Phage Display Antibody Library Research Articles

Isolation of single-chain variable fragment (scFv) antibodies for detection of Chickpea chlorotic dwarf virus (CpCDV) by phage display.

Chickpea chlorotic dwarf virus (CpCDV, genus Mastrevirus), has a wide host range and geographic distribution in many parts of the world, and it is one of the most important legume-infecting viruses. Detection of CpCDV-infected plants in the field and evaluation of viral resistance of plant cultivars are possible by conducting serological assays. Here, development and characterization of a specific recombinant monoclonal antibody for CpCDV as a diagnostic tool are described. For this purpose, the coat protein of CpCDV was expressed in Escherichia coli strain Rosetta (DE3) and used to screen a Tomlinson phage display antibody library to select a specific single-chain variable fragment (scFv). In each round of biopanning, the affinity of the phage for CpCDV-CP was tested by enzyme-linked immunosorbent assay (ELISA). The results showed that the specificity of the eluted phages increased after each round of panning. Testing of individual clones by ELISA showed that five clones of the monoclonal phage were more strongly reactive against CpCDV than the other clones. All selected positive clones contained the same sequence. The phage-displayed scFv antibody, which was named CpCDV-scFvB9, did not bind to other tested plant pathogens and showed high sensitivity in the detection of CpCDV. A Western blot assay demonstrated that CpCDV-scFvB9 reacted with the recombinant coat protein of CpCDV. Finally, the interaction CpCDV-scFvB9 and CpCDV-CP was analyzed in a molecular docking experiment. This is the first report on production of an scFv antibody against CpCDV, which could be useful for immunological detection of the virus.

Abstract 5653: Discovery of Anti-NKG2A antibody from immunized phage display approach

Abstract The inhibitory receptor NKG2A-CD94 is mainly expressed on NK cells and some T cell and mediates an inhibitory signal through association with a non-classical MHC class I molecule HLA-E, which is commonly over-expressed in human cancers. Targeting NKG2A with monoclonal antibody holds potentials in immunotherapy of cancer. However, proteins with homologous sequences such as NKG2C/CD94 or NKG2E/CD94 complexes generally deliver an activating signal upon ligand binding. NKG2A-specific antagonizing antibodies would be needed to further explore therapeutic potentials of targeting NKG2A for immunotherapy of cancer. We have developed and characterized panels of monoclonal antibodies (mAbs) that specifically recognize NKG2A. Balb/c and SJL mice had been immunized with recombinant NKG2A/CD94 protein, recombinant plasmids encoding hNKG2A or stable cell line with ectopic expression of hNKG2A. Splenocytes from such immunized animals were collected for construction of phage-displayed antibody library in a single chain variable fragment (scFv) format. The NKG2A-specific antibodies, that had no detectable binding to NKG2C, NKG2E or CD94, were isolated through multiple rounds of phage panning and ELISA-based screening. Such antibodies also block HLA-E ligand binding and enhanced cytotoxicity of NK cell lines and primary NK cells on cancer cells. The final lead is a humanized antibody which behaves well in stability test. Lab scale processes are successfully developed and confirmed. The safety and efficacy will be further validated. Citation Format: Teddy Yang, Jing Gao, Jingyun Yao, Dongxu Wang, Ken Dai. Discovery of Anti-NKG2A antibody from immunized phage display approach [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5653.

A panel of anti-influenza virus nucleoprotein antibodies selected from phage-displayed synthetic antibody libraries with rapid diagnostic capability to distinguish diverse influenza virus subtypes

