Human Single-chain Antibody Research Articles

Human monoclonal ScFv specific to NS1 protein inhibits replication of influenza viruses across types and subtypes

Currently, there is a need of new anti-influenza agents that target influenza virus proteins other than ion channel M2 and neuraminidase. Non-structural protein-1 (NS1) is a highly conserved multifunctional protein which is indispensable for the virus replication cycle. In this study, fully human single chain antibody fragments (HuScFv) that bound specifically to recombinant and native NS1 were produced from three huscfv-phagemid transformed Escherichia coli clones (nos. 3, 10 and 11) selected from a human ScFv phage display library. Western blot analysis, mimotope searching/epitope identification, homology modeling/molecular docking and phage mimotope ELISA inhibition indicated that HuScFv of clone no. 3 reacted with NS1 R domain important for host innate immunity suppression; HuScFv of clone nos. 10 and 11 bound to E domain sites necessary for NS1 binding to the host eIF4GI and CPSF30, respectively. The HuScFv of all clones could enter the influenza virus infected cells and interfered with the NS1 activities leading to replication inhibition of viruses belonging to various heterologous A subtypes and type B by 2–64-fold as semi-quantified by hemagglutination assay. Influenza virus infected cells treated with representative HuScFv (clone 10) had up-expression of IRF3 and IFN-β genes by 14.75 and 4.95-fold, respectively, in comparison with the controls, indicating that the antibodies could restore the host innate immune response. The fully human single chain antibodies have high potential for developing further as a safe (adjunctive) therapeutic agent for mitigating, if not abrogating, severe symptoms of influenza.

The Isolation of Novel Phage Display-Derived Human Recombinant Antibodies Against CCR5, the Major Co-Receptor of HIV

Selecting for antibodies against specific cell-surface proteins is a difficult task due to many unrelated proteins that are expressed on the cell surface. Here, we describe a method to screen antibody-presenting phage libraries against native cell-surface proteins. We applied this method to isolate antibodies that selectively recognize CCR5, which is the major co-receptor for HIV entry (consequently, playing a pivotal role in HIV transmission and pathogenesis). We employed a phage screening strategy by using cells that co-express GFP and CCR5, along with an excess of control cells that do not express these proteins (and are otherwise identical to the CCR5-expressing cells). These control cells are intended to remove most of the phages that bind the cells nonspecifically; thus leading to an enrichment of the phages presenting anti-CCR5-specific antibodies. Subsequently, the CCR5-presenting cells were quantitatively sorted by flow cytometry, and the bound phages were eluted, amplified, and used for further successive selection rounds. Several different clones of human single-chain Fv antibodies that interact with CCR5-expressing cells were identified. The most specific monoclonal antibody was converted to a full-length IgG and bound the second extracellular loop of CCR5. The experimental approach presented herein for screening for CCR5-specific antibodies can be applicable to screen antibody-presenting phage libraries against any cell-surface expressed protein of interest.

Human monoclonal ScFv that bind to different functional domains of M2 and inhibit H5N1 influenza virus replication

BackgroundNovel effective anti-influenza agent that tolerates influenza virus antigenic variation is needed. Highly conserved influenza virus M2 protein has multiple pivotal functions including ion channel activity for vRNP uncoating, anti-autophagy and virus assembly, morphogenesis and release. Thus, M2 is an attractive target of anti-influenza agents including small molecular drugs and specific antibodies.MethodsFully human monoclonal single chain antibodies (HuScFv) specific to recombinant and native M2 proteins of A/H5N1 virus were produced from huscfv-phagemid transformed E. coli clones selected from a HuScFv phage display library using recombinant M2 of clade 1 A/H5N1 as panning antigen. The HuScFv were tested for their ability to inhibit replication of A/H5N1 of both homologous and heterologous clades. M2 domains bound by HuScFv of individual E. coli clones were identified by phage mimotope searching and computerized molecular docking.ResultsHuScFv derived from four huscfv-phagemid transformed E. coli clones (no. 2, 19, 23 and 27) showed different amino acid sequences particularly at the CDRs. Cells infected with A/H5N1 influenza viruses (both adamantane sensitive and resistant) that had been exposed to the HuScFv had reduced virus release and intracellular virus. Phage peptide mimotope search and multiple alignments revealed that conformational epitopes of HuScFv2 located at the residues important for ion channel activity, anti-autophagy and M1 binding; epitopic residues of HuScFv19 located at the M2 amphipathic helix and cytoplasmic tail important for anti-autophagy, virus assembly, morphogenesis and release; epitope of HuScFv23 involved residues important for the M2 activities similar to HuScFv2 and also amphipathic helix residues for viral budding and release while HuScFv27 epitope spanned ectodomain, ion channel and anti-autophagy residues. Results of computerized homology modelling and molecular docking conformed to the epitope identification by phages.ConclusionsHuScFv that bound to highly conserved epitopes across influenza A subtypes and human pathogenic H5N1clades located on different functional domains of M2 were produced. The HuScFv reduced viral release and intracellular virus of infected cells. While the molecular mechanisms of the HuScFv await experimental validation, the small human antibody fragments have high potential for developing further as a safe, novel and mutation tolerable anti-influenza agent especially against drug resistant variants.

