Single-chain Antibody Fragment Research Articles

Antitumor Activity of DLX1008, an Anti-VEGFA Antibody Fragment with Low Picomolar Affinity, in Human Glioma Models

Angiogenesis mediated by vascular endothelial growth factor (VEGF) is a hallmark of glioblastoma. Based on the response rate and improved progression-free survival, although not on overall survival, the 149-kDa anti-VEGF-A IgG antibody bevacizumab (Avastin) has been approved in the United States and Japan for recurrent glioblastoma and in Japan for newly diagnosed glioblastoma; however, it is not approved in the EU. Here we characterize the biologic activity of DLX1008, a 26-kDa anti-VEGF-A single-chain antibody fragment that shows 30-fold stronger affinity to human VEGF-A than bevacizumab. The small molecular size of DLX1008 is predicted to result in improved target coverage over bevacizumab. DLX1008 showed superiority to bevacizumab in the inhibition of VEGF-A binding to VEGF receptor (VEGFR) 1 in enzyme-linked immunosorbent assay by a factor of around 10 and comparable efficacy for the inhibition of VEGF-A-stimulated VEGFR2 dimerization. In a tube-formation assay with human cerebral microvascular endothelial cells, DLX1008 was at least as active as bevacizumab. In vivo, DLX1008 delayed growth in a mouse subcutaneous U87 xenograft model (P = 0.0021) and improved survival in a mouse orthotopic U87 xenograft model (P = 0.00026). Given the exceptionally high affinity and small molecular size of DLX1008, these data warrant further clinical development of DLX1008 as an antiangiogenic agent in glioblastoma.

Generation of recombinant scFv antibody against Ochratoxin A (OTA)

Ochratoxin A (OTA) is a mycotoxin commonly found in agricultural products and can accumulate in the blood and tissues after that consuming contaminated food. Recombinant single-chain antibody fragments (scFv) against OTA were selected from phage display libraries. After of one round of biopanning against BSA-conjugated OTA (OTA-BSA), 52 and 6 phage clones displaying scFv antibodies were isolated from human (Yamo I.3) and rabbit (Bozmix I.2) libraries. Two phage clones (one from each libraries, i.e., yOTA1e3 and bOTA2a9) showed binding to free toxin by competitive ELISA. The soluble scFv antibodies were produced by superinfecting phage clones into E. coli suppressor strain HB2151. The scFv genes from these two phage clones were sub-cloned into pKP300ΔIII vectors to generate scFv-AP fusions. The binding affinity (IC50) of antibody derived from human library was higher than those from rabbit library. The binding property of recombinant antibody in the form of scFv-AP was better than those of soluble scFv form. Cross-reactivity analysis indicated that the two recombinant antibodies did not cross-react with other soluble toxins, namely AFB1, DON, ZEN and FB. The ability to use the recombinant scFv-AP to detect contaminated toxins in agricultural product (corn) was demonstrated.

Biocompatible coupling of therapeutic fusion proteins to human erythrocytes

AbstractCarriage of drugs by red blood cells (RBCs) modulates pharmacokinetics, pharmacodynamics, and immunogenicity. However, optimal targets for attaching therapeutics to human RBCs and adverse effects have not been studied. We engineered nonhuman-primate single-chain antibody fragments (scFvs) directed to human RBCs and fused scFvs with human thrombomodulin (hTM) as a representative biotherapeutic cargo (hTM-scFv). Binding fusions to RBCs on band 3/glycophorin A (GPA; Wright b [Wrb] epitope) and RhCE (Rh17/Hr0 epitope) similarly endowed RBCs with hTM activity, but differed in their effects on RBC physiology. scFv and hTM-scFv targeted to band 3/GPA increased membrane rigidity and sensitized RBCs to hemolysis induced by mechanical stress, while reducing sensitivity to hypo-osmotic hemolysis. Similar properties were seen for other ligands bound to GPA and band 3 on human and murine RBCs. In contrast, binding of scFv or hTM-scFv to RhCE did not alter deformability or sensitivity to mechanical and osmotic stress at similar copy numbers bound per RBCs. Contrasting responses were also seen for immunoglobulin G antibodies against band 3, GPA, and RhCE. RBC-bound hTM-scFv generated activated protein C (APC) in the presence of thrombin, but RhCE-targeted hTM-scFv demonstrated greater APC generation per bound copy. Both Wrb- and RhCE-targeted fusion proteins inhibited fibrin deposition induced by tumor necrosis factor-α in an endothelialized microfluidic model using human whole blood. RhCE-bound hTM-scFv more effectively reduced platelet and leukocyte adhesion, whereas anti-Wrb scFv appeared to promote platelet adhesion. These data provide a translational framework for the development of engineered affinity ligands to safely couple therapeutics to human RBCs.

Generation of high-affinity, internalizing anti-FGFR2 single-chain variable antibody fragment fused with Fc for targeting gastrointestinal cancers.

Fibroblast growth factor receptors (FGFRs) are promising targets for antibody-based cancer therapies, as their substantial overexpression has been found in various tumor cells. Aberrant activation of FGF receptor 2 (FGFR2) signaling through overexpression of FGFR2 and/or its ligands, mutations, or receptor amplification has been reported in multiple cancer types, including gastric, colorectal, endometrial, ovarian, breast and lung cancer. In this paper, we describe application of the phage display technology to produce a panel of high affinity single chain variable antibody fragments (scFvs) against the extracellular ligand-binding domain of FGFR2 (ECD_FGFR2). The binders were selected from the human single chain variable fragment scFv phage display libraries Tomlinson I + J and showed high specificity and binding affinity towards human FGFR2 with nanomolar KD values. To improve the affinity of the best binder selected, scFvF7, we reformatted it to a bivalent diabody format, or fused it with the Fc region (scFvF7-Fc). The scFvF7-Fc antibody construct presented the highest affinity for FGFR2, with a KD of 0.76 nM, and was selectively internalized into cancer cells overexpressing FGFR2, Snu-16 and NCI-H716. Finally, we prepared a conjugate of scFvF7-Fc with the cytotoxic drug monomethyl-auristatin E (MMAE) and evaluated its cytotoxicity. The conjugate delivered MMAE selectively to FGFR2-positive tumor cells. These results indicate that scFvF7-Fc-vcMMAE is a highly potent molecule for the treatment of cancers with FGFR2 overexpression.