Immunoassays based on sandwich immuno-complexes of capture and detection antibodies simultaneously binding to the target analytes have been powerful technologies in molecular analyses. Recent developments in single molecule detection technologies enable the detection limit of the sandwich immunoassays approaching femtomolar (10–15 M), driving the needs of developing sensitive and specific antibodies for ever-increasingly broad applications in detecting and quantifying biomarkers. The key components underlying the sandwich immunoassays are antibody-based affinity reagents, for which the conventional sources are mono- or poly-clonal antibodies from immunized animals. The downsides of the animal-based antibodies as affinity reagents arise from the requirement of months of development timespan and limited choices of antibody candidates due to immunodominance of humoral immune responses in animals. Hence, developing animal antibodies capable of distinguishing highly related antigens could be challenging. To overcome the limitation imposed by the animal immune systems, we developed an in vitro methodology based on phage-displayed synthetic antibody libraries for diverse antibodies as affinity reagents against closely related influenza virus nucleoprotein (NP) subtypes, aiming to differentiating avian influenza virus (H5N1) from seasonal influenza viruses (H1N1 and H3N2), for which the NPs are closely related by 90–94% in terms of pairwise amino acid sequence identity. We applied the methodology to attain, within four weeks, a panel of IgGs with distinguishable specificities against a group of representative NPs with pairwise amino acid sequence identities up to more than 90%, and the antibodies derived from the antibody libraries without further affinity refinement had comparable affinity of mouse antibodies to the NPs with the detection limit less than 1 nM of viral NP from lysed virus with sandwich ELISA. The panel of IgGs were capable of rapidly distinguishing infections due to virulent avian influenza virus from infections of seasonal flu, in responding to a probable emergency scenario where avian influenza virus would be transmissible among humans overlapping with the seasonal influenza infections. The results indicate that the in vitro antibody development methodology enables developing diagnostic antibodies that would not otherwise be available from animal-based antibody technologies.

Identification of Human Single-Domain Antibodies against SARS-CoV-2

SummaryThe worldwide spread of COVID-19 highlights the need for an efficient approach to rapidly develop therapeutics and prophylactics against SARS-CoV-2. The SARS-CoV-2 spike protein, containing the receptor-binding domain (RBD) and S1 subunit involved in receptor engagement, is a potential therapeutic target. We describe the development of a phage-displayed single-domain antibody library by grafting naive complementarity-determining regions (CDRs) into framework regions of a human germline immunoglobulin heavy chain variable region (IGHV) allele. Panning this library against SARS-CoV-2 RBD and S1 subunit identified fully human single-domain antibodies targeting five distinct epitopes on SARS-CoV-2 RBD with subnanomolar to low nanomolar affinities. Some of these antibodies neutralize SARS-CoV-2 by targeting a cryptic epitope located in the spike trimeric interface. Collectively, this work presents a versatile platform for rapid antibody isolation and identifies promising therapeutic anti-SARS-CoV-2 antibodies as well as the diverse immogneic profile of the spike protein.

Isolation of a human monoclonal antibody specific for the receptor binding domain of SARS-CoV-2 using a competitive phage biopanning strategy.

The infection of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused more than 200 000 deaths, but no vaccine or therapeutic monoclonal antibody is currently available. SARS-CoV-2 relies on its spike protein, in particular the receptor-binding domain (RBD), to bind human cell receptor angiotensin-converting enzyme 2 (ACE2) for viral entry, and thus targeting RBD holds the promise for preventing SARS-CoV-2 infection. In this work, a competitive biopanning strategy of a phage display antibody library was applied to screen blocking antibodies against RBD. High-affinity antibodies were enriched after the first round using a standard panning process in which RBD-His was immobilized as a bait. At the next two rounds, immobilized ACE2-Fc and free RBD-His were mixed with the enriched phage antibodies. Antibodies binding to RBD at epitopes different from ACE2-binding site were captured by the immobilized ACE2-Fc, forming a “sandwich” complex. Only antibodies competed with ACE2 can bind to the free RBD-His in the supernatant and be subsequently separated by the nickel-nitrilotriacetic acid magnetic beads. rRBD-15 from the competitive biopanning of our synthetic antibody library, Lib AB1, was produced as the full-length IgG1 format. It was proved to competitively block the binding of RBD to ACE2 and potently inhibit SARS-CoV-2 pseudovirus infection with IC50 values of 12 nM. Nevertheless, rRBD-16 from the standard biopanning can only bind to RBD in vitro, but not have the blocking or neutralization activity. Our strategy can efficiently isolate the blocking antibodies of RBD, and it would speed up the discovery of neutralizing antibodies against SARS-CoV-2.