Human 4F5 single-chain Fv antibody recognizing a conserved HA1 epitope has broad neutralizing potency against H5N1 influenza A viruses of different clades

Influenza A viruses present a significant threat to public health worldwide. High-affinity human scFv antibodies against a conserved epitope can potentially provide immunity to diverse viruses and protect against future pandemic viruses. A library of phage-displayed human scFv containing 6.0×108 members was generated from lymphocytes of H5N1 virus vaccinated individuals. Using the recombinant H5N1 virus hemagglutinin ectodomain (HA1), 4F5 scFv was identified with neutralizing activity against both clade 2 and 9 H5N1 viruses. In embryonated chicken eggs, the antiviral activity of 4F5 scFv conferred a 100% survival rate and at least a 62.5% survival rate against different clades of H5N1 viruses by pre-treatment and post-treatment, respectively. 4F5 scFv belongs to the VH-3-43 family according to the IMGT database, and a peptide (76)WLLGNP(81) containing half of an α-helix in HA1 was identified as the binding pocket. The conserved binding epitope of this novel broadly neutralizing scFv may become key in the design and implementation of vaccines or RNA interference against H5N1 viruses.

Potent killing of HBV-related hepatocellular carcinoma by a chimeric protein of anti-HBsAg single-chain antibody and truncated Bid

Targeted therapy is needed for hepatitis B virus (HBV)-mediated hepatocellular carcinoma (HCC) which shows overexpression of HBV surface antigen (HBsAg). We previously developed scFv15, a human single-chain antibody against HBsAg. Here we tested the strategic feasibility of scFv15-mediated delivery of apoptotic effectors for HBsAg-targeted HCC therapy and application of HA2 motif of influenza hemagglutinin to enhance endosome escape and antitumor effect. A class of HBsAg-targeted immunoproapoptotic molecule was generated by sequentially fusing scFv15, the furin-cleavable motif from diphtheria toxin (Fdt), HA2 and a truncated apoptotic protein Bid (tBid). The resulting scFv15-Fdt-HA2-tBid was prokaryotically expressed and functionally characterized for HBsAg-binding capacity, endosome escape activity and antitumor effect as compared with scFv15-Fdt-tBid. Both scFv15-Fdt-HA2-tBid and scFv15-Fdt-tBid retained affinity and specificity for HBsAg, and bound and selectively killed HBsAg-positive HCC cells via apoptosis. Notably, the IC50 of scFv15-Fdt-HA2-tBid in HBsAg-positive PLC/PRF/5 cells was 10 times lower than that of scFv15-Fdt-tBid. In vivo imaging of antitumor activity demonstrated 95% growth inhibition of orthotopic HCC by scFv15-Fdt-HA2-tBid compared with 75% suppression by scFv15-Fdt-tBid. This study represents an extended application of the immunoproapoptotic strategy in the treatment of HBsAg-positive HCC and shows significant potential of HA2 as a functional enhancer for endosome-encapsulated antibody-conjugates.

Design optimization and characterization of Her2/neu-targeted immunotoxins: comparative in vitro and in vivo efficacy studies

Targeted therapeutics are potential therapeutic agents because of their selectivity and efficacy against tumors resistant to conventional therapy. The goal of this study was to determine the comparative activity of monovalent, engineered anti-Her2/neu immunotoxins fused to recombinant gelonin (rGel) to the activity of bivalent IgG-containing immunoconjugates. Utilizing Herceptin and its derived humanized single-chain antibody (single-chain fragment variable, designated 4D5), we generated bivalent chemical Herceptin/rGel conjugate, and the corresponding monovalent recombinant immunotoxins in two orientations, 4D5/rGel and rGel/4D5. All the constructs showed similar affinity to Her2/neu-overexpressing cancer cells, but significantly different antitumor activities. The rGel/4D5 orientation construct and Herceptin/rGel conjugate were superior to 4D5/rGel construct in in vitro and in vivo efficacy. The enhanced activity was attributed to improved intracellular toxin uptake into target cells and efficient downregulation of Her2/neu-related signaling pathways. The Her2/neu-targeted immunotoxins effectively targeted cells with Her2/neu expression level >1.5 × 10(5) sites per cell. Cells resistant to Herceptin or chemotherapeutic agents were not cross-resistant to rGel-based immunotoxins. Against SK-OV-3 tumor xenografts, the rGel/4D5 construct with excellent tumor penetration showed impressive tumor inhibition. Although Herceptin/rGel conjugate demonstrated comparatively longer serum half-life, the in vivo efficacy of the conjugate was similar to the rGel/4D5 fusion. These comparative studies demonstrate that the monovalent, engineered rGel/4D5 construct displayed comparable in vitro and in vivo antitumor efficacy as bivalent Herceptin/rGel conjugate. Immunotoxin orientation can significantly impact the overall functionality and performance of these agents. The recombinant rGel/4D5 construct with excellent tumor penetration and rapid blood clearance may reduce the unwanted toxicity when administrating to patients, and warrants consideration for further clinical evaluation.

