Antibody Fragment scFv Research Articles

Enhancement of hERG channel activity by scFv antibody fragments targeted to the PAS domain

The human human ether-à-go-go-related gene (hERG) potassium channel plays a critical role in the repolarization of the cardiac action potential. Changes in hERG channel function underlie long QT syndrome (LQTS) and are associated with cardiac arrhythmias and sudden death. A striking feature of this channel and KCNH channels in general is the presence of an N-terminal Per-Arnt-Sim (PAS) domain. In other proteins, PAS domains bind ligands and modulate effector domains. However, the PAS domains of KCNH channels are orphan receptors. We have uncovered a family of positive modulators of hERG that specifically bind to the PAS domain. We generated two single-chain variable fragments (scFvs) that recognize different epitopes on the PAS domain. Both antibodies increase the rate of deactivation but have different effects on channel activation and inactivation. Importantly, we show that both antibodies, on binding to the PAS domain, increase the total amount of current that permeates the channel during a ventricular action potential and significantly reduce the action potential duration recorded in human cardiomyocytes. Overall, these molecules constitute a previously unidentified class of positive modulators and establish that allosteric modulation of hERG channel function through ligand binding to the PAS domain can be attained.

Abstract 3912: MM-310, a novel EphA2-targeted docetaxel nanoliposome

Abstract Taxanes are widely used to treat solid tumors either in the curative or palliative setting, in first or later lines of therapy. Analysis of docetaxel dose-response relationship strongly suggests that a higher dose would lead to high response, however will also lead to higher toxicity. This is likely related to the lack of organ and cellular specificity of docetaxel leading to high exposures in normal tissues and the relatively short circulation half-life which indirectly requires higher doses. With the goal of addressing the pharmacokinetic limitations of free docetaxel and the lack of cellular specificity, we developed a novel docetaxel-based nanoliposome (MM-310), targeted against Ephrin receptor A2 (EphA2) which is overexpressed in a wide range of tumors. MM-310 provides sustained release of docetaxel following accumulation in solid tumors. Preclinical models have demonstrated that MM-310 leverages tumor-specific accumulation through the enhanced permeability and retention effect, and cellular specificity through active targeting of EphA2 with specific scFv antibody fragments conjugated to the surface of the liposomes. Pharmacokinetic and biodistribution studies were performed in mice and rats to compare MM-310 to free docetaxel. Chronic tolerability studies were performed in rodent and non-rodent models with focus on overall animal health, as well as hematologic toxicities. Several cell-derived models of breast, lung and prostate xenografts were used to evaluate the differences betweenf MM-310 and free docetaxel. MM-310 had a significantly longer half-life than free docetaxel with prolonged exposure at the tumor site. In chronic tolerability studies, MM-310 was found to be 6-7 times better tolerated than free docetaxel with a maximum tolerated dose of at least 120 mpk, compared to 20 mpk for free docetaxel and no detectable hematological toxicity. At equitoxic dosing, MM-310 50 mpk showed greater activity than docetaxel 10 mpk in several breast, lung and prostate xenograft models. In conclusion, we developed a novel EphA2 targeted docetaxel nanoliposome with prolonged circulation time and slow and sustained drug release kinetics, to enable organ and cellular targeting. MM-310 was able to overcome hematologic toxicities observed upon treatment with free docetaxel in rodent and non-rodent models. MM-310 was also able to induce tumor regression or control tumor growth in several cell derived xenograft models, and was found to be more active than free docetaxel in most models. Citation Format: Dmitri B. Kirpotin, Suresh Tipparaju, Zhaohua Richard Huang, Walid S. Kamoun, Christine Pien, Tad Kornaga, Shinji Oyama, Ken Olivier, James D. Marks, Alexander Koshkaryev, Sarah S. Schihl, Gerald Fetterly, Birgit Schoeberl, Charles Noble, Mark Hayes, Daryl C. Drummond. MM-310, a novel EphA2-targeted docetaxel nanoliposome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3912.

Chimeric antigen receptor-engineered cytokine-induced killer cells overcome treatment resistance of pre-B-cell acute lymphoblastic leukemia and enhance survival.