Selection, preparation and characterization of scFv against human lipocalin 6 by phage display technology

Human lipocalin 6 (hLCN6) is a newly discovered epididymal-specific secreted protein, capable of binding to the head and tail of spermatozoa and involved in sperm maturation. Anti-hLCN6 monoclonal antibody coupled immunomagnetic beads (IMBs) can be effectively used for the separation and forensic identification of sperm cells from mixed stains. But the source of monoclonal antibody is limited. In this study, an immunized mouse phage display antibody library was constructed and the single-chain variable fragments (scFvs) against hLCN6 were screened. The selection was performed using four rounds of biopanning and positive clones were validated by phage ELISA. Two anti-hLCN6 scFv clones with highest affinity were selected and sequencing result showed that the two sequences were identical. After prokaryotic expression and purification, the purified scFv could specifically recognize the hLCN6 in the lysate of human sperm cells and epididymis by western blot analysis, without any cross-reactivity with cellular antigens in female epithelial cells. The dissociation constant (Kd) of anti-hLCN6 scFv was 6.69 × 10−7 mol/L measured by indirect ELISA. Therefore, our work not only provides a useful tool for further exploration of the biological functions of hLCN6, but also opens up new research avenues for the separation of sperm cells from mixed stains based on immuno-binding reaction.

Phage Display Libraries for Antibody Therapeutic Discovery and Development.

Phage display technology has played a key role in the remarkable progress of discovering and optimizing antibodies for diverse applications, particularly antibody-based drugs. This technology was initially developed by George Smith in the mid-1980s and applied by John McCafferty and Gregory Winter to antibody engineering at the beginning of 1990s. Here, we compare nine phage display antibody libraries published in the last decade, which represent the state of the art in the discovery and development of therapeutic antibodies using phage display. We first discuss the quality of the libraries and the diverse types of antibody repertoires used as substrates to build the libraries, i.e., naïve, synthetic, and semisynthetic. Second, we review the performance of the libraries in terms of the number of positive clones per panning, hit rate, affinity, and developability of the selected antibodies. Finally, we highlight current opportunities and challenges pertaining to phage display platforms and related display technologies.

Generation of a single-chain variable fragment phage display antibody library from naïve mice panned against Fasciola hepatica antigens

Monoclonal antibodies have a wide range of applications in basic and applied research as well as in the medical and pharmaceutical industries. Phage display antibody libraries offer an alternative to hybridoma technology for the generation of monoclonal antibodies and can be applied to high-throughput screening and facilitate the generation of novel antibodies. Despite their utility in several fields of research there has been limited application of antibody libraries in the study of trematode parasites.Fasciola hepatica causes considerable loss to the agriculture sector and is also a human pathogen. The parasite's excretory/secretory material contains numerous molecules that facilitate its invasion and survival within the mammalian host, including cathepsin B and L proteases. F. hepatica cathepsin B2 is expressed during the initial weeks of infection and has suspected roles in immune evasion and as a digestive enzyme in the parasite's gut; it is considered a good target for vaccination or therapeutic inhibitors.In this study, we produced a single-chain variable fragment (scFv) phage display library from naïve mice. The library was used to identify several scFv that can bind to antigens from adult F. hepatica homogenate, and a scFv that can bind to F. hepatica cathepsin B2. The results highlight the potential applicability of such a library to facilitate the study of F. hepatica and other parasites. This is the first report of the application of a naïve phage display antibody library to the study of F. hepatica.

A novel murine antibody and an open sandwich immunoassay for the detection of clenbuterol

Clenbuterol (CLEN) is a sympathomimetic amine used as a decongestant and bronchodilator while treating breathing disorders. It is also used in food-producing animals as it improves the rate of red meat production. However, it is prohibited in many countries nowadays due to human health and safety concerns. Unfortunately, the illegal use of CLEN is still rampant. Thus, monitoring it in food and livestock is important. Here, we report a novel murine antibody and an open sandwich enzyme linked immunosorbent assay (OS-ELISA) to detect CLEN based on antigen-antibody reactions. The genes of antibody variable regions in mice immunized with CLEN conjugated with bovine serum albumin were cloned into a phagemid (pDong1/Fab) to construct a phage-display antibody library, from which a novel antibody, A12, was selected. Then, an OS-ELISA was developed to detect CLEN using separated variable regions of the A12 antibody. The limit of detection of the assay was found to be 8 ng/mL, which was useful for monitoring CLEN usage.

Lesson from a Fab-enabled co-crystallization study of TDRD2 and PIWIL1.