Production of Human Monoclonal ScFv that Bind to Different Functional Domains of M2 and Inhibit H5N1 Influenza Virus Replication

Event Abstract Back to Event Production of Human Monoclonal ScFv that Bind to Different Functional Domains of M2 and Inhibit H5N1 Influenza Virus Replication Tippawan Pissawong1, Santi Maneewatch2, Potjanee Srimanote3, Kanyarat Thueng-In4, Fonthip Dong-Din-On5, Jeeraphong Thanongsaksrikul3, Thaweesak Songserm6 and Wanpen Chaicumpa7* 1 Mahidol University, Immunology, Thailand 2 Mahidol University, Molecular Tropical Medicine and Genetics, Thailand 3 Thammasat University, Graduate Program in Biomedical Sciences, Thailand 4 Kasetsart University, Microbiology and Immunology, Thailand 5 Kasetsart University, Veterinary Pathology, Thailand 6 Kasetsart University, Veterinary Pathology, Thailand 7 Mahidol University, Parasitology, Thailand Novel effective anti-influenza agent that tolerates influenza virus antigenic variation is needed. Highly conserved influenza virus M2 protein has multiple pivotal functions including ion channel activity for vRNP uncoating, anti-autophagy and virus assembly, morphogenesis and release. In this study, fully human monoclonal single chain antibodies (HuScFv) specific to recombinant and native M2 proteins of A/H5N1 viruses (both adamantane sentivie and resistant) were produced from four huscfv-phagemid transformed E. coli clones (no. 2, 19, 23 and 27) selected from a HuScFv phage display library. HuScFv of all clones show different amino acid sequences particularly at the CDRs. Influenza virus infected cells exposed to the HuScFv had reduced virus release and intracellular virus. Phage peptide mimotope searching revealed that conformational epitopes of HuScFv2 located at the residues important for ion channel activity, anti-autophagy and M1 binding; epitope of HuScFv19 located at the M2 amphipathic helix and cytoplasmic tail important for anti-autophagy, virus assembly, morphogenesis and release; epitope of HuScFv23 involved residues important for the M2 activities similar to HuScFv2 and also amphipathic helix residues for viral budding and release while HuScFv27 epitope spanned ectodomain, ion channel and anti-autophagy residues. Molecular docking results conformed to the epitope identification by phages. While the molecular mechanisms of the HuScFv await experimental confirmation, the small human antibody fragments have high potential for developing further as safe, novel and mutation tolerable anti-influenza agents especially against drug resistant variants. Acknowledgements The work was co-supported by the National Science and Technology Development Agency (NSTDA), the Thailand Research Fund (DPG5380001), and the Office of Higher Education Commission (RG and NRU projects), Ministry of Education, Thailand. Tippawan Pissawong received a scholarship from the Office of Higher Education Commission. References Maneewatch S, Thanongsaksrikul J, Songserm T, et al. Human single-chain antibodies that neutralize homologous and heterologous strains and clades of influenza A virus subtype H5N1. Antivir Ther. 2009;14(2):221-30. Pavoni E, Flego M, Dupuis ML, et al. Selection, affinity maturation, and characterization of a human scFv antibody against CEA protein. BMC Cancer 2006; 6:41. Skehel jj, Wiley DC. Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem 2000; 69:531-569. Keywords: Infuenza A viruses, Matrix protein-2 (M2), Human ScFv, phage display, Mimotopes, Homology Modeling and Molecular docking Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Translational immunology and immune intervention Citation: Pissawong T, Maneewatch S, Srimanote P, Thueng-In K, Dong-Din-On F, Thanongsaksrikul J, Songserm T and Chaicumpa W (2013). Production of Human Monoclonal ScFv that Bind to Different Functional Domains of M2 and Inhibit H5N1 Influenza Virus Replication. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01068 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 11 Jul 2013; Published Online: 22 Aug 2013. * Correspondence: Prof. Wanpen Chaicumpa, Mahidol University, Parasitology, Bangkok, Thailand, Wanpen.cha@mahidol.ac.th Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Tippawan Pissawong Santi Maneewatch Potjanee Srimanote Kanyarat Thueng-In Fonthip Dong-Din-On Jeeraphong Thanongsaksrikul Thaweesak Songserm Wanpen Chaicumpa Google Tippawan Pissawong Santi Maneewatch Potjanee Srimanote Kanyarat Thueng-In Fonthip Dong-Din-On Jeeraphong Thanongsaksrikul Thaweesak Songserm Wanpen Chaicumpa Google Scholar Tippawan Pissawong Santi Maneewatch Potjanee Srimanote Kanyarat Thueng-In Fonthip Dong-Din-On Jeeraphong Thanongsaksrikul Thaweesak Songserm Wanpen Chaicumpa PubMed Tippawan Pissawong Santi Maneewatch Potjanee Srimanote Kanyarat Thueng-In Fonthip Dong-Din-On Jeeraphong Thanongsaksrikul Thaweesak Songserm Wanpen Chaicumpa Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Cell penetrable human ScFv specific to influenza A virus matrix protein, M1, mitigates influenza severity

