalian cell lines. The hallmark of the vector is an engineered intron which houses the bacterial promoter and signal peptide for expression and secretion of scFv.Fc in E.coli. When the vector is transfected into a mammalian cell line, the intron is efficiently spliced out resulting in a functional operon for expression and secretion of the scFv.Fc fusion protein into the culture medium. By applying basic knowledge of mammalian introns and splisosome, we designed this vector to enable screening of phage libraries in a product like format. Like IgG, the scFv.Fc fusion protein is bi-valent for the antigen and possesses Fc effector functions. Expression in E.coli maintains the speed of the bacterial expression platform and is used to triage clones based on binding and other assays that are not sensitive to endotoxin. Triaged clones are then expressed in a mammalian cell line without the need for any additional cloning steps. Conditioned media from the mammalian cell line containing the fusion proteins are then used for different types of cell based assays. Thus this system retains the speed of the current screening system for phage libraries and adds additional functionality to it.

The Interface Between Iron Metabolism and Gene-Based Iron Contrast for MRI.

Using a gene-based approach to track cellular and molecular activity with magnetic resonance imaging (MRI) has many advantages. The strong correlation between transverse relaxation rates and total cellular iron content provides a basis for developing sensitive and quantitative detection of MRI reporter gene expression. In addition to biophysical concepts, general features of mammalian iron regulation add valuable context for interpreting molecular MRI predicated on gene-based iron labeling. With particular reference to the potential of magnetotactic bacterial gene expression as a magnetic resonance (MR) contrast agent for mammalian cell tracking, studies in different cell culture models highlight the influence of intrinsic iron regulation on the MRI signal. The interplay between dynamic regulation of mammalian iron metabolism and expression systems designed to sequester iron biominerals for MRI is presented from the perspective of their potential influence on MR image interpretation.

‘Engineered protein scaffolds: have they lived up to expectations?’

Monoclonal antibodies are currently the most successful class of therapeutic agents. However, the conventional immunoglobulin (Ig) format is not always optimally suited to meet clinical demands. First, immunological effector functions mediated by the Fc region can evoke undesired side effects. Second, poor tissue penetration due to the large molecular size hampers successful treatment of solid tumors. Also, the long circulation in blood resulting from both the large size and FcRn-mediated endosomal recycling is unfavorable both for therapies that require flexible adjustment of dosing and for in vivo imaging applications. Finally, due to their complex biomolecular architecture, including four polypeptide chains with around 1500 amino acids and at least two glycosylation sites, the production of full size antibodies is costly and requires mammalian expression systems. As a consequence, during the last two decades more than 50 alternative types of binding proteins have been proposed with the intention to overcome some of the inherent limitations of antibodies. However, only a minority of these ‘alternative scaffolds’ have reached the clinic so far, which can be seen as the ultimate success in pharmaceutical biotechnology. According to recent reviews, ten drug candidates based on seven different protein scaffolds have been tested in clinical trials while one biological received market approval (the engineered Kunitz domain/protease inhibitor ecallantide) [1,2]. At present, the biopharmaceutical development is dominated by the following protein scaffolds: • Affibodies based on the Z-domain of Staphylococcal protein A [3];

DEVELOPMENT OF AUTONOMOUSLY REPLICATING VIRAL RNA TO EXPRESS THE RECOMBINANT HUMAN GRANULOCYTE COLONY-STIMULATING FACTOR

Recombinant human granulocyte colony-stimulating factor (G-CSF) produced in cultured mammalian cells undergoes proper posttranslational modifications and, thereby, possesses better pharmacological properties in comparison with the homologous protein expressed in bacteria. Biopharmaceuticals derived from cell culture tend to be expensive because of lower yields compared to bacterially expressed competitors and numerous issues with the scalability of production. Particular limitation of scalability pertains to delivery of expression vectors to the cell culture. Natural and efficient way to deliver foreign DNA or RNA to cell is a viral infection. We intended to develop the viral genome capable of G-CSF expression.Objectives: To develop autonomously replicating viral RNA capable of heterologous expression of the G-CSF in cultured mammalian cells.Methods: Genetic engineering, cell culture, and virology.Results: Viable genome of the Venezuelan equine encephalomyelitis virus (VEE) was constructed. The G-CSF gene was synthesized de novo. Gene cassette GFP-2A-G-CSF was constructed for simultaneous expression of the fluorescent marker of viral replication (GFP) and the product of interest. Recombinant viral genome VEE/GFP-2A-G-CSF was assembled; its viability was confirmed upon transfection to the BHK-21 cells.Conclusion: Genetic engineering of the cDNA copies of the genomes of RNA viruses provides excessive opportunities to develop mammalian expression systems. We achieved production of recombinant proteins in cultured mammalian cells transfected with the VEE viral RNA carrying the inserted genes of marker protein and G-CSF.