Pre-emptive cancer immunotherapy by donor lymphocyte infusion (DLI) using cytokine-induced killer (CIK) cells may be beneficial to prevent relapse with a reduced risk of causing graft-versus-host-disease. CIK cells are a heterogeneous effector cell population including T cells (CD3(+) CD56(-) ), natural killer (NK) cells (CD3(-) CD56(+) ) and natural killer T (T-NK) cells (CD3(+) CD56(+) ) that exhibit non-major histocompatibility complex (MHC)-restricted cytotoxicity and are generated by ex vivo expansion of peripheral blood mononuclear cells in the presence of interferon (IFN)-γ, anti-CD3 antibody, interleukin-2 (IL-2) and interleukin-15 (IL-15). To facilitate selective target-cell recognition and enhance specific cytotoxicity against B-cell acute lymphoblastic leukemia (B-ALL), we transduced CIK cells with a lentiviral vector encoding a chimeric antigen receptor (CAR) that carries a composite CD28-CD3ζ domain for signaling and a CD19-specific scFv antibody fragment for cell binding (CAR 63.28.z). In vitro analysis revealed high and specific cell killing activity of CD19-targeted CIK/63.28.z cells against otherwise CIK-resistant cancer cell lines and primary B-ALL blasts, which was dependent on CD19 expression and CAR signaling. In a xenograft model in immunodeficient mice, treatment with CIK/63.28.z cells in contrast to therapy with unmodified CIK cells resulted in complete and durable molecular remissions of established primary pre-B-ALL. Our results demonstrate potent antileukemic activity of CAR-engineered CIK cells in vitro and in vivo, and suggest this strategy as a promising approach for adoptive immunotherapy of refractory pre-B-ALL.

Dual targeting of glioblastoma with chimeric antigen receptor-engineered natural killer cells overcomes heterogeneity of target antigen expression and enhances antitumor activity and survival

ABSTRACT Epidermal growth factor receptor (EGFR) and its mutant form EGFRvIII are overexpressed in a large proportion of glioblastomas (GBM). Immunotherapy with an EGFRvIII-specific vaccine has shown efficacy against GBM in clinical studies. However, immune escape by antigen-loss variants and lack of control of EGFR wild-type positive clones limit the usefulness of this approach. Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells may represent an alternative immunotherapeutic strategy. For targeting to GBM, we generated variants of the clinically applicable human NK cell line NK-92 that express CARs carrying a composite CD28-CD3ζ domain for signaling, and scFv antibody fragments for cell binding either recognizing EGFR, EGFRvIII, or an epitope common to both antigens. In vitro analysis revealed high and specific cytotoxicity of EGFR-targeted NK-92 against established and primary human GBM cells, which was dependent on EGFR expression and CAR signaling. EGFRvIII-targeted NK-92 only lysed EGFRvIII-positive GBM cells, while dual-specific NK cells expressing a cetuximab-based CAR were active against both types of tumor cells. In immunodeficient mice carrying intracranial GBM xenografts either expressing EGFR, EGFRvIII or both receptors, local treatment with dual-specific NK cells was superior to treatment with the corresponding monospecific CAR NK cells. This resulted in a marked extension of survival without inducing rapid immune escape as observed upon therapy with monospecific effectors. Our results demonstrate that dual targeting of CAR NK cells reduces the risk of immune escape and suggest that EGFR/EGFRvIII-targeted dual-specific CAR NK cells may have potential for adoptive immunotherapy of glioblastoma.

Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody.

Clostridium botulinum exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry.

CD19-CAR engineered NK-92 cells are sufficient to overcome NK cell resistance in B-cell malignancies.

Many B‐cell acute and chronic leukaemias tend to be resistant to killing by natural killer (NK) cells. The introduction of chimeric antigen receptors (CAR) into T cells or NK cells could potentially overcome this resistance. Here, we extend our previous observations on the resistance of malignant lymphoblasts to NK‐92 cells, a continuously growing NK cell line, showing that anti‐CD19‐CAR (αCD19‐CAR) engineered NK‐92 cells can regain significant cytotoxicity against CD19 positive leukaemic cell lines and primary leukaemia cells that are resistant to cytolytic activity of parental NK‐92 cells. The ‘first generation’ CAR was generated from a scFv (CD19) antibody fragment, coupled to a flexible hinge region, the CD3ζ chain and a Myc‐tag and cloned into a retrovirus backbone. No difference in cytotoxic activity of NK‐92 and transduced αCD19‐CAR NK‐92 cells towards CD19 negative targets was found. However, αCD19‐CAR NK‐92 cells specifically and efficiently lysed CD19 expressing B‐precursor leukaemia cell lines as well as lymphoblasts from leukaemia patients. Since NK‐92 cells can be easily expanded to clinical grade numbers under current Good Manufactoring Practice (cGMP) conditions and its safety has been documented in several phase I clinical studies, treatment with CAR modified NK‐92 should be considered a treatment option for patients with lymphoid malignancies.