Event Abstract Back to Event Cell penetrable human ScFv specific to influenza A virus matrix protein, M1, mitigates influenza severity Fonthip Dong-din-on1, Potjanee Srimanote2, Tippawan Pissawong3, Angkasiya Monkong4, Preeda Lertwatcharasarakul4, Thaweesak Songserm4 and Wanpen Chaicumpa5* 1 Kasetsart University, Center for Agricultural Biotechnology, Thailand 2 Thammasat University, Graduate Program in Biomedical Science, Faculty of Allied Health Sciences, Thailand 3 Mahidol University, Department of Immunology, Faculty of Medicine Siriraj Hospital, Thailand 4 Kasetsart University, Veterinary Midicine, Thailand 5 Mahidol University, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Thailand Matrix protein-1 (M1) is highly conserved across type A influenza viruses. This protein has many important functions in the viral replication cycle; thus it is one of the targets of novel anti-influenza. To study therapeutic efficacy of M1 specific, cell penetrable human single chain antibody (HuScFv) in mitigating severity of influenza in infected mice. E. coli derived-human single chain antibody fragments (HuScFv) specific to recombinant M1 of influenza A virus H5N1 (clade 1) was produced using a human antibody phage display library. HuScFv from selected huscfv-phagemid transformed E. coli clones were linked molecularly to a cell penetrating peptide, penetratin (PEN) and PEN-HuScFv were produced and purified. BALB/c mice were infected intranasally with mouse adapted-avian H5N1 virus (clade 2.3). They were then treated with M1 specific-PEN-HuScFv. Control infected mice received PBS treatment. Internal organs (lung, brain, spleen, liver and kidney) were collected. Tissue viral loads were determined by real time RT-PCR. Histopathology of the tissues was also examined. Infected BALB/c mice that received M1 specific-PEN-HuScFv had reduced viral loads and histopathological features in tissues compared to the controls. M1 specific-PEN-HuScFv could mitigate severity of influenza in mice infected with A H5N1 of the heterologous clade. Cross therapeutic efficacy of the transbodies on influenza caused by different virus subtypes remains to be evaluated. Acknowledgements This research work was financially supported by grant no. DPG5380001 of the Thailand Research Fund (TRF) and the National Research University (NRU) Project, Office of Higher Education Commission (CHE), Ministry of Education, Thailand. Fonthip is a TRF scholar of the Royal Golden Jubilee Ph.D. Program.

A humanized single-chain antibody against beta 3 integrin inhibits pulmonary metastasis by preferentially fragmenting activated platelets in the tumor microenvironment

AbstractPlatelets play a supportive role in tumor metastasis. Impairment of platelet function within the tumor microenvironment may provide a clinically useful approach to inhibit metastasis. We developed a novel humanized single-chain antibody (scFv Ab) against integrin GPIIIa49-66 (named A11) capable of lysing activated platelets. In this study, we investigate the effect of A11 on the development of pulmonary metastases. In the Lewis lung carcinoma (LLC) metastatic model, A11 decreases the mean number of surface nodules and mean volume of pulmonary nodules. It protects against lung metastases in a time window that extended 4 hours before and 4 hours after the IV injection of LLCs. Coinjection of GPIIIa49-66 albumin reverses the antimetastatic activity of A11 in the B16 melanoma model, consistent with the pathophysiologic relevance of the platelet GPIIIa49-66 epitope. Significantly, A11 had no effect on angiogenesis using both in vitro and in vivo assays. The underlying molecular mechanisms are a combination of inhibition of each of the following interactions: between activated platelets and tumor cells, platelets and endothelial cells, and platelets and monocytes, as well as disaggregation of an existing platelet/tumor thrombus. Our observations may provide a novel antimetastatic strategy through lysing activated platelets in the tumor microenvironment using humanized anti–GPIIIa49-66 scFv Ab.