Genetically Encoded Azide Containing Amino Acid in Mammalian Cells Enables Site-Specific Antibody-Drug Conjugates Using Click Cycloaddition Chemistry.

Antibody-drug conjugates (ADC) have emerged as potent antitumor drugs that provide increased efficacy, specificity, and tolerability over chemotherapy for the treatment of cancer. ADCs generated by targeting cysteines and lysines on the antibody have shown efficacy, but these products are heterogeneous, and instability may limit their dosing. Here, a novel technology is described that enables site-specific conjugation of toxins to antibodies using chemistry to produce homogeneous, potent, and highly stable conjugates. We have developed a cell-based mammalian expression system capable of site-specific integration of a non-natural amino acid containing an azide moiety. The azide group enables click cycloaddition chemistry that generates a stable heterocyclic triazole linkage. Antibodies to Her2/neu were expressed to contain N6-((2-azidoethoxy)carbonyl)-l-lysine at four different positions. Each site allowed over 95% conjugation efficacy with the toxins auristatin F or a pyrrolobenzodiazepine (PBD) dimer to generate ADCs with a drug to antibody ratio of >1.9. The ADCs were potent and specific in in vitro cytotoxicity assays. An anti Her2/neu conjugate demonstrated stability in vivo and a PBD containing ADC showed potent efficacy in a mouse tumor xenograph model. This technology was extended to generate fully functional ADCs with four toxins per antibody. The high stability of the azide-alkyne linkage, combined with the site-specific nature of the expression system, provides a means for the generation of ADCs with optimized pharmacokinetic, biological, and biophysical properties.

ID: 71: Ebola and Marburg virus proteins effectively interfere with the activation of TLR and RIG-I signaling pathways

Ebola (EBOV) and Marburg (MARV) filoviruses have caused several outbreaks in Africa. The latest Ebola virus outbreak which started in 2014 in Guinea progressed to a large epidemic which spread to several countries with ca 27 500 cases and 11 200 deaths (by June 2015). Basic knowledge in filovirus – host cell interactions should be increased in order to develop better modalities of treatment for these highly lethal viral pathogens. We have cloned six EBOV (strain from Sierra Leone, June 2014) and six MARV (strain from Uganda, October/November 2012) genes to be expressed in transient mammalian expression system. The effect of Ebola and Marburg virus proteins on Toll-like receptors (TLRs) and RIG-I signaling pathways was analyzed in detail by co-transfecting IFN-lambda1, IFN-beta and IFN-alpha promoter Luc-reporters and the expression constructs for various signaling molecules (MyD88, TRIF, MAVS, IKK-e, TBK1, NF-kB, IRF3, IRF7) and EBOV and MARV genes into primary human leukocytes and various cell lines. At least two EBOV proteins were found to interfere with the activation of RIG-I-mediated innate immune responses. VP35 interfered with the RNA induced activation of the RIG-I. VP24, instead, blocked the whole RIG-I pathway by interfering with IFN gene expression induced by all signaling component. VP24 targeted IRF3, whose nuclear accumulation and transcriptional activity for IFN genes was strongly inhibited. The data provides detailed information on the molecular mechanisms how filoviruses interfere with the activation of host IFN-mediated antiviral responses in human cell systems.

The Polymorphic Variant P24T of UDP-Glucuronosyltransferase 1A4 and Its Unusual Consequences.

The P24T polymorphic variant of the human UDP-glucuronosyltransferase 1A4 (UGT1A4*2, 70C>A) occurs within the signal peptide, five amino acids upstream of the cleavage site and the start of the mature protein. Bioinformatic analysis of the variant suggested that the signal peptide of part of the translated protein is cleaved two residues upstream of the regular site, whereas the rest is cleaved as usual. To test this, recombinant UGT1A4-P24T, with a C-terminal His-tag, was expressed in sf9 insect cells and affinity-purified for N-terminal protein sequencing. The results were in agreement with the in silico prediction. About half of the mutant protein was cleaved at the regular site, between S28 and G29, whereas the other half was cleaved two amino acids upstream, between A26 and E27. The glucuronidation of two substrates, dexmedetomidine and trifluoperazine, was assayed using membrane-enriched UGT1A4-P24T and wild-type UGT1A4. The variant exhibited much lower glucuronidation rates, but kinetic analyses revealed large differences between them only in the Vmax values. The Km values for both substrates were not affected by the mutation and its consequences. This might suggest that the unusual signal peptide cleavage in UGT1A4-P24T somehow disturbs protein folding. Moreover, it raises the possibility that the effect of UGT1A4-P24T on the glucuronidation rate in mammalian expression systems would be mild since they contain more effective post-translation protein control systems in the endoplasmic reticulum. In summary, our results reveal the effect of a polymorphic mutation on the signal sequence cleavage and thereby also the mature UGT.