The interaction of Tudor domain-containing proteins (TDRDs) with P-element-induced wimpy testis (PIWI) proteins plays critical roles in transposon silencing and spermatogenesis. Most human TDRDs recognize PIWI proteins in a methylarginine-dependent manner via their extended Tudor (eTudor) domains, except TDRD2, which prefers an unmethylated PIWI protein. In order to illustrate the recognition of unmethylated PIWI proteins by TDRD2, we extensively tried co-crystallization of the TDRD2 eTudor with different PIWIL1 peptides, but to no avail. Recombinant antigen-binding fragments (Fabs) have been used to crystallize some difficult proteins in the past, so we generated Fab against the TDRD2 eTudor protein using a phage-display antibody library, and one of these Fab fragments indeed facilitated the co-crystallization of TDRD2 and PIWIL1. Structural analysis of Fab, the TDRD2 eTudor domain in complex with an unmethylated PIWIL1-derived peptide revealed that the PIWIL1 residues G3 through R8 bound between the Tudor core and SN domain of TDRD2. The C-terminal residues of the PIWIL1 peptide were not resolved, presumably due to steric competition with the heavy chain of the Fab. We propose Fab-assisted crystallization as a tool not only for structural studies of single proteins, but also for analysis of interactions between proteins and their ligands in cases where co-crystallization of native protein complexes fails.

Abstract 2387: NKG2A-antagonizing monoclonal antibodies identified through phage display

Abstract The inhibitory receptor NKG2A-CD94 is mainly expressed on NK cells and some T cell and mediates an inhibitory signal through association with a non-classical MHC class I molecule HLA-E, which is commonly over-expressed in human cancers. Targeting NKG2A with monoclonal antibody holds potentials in immunotherapy of cancer. However, proteins with homologous sequences such as NKG2C/CD94 or NKG2E/CD94 complexes generally deliver an activating signal upon ligand binding. NKG2A-specific antagonizing antibodies would be needed to further explore therapeutic potentials of targeting NKG2A for immunotherapy of cancer. We have developed and characterized panels of monoclonal antibodies (mAbs) that specifically recognize NKG2A. Balb/c or SJL mice had been immunized with recombinant NKG2A/CD94 protein, recombinant plasmids encoding hNKG2A or stable cell line with ectopic expression of hNKG2A. Splenocytes from such immunized animals were collected for construction of phage-displayed antibody library in a single chain variable fragment (scFv) format. The NKG2A-specific antibodies, that had no binding to NKG2C, NKG2E or CD94, were isolated through multiple rounds of phage panning and ELISA-based screening. Such antibodies also block HLA-E ligand binding and enhanced cytotoxicity of NK cell lines and primary NK cells on cancer cells. In summary, isolation and characterization of a panel of NKG2A-specific antagonizing antibodies enable us to further determine the feasibility of NKG2A-targeted immunotherapy of cancer. Citation Format: Jing Gao, Lili Hu, Ruirui Sui, Cuicui Guo, Lina Wang, Teddy Yang, Jack Han, Qing Duan, Louis Liu. NKG2A-antagonizing monoclonal antibodies identified through phage display [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 2387.

Cognizance of Molecular Methods for the Generation of Mutagenic Phage Display Antibody Libraries for Affinity Maturation.

Antibodies leverage on their unique architecture to bind with an array of antigens. The strength of interaction has a direct relation to the affinity of the antibodies towards the antigen. In vivo affinity maturation is performed through multiple rounds of somatic hypermutation and selection in the germinal centre. This unique process involves intricate sequence rearrangements at the gene level via molecular mechanisms. The emergence of in vitro display technologies, mainly phage display and recombinant DNA technology, has helped revolutionize the way antibody improvements are being carried out in the laboratory. The adaptation of molecular approaches in vitro to replicate the in vivo processes has allowed for improvements in the way recombinant antibodies are designed and tuned. Combinatorial libraries, consisting of a myriad of possible antibodies, are capable of replicating the diversity of the natural human antibody repertoire. The isolation of target-specific antibodies with specific affinity characteristics can also be accomplished through modification of stringent protocols. Despite the ability to screen and select for high-affinity binders, some ‘fine tuning’ may be required to enhance antibody binding in terms of its affinity. This review will provide a brief account of phage display technology used for antibody generation followed by a summary of different combinatorial library characteristics. The review will focus on available strategies, which include molecular approaches, next generation sequencing, and in silico approaches used for antibody affinity maturation in both therapeutic and diagnostic applications.