Targeted Enhancement of hERG K+ Channel Activity with scFv Antibody Fragments

Cardiac IKr is a repolarizing current comprising subunits encoded by the human ether-a-go-go related gene (hERG). A defining feature of hERG and related channel types is an N-terminal PAS domain that, through its effects on gating, reduces current amplitude. To probe the functional consequences of disrupting this natural channel modulation, we screened for single chain fragment variable (scFv) antibodies against the PAS domain using phage display. We assessed the binding properties to PAS and functional consequences of these antibodies on the hERG channel. Two isolates affecting hERG 1a current properties were shown by GST pull-down assays to interact with different regions of the PAS domain with submicromolar affinities. In HEK-293 cells stably expressing hERG 1a, one antibody slowed inactivation onset and the other accelerated recovery from inactivation. Both significantly increased repolarizing current during a voltage protocol mimicking a ventricular action potential. Because native IKr channels are heteromers of the original hERG 1a isolate and hERG 1b, an isoform from which the PAS domain is transcriptionally omitted, we tested the effect of the scFv antibodies on cardiomyocytes derived from human induced pluripotent stem cells. Similar to the HEK-293 findings, the antibodies diminished channel rectification and significantly increased the repolarizing charge during a ventricular action potential voltage clamp. Here, we demonstrate the versatility of scFv antibodies as a site-specific probe of ion channel function. Given their ability to increase IKr, we propose the PAS-channel interface as a therapeutic target and the use of scFv antibodies as a novel treatment for diseases of excitability where action potential duration is prolonged.

Systematic screening of soluble expression of antibody fragments in the cytoplasm of E. coli.

BackgroundDisulfide bonds are the most common structural, post-translational modification found in proteins. Antibodies contain up to 25 disulfide bonds depending on type, with scFv fragments containing two disulfides and Fab fragments containing five or six disulfide bonds. The production of antibody fragments that contain native disulfide bonds can be challenging, especially on a large scale. The protein needs to be targeted to prokaryotic periplasm or the eukaryotic endoplasmic reticulum. These compartments are specialised for disulfide bond formation, but both compartments have limitations.ResultsHere we show that the introduction into the cytoplasm of a catalyst of disulfide bond formation and a catalyst of disulfide bond isomerization allows the efficient formation of natively folded scFv and Fab antibody fragments in the cytoplasm of Escherichia coli with intact reducing pathways. Eleven scFv and eleven Fab fragments were screened and ten of each were obtained in yields of >5 mg/L from deep-well plates. Production of eight of the scFv and all ten of the Fab showed a strong dependence on the addition of the folding factors. Yields of purified scFv of up to 240 mg/L and yields of purified Fab fragments of up to 42 mg/L were obtained. Purified fragments showed circular dichroism spectra consistent with being natively folded and were biologically active.ConclusionsOur results show that the efficient production of soluble, biologically active scFv and Fab antibody fragments in the cytoplasm of E. coli is not only possible, but facile. The required components can be easily transferred between different E. coli strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0419-5) contains supplementary material, which is available to authorized users.