Expression, purification and characterization of a human single-chain Fv antibody fragment fused with the Fc of an IgG1 targeting a rabies antigen in Pichia pastoris

Because the demand for rabies post exposure prophylaxis (PEP) treatment has increased exponentially in recent years, the limited supply of human and equine rabies immunoglobulin (HRIG and ERIG) has failed to provide an adequate amount of the required passive immune component in PEP in countries where canine rabies is endemic. The replacement of HRIG and ERIG with a potentially cheaper and efficacious alternative biological for the treatment of rabies in humans, therefore, remains a high priority. In this study, we set out to assess a human single-chain Fv antibody fragment fused with the Fc of an IgG1 targeting a rabies antigen to develop a product that can be used as a component of the PEP cocktail. We cloned the ScFv fragment from a human ScFv library that was established previously and inserted this fragment into the expression vector pPICZαC/Fc. An active recombinant ScFv–Fc fusion protein was successfully expressed in Pichia pastoris. The production of ScFv–Fc was optimized and scaled up in an 80L fermentor with yields exceeding 60mg/L. The ScFv–Fc protein was purified to more than 95% purity using a two-step scheme: ammonium sulfate fractionation and Protein A Sepharose CL-4B. The ScFv–Fc fusion protein neutralized rabies virus in a standard in vivo neutralization assay in which the virus was incubated with the ScFv–Fc molecules before intracranial inoculation in mice. Our results suggest that functional antibodies can be produced in P. pastoris and that ScFv–Fc fusion proteins have the potential to serve as therapeutic candidates.

New Human Antibody Fragments Homing to Atherosclerotic Endothelial and Subendothelial Tissues: An in Vivo Phage Display Targeting Human Antibodies Homing to Atherosclerotic Tissues

In vivo phage display selection is a powerful strategy for directly identifying agents that target the vasculature of normal or diseased tissues in living animals. We describe here a new in vivo biopanning strategy in which a human phage single-chain antibody (scFv) library was injected into high-fat diet-fed ApoE(-/-) mice. Extracellular and internalized phage scFvs were selectively recovered from atherosclerotic vascular endothelium and subjacent tissues. After three successive biopanning rounds, a panel of six clones with distinct gene sequences was isolated. Four scFvs produced and purified in soluble form were shown to interact in vitro with a rabbit atheromatous protein extract by time-resolved fluorescence resonance energy transfer and to target the endothelial cell surface and inflamed intima-related regions of rabbit and human tissue sections ex vivo. These new scFvs selected in a mouse model recognized both rabbit and human tissue, underlying the interspecies similarities of the recognized epitopes. By combining immunoprecipitation and mass spectrometry, one of the selected scFvs was shown to recognize carbonic anhydrase II, an up-regulated enzyme involved in resorption of ectopic calcification. These results show that in vivo biopanning selection in hypercholesterolemic animals makes it possible to identify both scFvs homing to atherosclerotic endothelial and subendothelial tissues, and lesion-associated biomarkers. Such scFvs offer promising opportunities in the field of molecular targeting for the treatment of atherosclerosis.

Abstract 1959: Surface modifications and drug-loading affect immunogenicity of targeted nanoparticles

Abstract Introduction: Targeted nanoparticles offer great promise for developing new cancer imaging and targeted therapy approaches. For future clinical translation, one of the major concerns is that repeated administrations of the targeted nanoparticles may induce a strong immune response to the targeting ligands. The production of a high titer of neutralizing antibodies will block the receptor-binding site and reduce efficiency of targeted delivery of the nanoparticles. Methods: We have examined the titers of targeting ligand-specific antibodies in the blood and the antibody producing B lymphocytes in the spleen of Balb/c mice, bearing mouse 4T1 mammary tumors, following injections of magnetic iron oxide nanoparticles (IONPs) conjugated with targeting ligands derived from mice or humans. Results: We found that conjugation of targeting ligands onto IONPs increases immunogenicity of the ligand and results in a higher antibody titer compared to free targeting ligand injected mice. Furthermore, targeting ligands from different species, such as a human single chain antibody to epidermal growth factor receptor (scFvEGFR), induce a much higher level of the antibody in the mice compared to the mice that received a recombinant receptor binding domain of the mouse urokinase plasminogen activator (uPA), which targets uPA receptor. Surface coating of polyethylene glycol (PEG) reduces nonspecific uptake of the nanoparticles by macrophages in the liver and spleen. However, we found higher antibody titers in the mice that received PEG-coated targeting IONPs compared to the mice received targeting IONPs without PEG. We further elucidated the mechanism of the enhanced antibody response and found that although PEG-coated IONPs avoided uptake by mouse macrophages, they efficiently enter into mouse dendritic cells. High levels of the PEG-coated IONPs and longer circulation time in the blood allow uptake of more IONPs in the antigen-presenting dendritic cells, thereby stimulating antibody production. However, for targeted therapy using nanoparticles carrying chemotherapy drugs, the antibody response may not be significant since we found that the antibody response was significantly inhibited in mice that received the targeted nanoparticles carrying a chemotherapy drug, doxorubicin. Results of ELISPOT assay of the spleen cells from the mice that received systemic delivery of the targeted nanoparticles with or without doxorubicin revealed that the nanoparticles carrying the drug were able to selectively eliminate ligand-specific, antibody-producing B lymphocytes in the spleen, with no overall decrease in the total number of antibody producing cells. Results of our study provide important information for the development of targeted nanoparticle drug carriers for effective treatment of human cancer. Our study further supports the feasibility of translation of targeted theranostic nanoparticles for clinic applications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1959. doi:1538-7445.AM2012-1959