Construction of a novel phage display antibody library against Fasciola hepatica, and generation of a single-chain variable fragment specific for F. hepatica cathepsin L1

Phage display technology to produce recombinant monoclonal antibodies or antibody fragments permits the identification of sought after antibodies in short time frames at low cost along with direct and rapid selection for antibody characteristics. Monoclonal antibodies can facilitate the identification and characterisation of parasite molecules that function at the host-parasite interface to help understand at the molecular level the biology of the parasite and disease progression, which often leads to new drug targets, diagnostic antigens or vaccine candidates.The trematode Fasciola hepatica is an important veterinary and human parasite. In this work, we infected rats with F. hepatica and amplified the generated antibody repertoire to produce a single-chain variable fragment (scFv) phage display library. The library was used to identify a scFv that recognises cathepsin L1, a major component of the adult parasites excretory/secretory material and an important vaccine candidate.This is the first report of the construction of a phage display antibody library from a F. hepatica infected host, and also the first instance such a library has been used to identify an affinity-matured monoclonal antibody fragment that binds to a F. hepatica antigen. The scFv library and methods detailed should facilitate future research characterising F. hepatica antigens.

An anti-Gn glycoprotein antibody from a convalescent patient potently inhibits the infection of severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease localized to China, Japan, and Korea that is characterized by severe hemorrhage and a high fatality rate. Currently, no specific vaccine or treatment has been approved for this disease. To develop a therapeutic agent for SFTS, we isolated antibodies from a phage-displayed antibody library that was constructed from a patient who recovered from SFTS virus (SFTSV) infection. One antibody, designated as Ab10, was reactive to the Gn envelope glycoprotein of SFTSV and protected host cells and A129 mice from infection in both in vitro and in vivo experiments. Notably, Ab10 protected 80% of mice, even when injected 5 days after inoculation with a lethal dose of SFTSV. Using cross-linker assisted mass spectrometry and alanine scanning, we located the non-linear epitope of Ab10 on the Gn glycoprotein domain II and an unstructured stem region, suggesting that Ab10 may inhibit a conformational alteration that is critical for cell membrane fusion between the virus and host cell. Ab10 reacted to recombinant Gn glycoprotein in Gangwon/Korea/2012, HB28, and SD4 strains. Additionally, based on its epitope, we predict that Ab10 binds the Gn glycoprotein in 247 of 272 SFTSV isolates previously reported. Together, these data suggest that Ab10 has potential to be developed into a therapeutic agent that could protect against more than 90% of reported SFTSV isolates.

Recombinant Antibody Selections by Combining Phage and Yeast Display.

In vitro display technologies have put together the generation of large antibody libraries with selection and screening procedures to identify lead candidates. Phage display antibody libraries allow selecting and identifying binders for a variety of antigens. Nonetheless, the procedure is limited by the possibility to quantitatively follow the enrichment during selection cycles and tune up the clones for specific binding proprieties (i.e., affinity). Yeast display allows the expression of thousands of copies of the antibody on each cell, simultaneously carrying the plasmid encoding that antibody, moreover the selection parameters can be accurately controlled by flow cytometry-based analysis and sorting.The combination of phage and yeast display takes advantage of both platforms by starting with a vast number of antibodies in the phage display selections followed by the precise sorting of the clones specifically recognizing the target of interest.In the present chapter, we illustrate protocols to generate and enrich - usingfluorescence-activated cell sorting (FACS) -yeast display antibody libraries, using selection outputs obtained from phage antibody display libraries as starting material. The present methods can be easily applicable for the identification of monoclonal antibodies with desired binding properties.