Abstract A164: Genetically modified cytokine-induced killer (CIK) cells for targeted cancer therapy

Abstract Pre-emptive immunotherapy after HSC transplantation based on minimal residual disease (MRD) status with donor lymphocyte infusions (DLI) using cytokine-induced killer (CIK) cells may be beneficial to prevent relapse with a reduced risk of causing graft-versus-host-disease (GvHD) compared to conventional T cell infusion. CIK cells are a heterogeneous effector cell population including T cells (CD3+CD56-), natural killer (NK) cells (CD3-CD56+) and natural killer T (T-NK) cells (CD3+CD56+), that exhibit non-MHC-restricted cytotoxic activity and are generated by ex vivo expansion of peripheral blood mononuclear cells (PBMC) through the addition of interferon (IFN)-γ, anti-CD3 antibody, IL-2 and IL-15. While CIK cells have shown potent in vivo activity against various cancer types such as lymphomas or colorectal cancer, their cytotoxicity against B-ALL, characterized by the expression of CD19, has been limited. Hence, retargeting of CIK cells using chimeric antigen receptors (CARs) to facilitate selective target cell recognition and enhance specific cytotoxicity represents a promising approach. CAR comprise an extracellular scFv antibody fragment as an antigen-binding domain, linked via a flexible hinge region and a transmembrane domain to an intracellular signaling moiety such as CD3 zeta chain (first generation CAR), or zeta chain fused to a co-stimulatory protein domain such as CD28 (second generation CAR). We established an optimized protocol for transduction of CIK cells with CD19-specific lentiviral CAR constructs, and characterized cells for expression of an EGFP marker gene and CAR surface expression. Effects of exposure to lentiviral vector particles on the development of CIK cell subpopulations were monitored. In in vitro cytotoxicity assays we could demonstrate potent and selective cytotoxicity of retargeted CIK cells towards established cancer cell lines expressing CD19 and primary pre-B-ALL blasts. In addition, we observed significantly enhanced degranulation of CAR-CIK cells upon target cell contact and showed increased secretion of pro-inflammatory cytokines, while no secretion of immunosuppressive IL-10 could be detected. Cytotoxity towards non-malignant allo-PBMCs remained low. In addition, we investigated anti-leukemic activity of retargeted CIK cells in vivo in NOD/SCID common gamma chain knockout (NSG) mouse models using bioluminescence and fluorescence imaging. We observed potent reduction of tumor load after only two CIK cell applications in mice with an engrafted primary pre-B-ALL yielding long-term survivors with a negative MRD status (4/6). In a separate model, we demonstrated migration of CD19-specific CIK cells into local, subcutaneous tumor sites. In conclusion, CAR-CIK cells represent a promising alternative to CAR-T cell therapy, as relatively high numbers of conventional CIK cells have already been infused in clinical trials, which demonstrated a low risk of causing severe side effects. Citation Format: Sarah Oelsner, Juliane Wagner, Miriam E. Friede, Verena Pfirrmann, Eva Rettinger, Ralf Schubert, Heike Pfeifer, Evelyn Ullrich, Peter Bader, Winfried S. Wels. Genetically modified cytokine-induced killer (CIK) cells for targeted cancer 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 A164.

Optimized Lentiviral Transduction Protocols by Use of a Poloxamer Enhancer, Spinoculation, and scFv-Antibody Fusions to VSV-G.

Lentiviral vectors (LV) are widely used to successfully transduce cells for research and clinical applications. This optimized LV infection protocol includes a nontoxic poloxamer-based adjuvant combined with antibody-retargeted lentiviral particles. The novel poloxamer P338 demonstrates superior characteristics for enhancing lentiviral transduction over the best-in-class polybrene-assisted transduction. Poloxamer P338 exhibited dual benefits of low toxicity and high efficiency of lentiviral gene delivery into a range of different primary cell cultures. One of the major advantages of P338 is its availability in pharma grade and applicability as cell culture medium additive in clinical protocols. Lentiviral vectors pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G) can be produced to high titers and mediate high transduction efficiencies in vitro. For clinical applications the need for optimized transduction protocols, especially for transduction of primary T and stem cells, is high. The successful use of retronectin, the second lentivirus enhancer available as GMP material, requires the application of specific coating protocols not applicable in all processes, and results in the need of a relatively high multiplicity of infection (MOI) to achieve effective transduction efficiencies for hematopoietic cells (e.g., CD34+ hematopoietic stem cells). Cell specificity of lentiviral vectors was successfully increased by displaying different ratios of scFv-fused VSV-G glycoproteins on the viral envelope. The system has been validated with human CD30+ lymphoma cells, resulting in preferential gene delivery to CD30+ cells, which was increased fourfold in mixed cell cultures, by presenting scFv antibody fragments binding to respective surface markers. A combination of spinoculation and poloxamer-based chemical adjuvant increases the transduction of primary T-cells by greater than twofold. The combination of poloxamer-based and scFv-retargeted LVs increased transduction of CD30+ lymphoma cells more than tenfold, and has the potential to improve clinical protocols.