Redirected Antitumor Activity of Primary Human Lymphocytes Transduced With a Fully Human Anti-mesothelin Chimeric Receptor

Cancer regression by gene-modified T cells bearing a chimeric antigen receptor (CAR) exodomain of mouse origin can be limited by the induction of transgene immunogenicity resulting in poor persistence and function in vivo. The development of functionally-active CAR of human origin can address this issue. Here, we constructed and evaluated fully human anti-mesothelin CARs comprised of a human mesothelin-specific single-chain antibody variable fragment (P4 scFv) coupled to T cell signaling domains. Primary human T cells expressing P4 CAR specifically produced proinflammatory cytokines, degranulated and exerted potent cytolytic functions when cultured with mesothelin-expressing tumors in vitro. P4 CAR T cells also mediated bystander killing of mesothelin-negative cancer cells during coculture. CAR reactivity was not abrogated by soluble tumor-secreted or recombinant mesothelin protein even at supraphysiological levels. Importantly, adoptive transfer of P4 CAR-expressing T cells mediated the regression of large, established tumor in the presence of soluble mesothelin in a xenogenic model of human ovarian cancer. Thus, primary human T cells expressing fully human anti-mesothelin CAR efficiently kill mesothelin-expressing tumors in vitro and in vivo and have the potential to overcome the issue of transgene immunogenicity that may limit CAR T cell trials that utilize scFvs of mouse origin.

Expression of a humanized single-chain variable fragment antibody targeting chronic myeloid leukemia cells in Escherichia coli and its characterization

Chronic myeloid leukemia (CML) is a malignant blood disease originating from hematopoietic stem cells. Drug resistance and tumor recurrence have become major problems for the treatment of this disease. Therefore, new therapeutic methods need to be developed. Antigens expressed on the surface of cancer cells are potential targets for antibody-mediated drug delivery. In our study, an anti-CML cell single-chain variable fragment (scFv) antibody has been produced and characterized because it is the first step towards the construction of a novel cancer-targeted agent for cancer diagnosis and treatment. Here, a 46kDa antibody derivative was produced by genetic fusion of a humanized scFv antibody against a CML cell surface antigen with the 6xHis-tag, which can specifically bind to CML cells. The recombinant scFv against CML cells was expressed as a fusion protein containing the 6xHis-tag at its N-termini, and purified by Ni2+-NTA column chromatography. The recombinant scFv, which was soluble, was expressed and produced in bacteria, and was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot assays. Finally, its cell-binding activity and immunoactivity were demonstrated by enzyme-linked immunosorbent assay (ELISA). Furthermore, flow cytometry analysis demonstrated that this scFv specifically targeted CML cells expressing the associated antigen (47.9 and 34.4%) other than non-expressing tumor cells (1.25%) invitro. The results presented in this study illustrate that the humanized anti-CML cell scFv antibody may function as a novel therapeutic tool for CML.