Construction of an immunized rabbit phage display antibody library for screening microcystin-LR high sensitive single-chain antibody

Microcystin-LR (MC-LR) is one of the most common biotoxin that pollutes water and agricultural products. The study aims to obtain the high sensitive anti-MC-LR single-chain antibody (scFv) for detecting MC-LR. Here, a MC-LR-immunized rabbit phage display scFv library with its capacity of 3.26×109CFU/mL was constructed and used for anti-MC-LR phage scFv particles screening. After four rounds of biopanning, 18 positives were isolated and identified successfully. The most positive scFv (RscFv3) was expressed in Escherichia coli HB2151 and purified with a concentration of 796.7μg/mL, and the purified anti-MC-LR polyclonal antibodies (PAbs) were 3.6mg/mL. The PAbs and scFv based indirect competitive enzyme linked immunosorbent assay (IC-ELISAs) were established for MC-LR and its analogues, and the results showed they all had high sensitive for MC-LR with detection limits of 0.03 and 0.05μg/L, and had strong cross-reactivity for MC-RR, MC-WR and MC-YR, respectively. The average recovery rate was 91.9% with coefficient of variation <6.8% for scFv-based IC-ELISA to detect MC-LR spiked in water samples, which met the requirement of indoor testing. The present results indicate that we have obtained a high sensitive anti-MC-LR scFv by the MC-LR-immunized phage library construction and screening, and the scFv-based IC-ELISA can be used for monitoring MC-LR in water samples.

Construction and next-generation sequencing analysis of a large phage-displayed VNAR single-domain antibody library from six naïve nurse sharks.

ABSTRACTBackgroundShark new antigen receptor variable domain (VNAR) antibodies can bind restricted epitopes that may be inaccessible to conventional antibodies.MethodsHere, we developed a library construction method based on polymerase chain reaction (PCR)-Extension Assembly and Self-Ligation (named “EASeL”) to construct a large VNAR antibody library with a size of 1.2 × 1010 from six naïve adult nurse sharks (Ginglymostoma cirratum).ResultsThe next-generation sequencing analysis of 1.19 million full-length VNARs revealed that this library is highly diversified because it covers all four classical VNAR types (Types I–IV) including 11% of classical Type I and 57% of classical Type II. About 30% of the total VNARs could not be categorized as any of the classical types. The high variability of complementarity determining region (CDR) 3 length and cysteine numbers are important for the diversity of VNARs. To validate the use of the shark VNAR library for antibody discovery, we isolated a panel of VNAR phage binders to cancer therapy-related antigens, including glypican-3, human epidermal growth factor receptor 2 (HER2), and programmed cell death-1 (PD1). Additionally, we identified binders to viral antigens that included the Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS) spike proteins. The isolated shark single-domain antibodies including Type I and Type II VNARs were produced in Escherichia coli and validated for their antigen binding. A Type II VNAR (PE38-B6) has a high affinity (Kd = 10.1 nM) for its antigen.ConclusionsThe naïve nurse shark VNAR library is a useful source for isolating single-domain antibodies to a wide range of antigens. The EASeL method may be applicable to the construction of other large diversity gene expression libraries.

High-throughput reformatting of phage-displayed antibody fragments to IgGs by one-step emulsion PCR.

Single-chain variable fragment (scFv) is the most common format for phage display antibody library. The isolated scFvs need to be reformatted to full-length IgGs for further characterization. High throughput reformatting of scFv to IgG without disrupting VH-VL pairing is of great demanding for exhaustive screening of all antibodies in IgG format. Herein, we developed a strategy based on the overlap extension PCR in emulsion to reformat scFv to IgG while maintain the accuracy and complexity of variable region pairing. Using CD40 as an example target, we reformatted phage display derived CD40 binding scFv library to IgG mammalian display library and isolated high affinity CD40 binding IgGs. This robust and reliable antibody reformatting approach could be integrated into any phage display based antibody drug discovery.

Abstract 830: ISU104, a fully human antibody targeting a specific epitope on the ErbB3, displays potent inhibition of tumor growth in multiple xenograft tumor models