Generation of a Novel Bacteriophage Library Displaying scFv Antibody Fragments from the Natural Buffalo Host to Identify Antigens from Adult Schistosoma japonicum for Diagnostic Development.

The development of effective diagnostic tools will be essential in the continuing fight to reduce schistosome infection; however, the diagnostic tests available to date are generally laborious and difficult to implement in current parasite control strategies. We generated a series of single-chain antibody Fv domain (scFv) phage display libraries from the portal lymph node of field exposed water buffaloes, Bubalus bubalis, 11–12 days post challenge with Schistosoma japonicum cercariae. The selected scFv-phages showed clear enrichment towards adult schistosomes and excretory-secretory (ES) proteins by immunofluorescence, ELISA and western blot analysis. The enriched libraries were used to probe a schistosome specific protein microarray resulting in the recognition of a number of proteins, five of which were specific to schistosomes, with RNA expression predominantly in the adult life-stage based on interrogation of schistosome expressed sequence tags (EST). As the libraries were enriched by panning against ES products, these antigens may be excreted or secreted into the host vasculature and hence may make good targets for a diagnostic assay. Further selection of the scFv library against infected mouse sera identified five soluble scFv clones that could selectively recognise soluble whole adult preparations (SWAP) relative to an irrelevant protein control (ovalbumin). Furthermore, two of the identified scFv clones also selectively recognised SWAP proteins when spiked into naïve mouse sera. These host B-cell derived scFvs that specifically bind to schistosome protein preparations will be valuable reagents for further development of a cost effective point-of-care diagnostic test.

Engineering recombinant antibodies for polymer biofunctionalization

The attachment of recognition elements such as antibody fragments to polymeric substrates can be used to mediate cell‐ or protein‐specific interactions. In this work, single‐chain Fv (scFv) antibody fragments were isolated against two cell types of interest and expressed in an Escherichia coli expression platform. The scFvs were engineered at their C‐terminus to incorporate a cysteine‐containing linker, for reaction with maleimide‐linked polymers, or a heptasaccharide glycan for complexation with surface amine moieties. Antigen binding of the modified scFvs was unchanged, and expression yields of the glyco‐engineered scFvs were similar to the unmodified molecules, while cys‐tagged scFv yields varied between scFv variants. Targeted immobilization of the scFvs via either modification resulted in three‐ to five‐fold higher binding of ligands over adsorbed molecules. The study demonstrates a simple and efficient antibody engineering and modification approach for effective targeted immobilization on polymeric substrates. Copyright © 2015 John Wiley & Sons, Ltd.

Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display

Xylella fastidiosa is a member of the gamma proteobacteria. It is fastidious, insect-vectored and xylem-limited and causes a variety of diseases, some severe, on a wide range of economically important perennial crops, including grape and citrus. Antibody based detection assays are commercially available for X. fastidiosa, and are effective at the species, but not at the subspecies level. We have made a library of scFv antibody fragments directed against X. fastidiosa subsp. pauca strain 9a5c (citrus) by using phage display technology. Antibody gene repertoires were PCR-amplified using 23 primers for the heavy chain variable region (VH) and 21 primers for the light chain variable region (VL). The VH and VL were joined by overlap extension PCR, and then the genes of the scFv library were ligated into the phage vector pKM19. The library contained 1.2×107 independent clones with full-length scFv inserts. In each of 3cycles of affinity-selection with 9a5c, about 1.0×1012 phage were used for panning with 4.1×106, 7.1×106, 2.1×107 phage recovered after the first, second and third cycles, respectively. Sixty-six percent of clones from the final library bound X. fastidiosa 9a5c in an ELISA. Some of these scFv antibodies recognized strain 9a5c and did not recognize X. fastidiosa strains that cause Pierce's disease of grapevine.

Immunoliposomes for Targeted Delivery of an Antifibrotic Drug.