Structural insights into a human anti-IFN antibody exerting therapeutic potential for systemic lupus erythematosus

Increasing evidences suggest that the type I interferon α (IFN α) plays a critical role in the etiopathogenesis of systemic lupus erythematosus (SLE), which makes it a promising therapeutic target for the treatment of the disease. By screening a large size non-immune human antibody library, we have developed a human single-chain antibody (ScFv) AIFN α 1bScFv01 and corresponding whole antibody AIFN α 1bIgG01 to human interferon α 1b (IFN α 1b) with high specificity and high affinity. The IgG antibody could down-regulate the expression of ISG15 and IFIT-1 induced by either recombinant IFN α 1b or naïve IFN α from SLE patients' sera, and reduced total serum IgG and IgM antibodies level in a pristane-primed lupus-like mouse model. The crystal structure of AIFN α 1bScFv01-IFN α 1b complex solved to 2.8 Å resolution revealed that both Pro26-Gln40 region in loop AB and Glu147-Arg150 region in helix E of IFN α 1b contribute to binding with AIFN α 1bScFv01. Four residues of above two regions (Leu30, Asp32, Asp35 and Arg150) are critical for the formation of antigen-antibody complexes. AIFN α 1bScFv01 shares partial epitopes of IFN α 1b with its receptor IFNAR2 but with much higher binding affinity to IFN α 1b than IFNAR2. Thus, AIFN α 1bIgG01 exhibits its neutralizing activity through competition with IFNAR2 to bind with IFN α and prevents the activation of IFN α-mediated signaling pathway. Our results highlight the potential use of the human antibody for modulating the activity of IFN α in SLE.

In Vivo Detection of Oxidation-Specific Epitopes in Atherosclerotic Lesions Using Biocompatible Manganese Molecular Magnetic Imaging Probes

This study sought to evaluate the in vivo magnetic resonance imaging (MRI) efficacy of manganese [Mn(II)] molecular imaging probes targeted to oxidation-specific epitopes (OSE). OSE are critical in the initiation, progression, and destabilization of atherosclerotic plaques. Gadolinium [Gd(III)]-based MRI agents can be associated with systemic toxicity. Mn is an endogenous, biocompatible, paramagnetic metal ion that has poor MR efficacy when chelated, but strong efficacy when released within cells. Multimodal Mn micelles were generated to contain rhodamine for confocal microscopy and conjugated with either the murine monoclonal IgG antibody MDA2 targeted to malondialdehyde (MDA)-lysine epitopes or the human single-chain Fv antibody fragment IK17 targeted to MDA-like epitopes ("targeted micelles"). Micelle formulations were characterized in vitro and in vivo, and their MR efficacy (9.4-T) evaluated in apolipoprotein-deficient (apoE(-/-)) and low-density lipoprotein receptor negative (LDLR(-/-)) mice (0.05 mmol Mn/kg dose) (total of 120 mice for all experiments). In vivo competitive inhibition studies were performed to evaluate target specificity. Untargeted, MDA2-Gd, and IK17-Gd micelles (0.075 mmol Gd/kg) were included as controls. In vitro studies demonstrated that targeted Mn micelles accumulate in macrophages when pre-exposed to MDA-LDL with ∼10× increase in longitudinal relativity. Following intravenous injection, strong MR signal enhancement was observed 48 to 72 h after administration of targeted Mn micelles, with colocalization within intraplaque macrophages. Co-injection of free MDA2 with the MDA2-Mn micelles resulted in full suppression of MR signal in the arterial wall, confirming target specificity. Similar MR efficacy was noted in apoE(-/-) and LDLR(-/-) mice with aortic atherosclerosis. No significant differences in MR efficacy were noted between targeted Mn and Gd micelles. This study demonstrates that biocompatible multimodal Mn-based molecular imaging probes detect OSE within atherosclerotic plaques and may facilitate clinical translation of noninvasive imaging of human atherosclerosis.

Engineering Antibody Fitness and Function Using Membrane-Anchored Display of Correctly Folded Proteins

A hallmark of the bacterial twin-arginine translocation (Tat) pathway is its ability to export folded proteins. Here, we discovered that overexpressed Tat substrate proteins form two distinct, long-lived translocation intermediates that are readily detected by immunolabeling methods. Formation of the early translocation intermediate Ti-1, which exposes the N- and C-termini to the cytoplasm, did not require an intact Tat translocase, a functional Tat signal peptide, or a correctly folded substrate. In contrast, formation of the later translocation intermediate, Ti-2, which exhibits a bitopic topology with the N-terminus in the cytoplasm and C-terminus in the periplasm, was much more particular, requiring an intact translocase, a functional signal peptide, and a correctly folded substrate protein. The ability to directly detect Ti-2 intermediates was subsequently exploited for a new protein engineering technology called MAD-TRAP (membrane-anchored display for Tat-based recognition of associating proteins). Through the use of just two rounds of mutagenesis and screening with MAD-TRAP, the intracellular folding and antigen-binding activity of a human single-chain antibody fragment were simultaneously improved. This approach has several advantages for library screening, including the unique involvement of the Tat folding quality control mechanism that ensures only native-like proteins are displayed, thus eliminating poorly folded sequences from the screening process.