Abstract Members of the epidermal growth factor receptor family (ErbB family) are known as potent mediators in the development and progression of cancer. Activated ErbBs recruit various adaptors and signaling molecules through the phosphorylated cytoplasmic domain, which further leads to activation of downstream oncogenic signaling pathways. There are approved therapeutics for ErbB1 (EGFR) and ErbB2 (HER2) in the treatment of human cancers, while monoclonal antibodies targeting ErbB3 are just undergoing clinical trials. ISU104 is a fully human anti-ErbB3 antibody isolated from phage display antibody library. We performed the hydrogen/deuterium exchange mass spectrometry (HDX-MS) analysis with ErbB3 extracellular domain and ISU104, and that indicated that ISU104 mainly binds with domain 3 and weakly interacts with domain 1 of ErbB3. The binding property of ISU104 induced dose-dependent inhibition of ligand (heregulin, HRG) binding, blocking of dimerization of ErbB3 with other ErbBs and subsequently inactivated the downstream signaling of ErbB3. Also, ISU104 occasioned internalization of ErbB3 from plasma membrane, and downregulated the expression level of ErbB3. We demonstrated the biologic effect of ISU104 in several ErbB3-expressing cancer cell lines, including head and neck squamous cell carcinoma (HNSCC) and breast cancers. ISU104 completely suppressed the HRG-induced ErbB3/AKT phosphorylation, reduced cell proliferation and survival. Next, we evaluated efficacy of ISU104 in multiple xenograft models. Mice were treated with 10 mg/kg of ISU104 twice weekly. ISU104 regressed tumor growth in FaDu HNSCC model, and showed more than 70% tumor growth inhibition (TGI) in CAL27 (HNSCC), BxPC3 (pancreatic cancer), MDA-MB-468 (breast cancer), A549 (skin cancer), and BT474 (breast cancer) models. Our results suggest that ISU104 effectively blocks activation of ErbB3 and the downstream pathway by ErbB3, and may provide clinical benefit to ErbB3-activated patients. Citation Format: Miyoung Kim, Youngmi Hur, Sohyeon Seo, Heynjeong Lim, Kyungyong Kim, Youngsoo Sohn, Seung-Beom Hong, Donggoo Bae. ISU104, a fully human antibody targeting a specific epitope on the ErbB3, displays potent inhibition of tumor growth in multiple xenograft tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 830.

Abstract 1763: BiTEs vs CAR-Ts: Preclinical targeting of CD133+ brain tumor initiating cells using immunotherapy-based treatment strategies

Abstract Glioblastoma (GBM) is a uniformly fatal primary brain tumor, characterized by extensive cellular heterogeneity. Numerous studies have implicated CD133+ brain tumor initiating cells (BTICs) as drivers of chemo- and radio-resistance in GBM. We have recently demonstrated that a CD133-driven gene signature is predictive of poor overall survival and targeting CD133+ treatment-refractory cells may be an effective strategy to block GBM recurrence. Bispecific T-Cell engaging antibodies (BiTEs) and Chimeric antigen receptors (CARs) present promising immunotherapeutic approaches that have not yet been validated for recurrent GBM. Using CellectSeq, a novel methodology that combines the use of phage-displayed synthetic antibody libraries and DNA sequencing, we developed the CD133-specific monoclonal antibody ‘RW03'. We constructed CD133-specific BiTEs that consist of two arms; one recognizes the tumor antigen (CD133) and the second is specific to the CD3 antigen that binds to human GBMs and PBMC-derived T cells, respectively. We observed BiTEs redirecting T cells to kill GBMs, with greater efficiency observed in CD133high GBMs, validating BiTE target specificity. Incubating T-cells with BiTEs and the CD133high GBMs resulted in increased expression of T cell activation markers. In parallel, we derived the single chain variable fragment (scFv) from RW03 we engineered a second-generation CAR T cell. CD133-specific CAR-T cells were cytotoxic to CD133+ GBMs. Co-culturing CD133 CAR-T cells with GBMs triggered T cell activation and proliferation. Treatment of GBM tumor-bearing mice with CD133-specific CAR-T cells yielded extended survival in mice and significant reductions in brain tumor burden. Furthermore, we uniquely adapted the existing chemoradiotherapy protocol for GBM patients for treatment of immunocompromised mice engrafted with human GBMs. Within this model, we have initiated treatment of recurrent GBM directed against CD133+ BTICs, to allow for a direct prospective comparison of toxicity and efficacy of BiTEs and CAR-T cell strategies. Citation Format: Parvez Vora, Jarrett Adams, Mohini Singh, Chitra Venugopal, Nazanin Tatari, Chirayu Chokshi, Maleeha Qazi, Sabra Salim, Sujeivan Mahendram, David Bakhshinyan, Max London, Neil Savage, Minomi Subapanditha, Nicole McFarlane, James Pan, Jonathan Bramson, Sachdev Sidhu, Jason Moffat, Sheila Singh. BiTEs vs CAR-Ts: Preclinical targeting of CD133+ brain tumor initiating cells using immunotherapy-based treatment strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1763.