Excessive extracellular matrix formation in organs and tissues arises from an imbalance between the synthesis and degradation of matrix proteins, especially collagen. This condition interferes with proper wound healing and regeneration, and to date, no specific treatment is available. In the present study, we propose a targeted drug delivery system consisting of cell-specific immunoliposomes (ILs) loaded with deferoxamine (DFO) as an antifibrotic drug. ILs were functionalized with polyethylene glycol (PEG) to improve the steric stability and prolong their half-life. In addition, a single-chain Fv (scFv) antibody fragment that specifically targets fibroblast activation protein (FAP) was incorporated. An in vitro fibrosis model was employed to test this construct. This model consisted of highly activated pro-fibrotic fibroblasts with 2- to 6-fold induction of selected fibrosis markers: cell/matrix deposited collagen I, total soluble collagen, and α smooth muscle actin. The activation was accompanied by a significant and cell-specific elevation of FAP expression and activity, thereby confirming that FAP is an adequate target for antifibrotic drug delivery. Purified anti-FAP scFv was shown to bind specifically to these cells without influencing the FAP enzymatic activity. DFO was demonstrated to have a dose-dependent antifibrotic activity as quantified by collagen deposition. Specific binding and intracellular uptake of DiI-labeled ILs into the activated fibroblasts were shown by flow cytometry and microscopy. Finally, DFO-loaded ILs targeted to FAP caused a significant reduction in the collagen deposition, whereas no effect was observed using liposomes that lacked the targeting antibody fragment. These results suggest that the FAP-specific scFv-conjugated liposomes have considerable potential for cell-specific targeting applicable as a therapy for excessive collagen deposition during fibrosis. In general, through liposome encapsulation, bioactive molecules, such as DFO, that have broad effects and poor cell penetration can be converted into cell-specific composites for targeted drug delivery.

A new expression vector facilitating production and functional analysis of scFv antibody fragments selected from Tomlinson I + J phagemid libraries

Tomlinson I+J are synthetic phagemid human scFv libraries widely employed to obtain specific antibody fragments via a phage display method. The pIT2/HB2151 expression system proposed by the designers of the libraries has certain drawbacks which result in the lack of expression or low expression levels of numerous soluble scFvs. At the stage of scFv screening, this may lead to losing some excellent antibodies, which can be avoided but requires laborious and expensive work. Here we present a new, pET-30-based vector, which is compatible with Tomlinson libraries, retains all virtues of pIT2 used as a plasmid and eliminates all its flaws. We demonstrate that pET-scFv-T is frequently superior to pIT2 in terms of efficient scFv expression. Moreover, an amber suppressor bacterial strain, RosettaBlue(DE3)pLysS, transformed with the new vector, pET-scFv-T, coding for a number of scFvs, produces substantial amounts of functional, easy to purify recombinant antibody fragments, regardless of whether their coding sequences contain amber codons. Thus, pET-scFv-T/RosettaBlue(DE3)pLysS expression system seems to be a perfect tool for screening for the finest soluble scFvs selected from Tomlinson I+J, as well as from many other phagemid libraries.

Advances in Drug Delivery

Advances in Drug Delivery

Structural and functional characterization of a novel scFv anti-HSP60 of Strongyloides sp.

Phage display is a powerful technology that selects specific proteins or peptides to a target. We have used Phage Display to select scFv (single-chain variable fragment) clones from a combinatorial library against total proteins of Strongyloides venezuelensis. After scFv characterization, further analysis demonstrated that this recombinant fragment of antibody was able to bind to an S. venezuelensis antigenic fraction of ~65 kDa, present in the body periphery and digestive system of infective larvae (L3), as demonstrated by immunofluorescence. Mass spectrometry results followed by bioinformatics analysis showed that this antigenic fraction was a heat shock protein 60 (HSP60) of Strongyloides sp. The selected scFv was applied in serodiagnosis by immune complexes detection in serum samples from individuals with strongyloidiasis using a sandwich enzyme-linked immunosorbent assay (ELISA), showing sensitivity of 97.5% (86.84–99.94), specificity of 98.81 (93.54–99.97), positive likelihood ratio of 81.60 and an area under the curve of 0.9993 (0.9973–1.000). Our study provided a novel monoclonal scFv antibody fragment which specifically bound to HSP60 of Strongyloides sp. and was applied in the development of an innovative serodiagnosis method for the human strongyloidiasis.