Functional Characterization of an scFv-Fc Antibody that Immunotherapeutically Targets the Common Cancer Cell Surface Proteoglycan CSPG4

Cell surface chondroitin sulfate proteoglycan 4 (CSPG4) is an attractive target for antibody-based cancer immunotherapy because of its role in tumor cell biology, its high expression on malignant cells including cancer-initiating cells, and its restricted distribution in normal tissues. The clinical use of CSPG4 has been hampered by the lack of a CSPG4-specific chimeric, humanized, or fully human monoclonal antibody. To overcome this limitation, we generated a CSPG4-specific fully human single-chain antibody termed scFv-FcC21 and characterized its specificity and antitumor activity. Viable CSPG4(+) melanoma cells were used in a screen of a human scFv phage display library that included CDR3 engineered to optimize antibody binding sites. The scFv antibody isolated was then recombinantly engineered with a human immunoglobulin G1 Fc region to construct the fully human antibody scFv-FcC21, which recognized tumors of neuroectodermal origin, various types of carcinomas, mesotheliomas, and sarcomas as well as myeloid leukemias. scFv-FcC21 inhibited in vitro growth and migration of tumor cells and in vivo growth of human tumor xenografts. These effects were mediated by inhibition of the activation of extracellular signal-regulated kinase and focal adhesion kinase signaling pathways that are critical for tumor cell growth and migration, respectively. Our findings define the CSPG4-specific fully human scFv-FcC21 antibody as a candidate therapeutic agent to target the many types of tumors that express CSPG4.

High-level secretion of recombinant monomeric murine and human single-chain Fv antibodies from Drosophila S2 cells

Single-chain variable fragment (scFvs) antibodies are small polypeptides (∼26 kD) containing the heavy (VH) and light (VL) immunoglobulin domains of a parent antibody connected by a flexible linker. In addition to being frequently used in diagnostics and therapy for an increasing number of human diseases, scFvs are important tools for structural biology as crystallization chaperones. Although scFvs can be expressed in many different organisms, the expression level of an scFv strongly depends on its particular amino acid sequence. We report here a system allowing for easy and efficient cloning of (i) scFvs selected by phage display and (ii) individual heavy and light chain sequences from hybridoma cDNA into expression plasmids engineered for secretion of the recombinant fragment produced in Drosophila S2 cells. We validated the method by producing five scFvs derived from human and murine parent antibodies directed against various antigens. The production yields varied between 5 and 12 mg monomeric scFv per liter of supernatant, indicating a relative independence on the individual sequences. The recombinant scFvs bound their cognate antigen with high affinity, comparable with the parent antibodies. The suitability of the produced recombinant fragments for structural studies was demonstrated by crystallization and structure determination of one of the produced scFvs, derived from a broadly neutralizing antibody against the major glycoprotein E2 of the hepatitis C virus. Structural comparison with the Protein Data Bank revealed the typical spatial organization of VH and VL domains, further validating the here-reported expression system.

Mucosal Immunization with an Unadjuvanted Vaccine That Targets Streptococcus pneumoniae PspA to Human Fcγ Receptor Type I Protects against Pneumococcal Infection through Complement- and Lactoferrin-Mediated Bactericidal Activity

Targeting an antigen to Fc receptors (FcR) can enhance the immune response to the antigen in the absence of adjuvant. Furthermore, we recently demonstrated that intranasal immunization with an FcγR-targeted antigen enhances protection against a category A intracellular mucosal pathogen, Francisella tularensis. To determine if a similar strategy could be applied to the important pathogen Streptococcus pneumoniae, we used an improved mucosal FcR-targeting strategy that specifically targets human FcγR type I (hFcγRI). A humanized single-chain antibody component in which the variable domain binds to hFcγRI [anti-hFcγRI (H22)] was linked in a fusion protein with the pneumococcal surface protein A (PspA). PspA is known to elicit protection against pneumococcal sepsis, carriage, and pneumonia in mouse models when administered with adjuvants. Anti-hFcγRI-PspA or recombinant PspA (rPspA) alone was used to intranasally immunize wild-type (WT) and hFcγRI transgenic (Tg) mice in the absence of adjuvant. The hFcγRI Tg mice receiving anti-hFcγRI-PspA exhibited elevated S. pneumoniae-specific IgA, IgG2c, and IgG1 antibodies in serum and bronchoalveolar lavage fluid. Neither immunogen was effective in protecting WT mice in the absence of adjuvant, but when PspA was targeted to hFcγRI as the anti-hFcγRI-PspA fusion, enhanced protection against lethal S. pneumoniae challenge was observed in the hFcγRI Tg mice compared to mice given nontargeted rPspA alone. Immune sera from the anti-hFcγRI-PspA-immunized Tg mice showed enhanced complement C3 deposition on bacterial surfaces, and protection was dependent upon an active complement system. Immune serum also showed an enhanced bactericidal activity directed against S. pneumoniae that appears to be lactoferrin mediated.