Supported Lipid Bilayer Technology for the Study of Cellular Interfaces.

Glass-supported lipid bilayers presenting freely diffusing proteins have served as a powerful tool for studying cell-cell interfaces, in particular, T cell-antigen presenting cell (APC) interactions, using optical microscopy. Here we expand upon existing protocols and describe the preparation of liposomes by an extrusion method, and describe how this system can be used to study immune synapse formation by Jurkat cells. We also present a method for forming such lipid bilayers on silica beads for the study of signaling responses by population methods, such as western blotting, flow cytometry, and gene-expression analysis. Finally, we describe how to design and prepare transmembrane-anchored protein-laden liposomes, following expression in suspension CHO (CHOs) cells, a mammalian expression system alternative to insect and bacterial cell lines, which do not produce mammalian glycosylation patterns. Such transmembrane-anchored proteins may have many novel applications in cell biology and immunology.

Abstract 348: Amino acid-based prodrugs of gemcitabine - a therapeutic option to overcome chemoresistance in pancreatic cancer

Abstract Pancreatic cancer is the deadliest of all cancers. Currently, gemcitabine is the only drug of choice to treat this cancer but the results are modest as the disease itself is diagnosed at an advanced stage and often times develops resistance to gemcitabine via silencing of the influx transporters for the drug. We hypothesized that the delivery of gemcitabine into pancreatic cancer cells in a different form via some other transporters would provide a logical alternative approach. It would even be better if we can exploit for this purpose the transporters that are selectively upregulated in pancreatic cancer. Several nucleoside drugs are transportable substrates for the amino acid transporter SLC6A14 if used in the form of amino acid-based prodrugs. Such strategy would work for gemcitabine if SLC6A14 is upregulated in pancreatic cancer. With this in mind, we analyzed publically available microarray datasets for the amino acid transporters that are upregulated in pancreatic cancer. We found four transporters (SLC1A5, SLC7A5, SLC7A11, and SLC6A14) overexpressed in tumors compared to normal tissue. The fold-increase in expression is the highest for SLC6A14 in all datasets (range, 1.3 - 163.3; p, 0.05 - 2×10−14). The fold-increase for the other three transporters is 2 at the maximum. We confirmed these findings using primary pancreatic cancer tissues and cancer cell lines. First, we used pancreatic cancer tissues and compared with the normal tissues. SLC6A14 mRNA is increased at least by 7-fold in tumors versus normal tissue. We then evaluated the expression of SLC6A14 mRNA and protein in normal and pancreatic cancer cell lines. The expression is higher in all cancer cell lines than in a normal cell line. This was further confirmed with a tissue microarray containing pancreatic tumor tissues from 52 patients. The delivery of gemcitabine in the form of amino acid-based prodrugs via SLC6A14, an exceptionally energy-coupled transporter, as a means to overcome gemcitabine resistance is a novel idea that has never been tested. As a proof of principle, we have shown that SLC6A14 can transport several nucleoside drugs (acyclovir, ganciclovir, and cytarabine) in the form of amino acid-based prodrugs. The parent nucleosides are not substrates for the transporter. To specifically assess the interaction of amino acid-based prodrugs of gemcitabine with SLC6A14, we synthesized the valyl ester of gemcitabine and investigated its transport via SLC6A14 using two different heterologous expression systems: a mammalian cell expression system and the Xenopus oocyte expression system. In both experimental approaches, we found that the valyl ester of gemcitabine, but not the parent drug, is a transportable substrate for SLC6A14. The transport process is Na/Cl-coupled and electrogenic. We conclude that amino acid-based prodrugs of gemcitabine represent a viable alternative to overcome resistance to gemcitabine in the treatment of pancreatic cancer. Citation Format: Yangzom D. Bhutia, Ellappan Babu, Pankaj K. Singh, Vadivel Ganapathy. Amino acid-based prodrugs of gemcitabine - a therapeutic option to overcome chemoresistance in pancreatic cancer. [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 348. doi:10.1158/1538-7445.AM2015-348

Abstract 1566: Association of a serum HPV16 IgG signature and risk of oropharyngeal cancer