Production of biologically active scFv and VHH antibody fragments in Bifidobacterium longum.

Bifidobacteria constitute a significant part of healthy intestinal microbiota in adults and infants and present a promising platform for construction of genetically modified probiotic agents for treatment of gastrointestinal disorders. In this study, three strains of Bifidobacterium longum were constructed that express and secrete biologically active single-chain antibodies against human TNF-α and Clostridium difficile exotoxin A. Anti-TNF-α scFv antibody D2E7 was produced at the level of 25 μgL(-1) in broth culture and was mostly retained in the cytoplasm, while VHH-type antibodies A20.1 and A26.8 against C. difficile exotoxin A were produced at the levels of 0.3-1 mgL(-1) and secreted very efficiently. The biological activity of both antibody types was demonstrated in the mammalian cell-based assays. Expression of A20.1 and A26.8 was also observed in vivo after intragastric administration of transformed B. longum strains to (C57/BL6 × DBA/2)F1 mice. The obtained B. longum strains may serve as prototypes for construction of novel probiotic medications against inflammatory bowel disease and C. difficile-associated disease.

Selection of scFv Antibody Fragments Binding to Human Blood versus Lymphatic Endothelial Surface Antigens by Direct Cell Phage Display.

The identification of marker molecules specific for blood and lymphatic endothelium may provide new diagnostic tools and identify new targets for therapy of immune, microvascular and cancerous diseases. Here, we used a phage display library expressing human randomized single-chain Fv (scFv) antibodies for direct panning against live cultures of blood (BECs) and lymphatic (LECs) endothelial cells in solution. After six panning rounds, out of 944 sequenced antibody clones, we retrieved 166 unique/diverse scFv fragments, as indicated by the V-region sequences. Specificities of these phage clone antibodies for respective compartments were individually tested by direct cell ELISA, indicating that mainly pan-endothelial cell (EC) binders had been selected, but also revealing a subset of BEC-specific scFv antibodies. The specific staining pattern was recapitulated by twelve phage-independently expressed scFv antibodies. Binding capacity to BECs and LECs and differential staining of BEC versus LEC by a subset of eight scFv antibodies was confirmed by immunofluorescence staining. As one antigen, CD146 was identified by immunoprecipitation with phage-independent scFv fragment. This antibody, B6-11, specifically bound to recombinant CD146, and to native CD146 expressed by BECs, melanoma cells and blood vessels. Further, binding capacity of B6-11 to CD146 was fully retained after fusion to a mouse Fc portion, which enabled eukaryotic cell expression. Beyond visualization and diagnosis, this antibody might be used as a functional tool. Overall, our approach provided a method to select antibodies specific for endothelial surface determinants in their native configuration. We successfully selected antibodies that bind to antigens expressed on the human endothelial cell surfaces in situ, showing that BECs and LECs share a majority of surface antigens, which is complemented by cell-type specific, unique markers.

Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization.

Crystallization chaperones are attracting increasing interest as a route to crystal growth and structure elucidation of difficult targets such as membrane proteins. While strategies to date have typically employed protein-specific chaperones, a peptide-specific chaperone to crystallize multiple cognate peptide epitope-containing client proteins is envisioned. This would eliminate the target-specific chaperone-production step and streamline the co-crystallization process. Previously, protein engineering and directed evolution were used to generate a single-chain variable (scFv) antibody fragment with affinity for the peptide sequence EYMPME (scFv/EE). This report details the conversion of scFv/EE to an anti-EE Fab format (Fab/EE) followed by its biophysical characterization. The addition of constant chains increased the overall stability and had a negligible impact on the antigen affinity. The 2.0 Å resolution crystal structure of Fab/EE reveals contacts with larger surface areas than those of scFv/EE. Surface plasmon resonance, an enzyme-linked immunosorbent assay, and size-exclusion chromatography were used to assess Fab/EE binding to EE-tagged soluble and membrane test proteins: namely, the β-barrel outer membrane protein intimin and α-helical A2a G protein-coupled receptor (A2aR). Molecular-dynamics simulation of the intimin constructs with and without Fab/EE provides insight into the energetic complexities of the co-crystallization approach.