Abstract Introduction: Human papillomavirus type 16 (HPV16) infection is associated with an increasing fraction of oropharyngeal cancer. The aim of our study was to determine the association of HPV16 antibodies (Abs) and oropharyngeal cancer (OPC) risk in sera obtained prior to clinical diagnosis. Methods: We identified 92 participants with incident oropharyngeal cancers and 460 age-, gender-, region- and year- matched controls from the Janus Serumbank in Norway. Serum samples were collected from cases, on average, 9.2 years before diagnosis (range, -0.6-14.9 years). 11 cases had serum samples from multiple time points. IgG Abs to the HPV16 antigens E1, E4, E5, E6, E7, L1, L2, and the N-terminal and C-terminal fragments of E2 (NE2, CE2) were quantified in blinded fashion using a custom RAPID ELISA assay. Full-length genes were expressed as GST fusion proteins with a mammalian protein expression system, incubated with sera, and bound IgG was measured. The ratio of relative luminescence units (RLU) values for each antigen to control GST protein was determined. Cutoff values were pre-established as 3 SD>median of 247 healthy controls. A logistic regression classifier of a 7-Ab biomarker panel (E1, NE2, CE2, E4, E5, E6, E7) for the predictive diagnosis of OPC was trained on an independent sample set, and applied to these samples. P-values were calculated by Fisher's exact test. Odds ratios (ORs) of cancer and 95% CIs were calculated. Results: HPV16 early antigen seropositivity was present in 13% of patients with OPC and 1% of controls (OR, 17.0; 95% CI, 5.0-74.0, p<0.0001). The probability of seropositivity was inversely associated with the time from serum collection and diagnosis (0-2 years, 84.9; >10 years, 5.4). Abs to CE2 were strongly associated with cases 0-2 years pre-diagnosis (OR, 112.0; 95% CI, 15.0-971.2, p<0.0001). Conclusion: HPV16 Abs to multiple early viral antigens are detectable years prior to diagnosis of oropharyngeal cancer, and the probability of seropositivity increases closer to diagnosis. A model for screening would help identify individuals at risk for whom interventions and surveillance may be evaluated. Citation Format: Karen S. Anderson, Hilde Langseth, Garrick Wallstrom, Marshall Posner, Julia N. Cheng, Rizwan Alam, Diego Chowell, Jon Mork. Association of a serum HPV16 IgG signature and risk of oropharyngeal cancer. [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 1566. doi:10.1158/1538-7445.AM2015-1566

Abstract 643: Identification of novel pancreatic cancer-specific antibodies and their target antigens through a next generation immune sequencing platform

Abstract One of the major challenges in developing new therapies for pancreatic cancer is the lack of known specific and sensitive targets to the cancer cells. In this study, we present a powerful way to identify novel specific targets to pancreatic cancer by leveraging the antibody responses of tumor infiltrating lymphocytes (TILs) using a novel next-generation immune sequencing approach. We first deeply characterize the antibody repertoire produced by TILs, followed by antibody expression, screening and functional characterization to identify their biological targets. We aim to manipulate the antibody cancer targeting specificity into potential therapeutics by utilizing antibody-drug conjugation (ADC) technology. We obtained tumor resections and matched normal adjacent tissue from patients diagnosed with pancreatic ductal adenocarcinoma or acinar cell carcinoma, and characterized each sample's TIL immune repertoire using AbVitro's proprietary Next-Generation Immune Sequencing platform. Interestingly, results indicated the presence of millions of B and T cells in both tumor and normal tissues. Strikingly, tumor tissues across all patients were characterized by an abundance of expanded B-cell lineages expressing IgG, whereas normal adjacent tissue and healthy control samples contained almost exclusively IgA expressing cells. Antibody candidates were selected from the sequencing data and heavy and light chains were synthesized and expressed in a mammalian cell-based expression system. The antibodies were then screened for cell surface binding, tissue specificity and cell killing potential against pancreatic cancer cell lines and FFPE human cancer tissues. Selected antibodies from these screens were then chosen as candidates for antigen identification. Our study uncovered multiple antibodies that specifically bind to pancreatic cancer tissues but not to healthy tissues. Interestingly, some of the antibodies also showed strong binding to other types of cancer such as lung squamous cell carcinoma. Initial target identification efforts for these antibodies yielded a short list of antigens which are known to be expressed at high levels in the pancreas or pancreatic cancer cell lines. We are currently performing studies to further understand the antigen specificity and sensitivity of these antibodies and evaluate their cell killing potential as ADCs, as well as investigating the diagnostic value of the novel antigen targets. In summary, our study demonstrates the potential of next-generation sequencing in TIL analysis for the discovery of novel cancer-specific targets of potential therapeutic value. Citation Format: David A. Fabrizio, Sonia Timberlake, Brian Belmont, Stephen J. Goldfless, Adrian W. Briggs, Teresa J. Broering, Francois Vigneault. Identification of novel pancreatic cancer-specific antibodies and their target antigens through a next generation immune sequencing platform. [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 643. doi:10.1158/1538-7445.AM2015-643