DNA Origami as a Nanoscale Platform for T-Cell Activation

In the human immune system, the recognition of an antigen by the T-cell receptor (TCR) takes place within the contact area between the T-cell and the antigen-presenting cell (APC). It is thought that the nanoscale spatial distribution of proteins within this contact zone plays an essential role in the initiation of an immune response. Despite extensive studies, the molecular details of this process, in particular the structural requirements for TCR triggering and how nanoscale events are translated into T-cell activation, are still poorly understood. Here, we use DNA origami decorated with TCR ligands anchored to a planar glass-supported lipid bilayer to assess the effects of local ligand density and arrangement on T-cell activation. Thus, our experimental setup allows for the precise nanoscale arrangement of TCR ligands on the DNA origami scaffold, while at the same time permitting the re-organization of ligand and TCR during T-cell activation. We used either fluorescently labeled CD3 antibody or recombinant TCRβ-reactive single chain antibody fragment (scFv) as stimulatory ligands that were placed on the DNA origami at one to 20 engineered capture sites in different layouts and densities. The actual number of ligands per origami was determined using several single molecule fluorescence microcopy methods and atomic force microscopy. The activation of T-cells interfaced with the APC-mimicking surfaces was measured using a calcium-sensitive fluorescent dye and the effects of local ligand density, nanoscale ligand arrangement as well as the nature of the ligand were assessed. Further, the rearrangement of TCR and ligands in the process of T-cell activation was monitored by single molecule microscopy.

Ultrasensitive direct impedimetric immunosensor for detection of serum HER2

Assesment of human epidermal growth factor receptor 2 status is a key factor prompting definitive treatment decisions that help in reducing mortality rates associated with breast cancer. In this article, highly sensitive and low-cost impedimetric immunosensor using single-chain fragment variable antibody fragments was developed for quantitative detection of human epidermal growth factor receptor 2 from serum employing gold nanoparticle-modified disposable screen-printed carbon electrodes. The gold nanoparticles facilitate fast electron transfer and offer a biocompatible surface for immobilization of small antibody fragments in an oriented manner, resulting in improved antigen binding efficiency. The single-chain fragment variable antibody fragment-modified screen printed immunosensor exhibits wide dynamic range of 0.01–100 ng mL−1 and detection limit of 0.01 ng mL−1. The advantages offered by this platform in terms of high sensitivity, broad dynamic range and low-cost demonstrates great potential for improved monitoring of human epidermal growth factor receptor 2 levels for the management of breast and other cancers.

Evaluation of METase-pemetrexed-loaded PEG-PLGA nanoparticles modified with anti-CD133-scFV for treatment of gastric carcinoma.

PEG–PLGA nanoparticles (NPs) modified with anti-CD133 and tumor-targeting single-chain antibody fragment (scFV–NPs) for systemic delivery of methioninase (METase) and pemetrexed for gastric carcinoma were successfully formulated. The structure characterization and biological functions of METase-pemetrexed-loaded scFV–PEG–PLGA NPs (scFV–METase/pemetrexed–NPs) in vitro were investigated. Functional scFV–PEG–PLGA NPs or PEG–PLGA NPs present low cell cytoxicity in CD133+ SGC7901 cells. scFV–METase/pemetrexed–NPs (scFv–M/P–NP) was more effective in inhibiting tumor growth (including cell growth and migration ability) in CD133 positive expressed gastric cancer cells than METase/pemetrexed-NPs (M/P–NP). Moreover, METase enhanced the inhibitory effect of pemetrexed on thymidylate synthase (TS) synthesis and cell apoptosis. We have demonstrated the application of scFV-targeted PEG–PLGA NPs as a new potential strategy to enhance treatment benefits for gastric carcinoma.

Production of human single-chain fragment antibody (ScFv) against human epidermal growth factor receptor-2 (HER-2) by phage display technology.

Breast cancer with more than 1.7 million diagnoses per year has been known as one of the most prevalent cancers among women worldwide. Despite the availability of advanced treatment options, cancer progression and metastasis is observed in 20% of patients. Human epidermal growth factor receptor-2 (HER-2) is considered as an important prognostic and diagnostic tumor marker for breast cancer. While HER-2 is expressed on the surface of normal cells, its overexpression occurs in 20-25% on breast cancer tumor cells. This type of tumor which is referred to as HER-2+ is the most aggressive type of breast cancer and shows more resistance to radiotherapy. Single-chain fragment antibodies (ScFvs) offer several advantages in comparison to conventional whole antibodies due to their small size. Particularly, ScFv fragments show improved diffusion and solid tumor penetration. In this study, a human ScFv antibody library was used to isolate anti-HER-2 ScFv antibodies through cell panning and mix antigen-cell panning strategies. Analysis of the binding activity and specificity of isolated ScFv antibodies against HER-2-expressing cell lines and recombinant HER-2 antigen indicated the higher efficiency of the cell panning strategy in isolation of ScFv antibody fragments.

In Vitro Evaluation of Humanized/De-immunized Anti-PSMA Immunotoxins for the Treatment of Prostate Cancer.

We generated humanized/de-immunized immunotoxins targeting the prostate-specific membrane antigen (PSMA) and tested their cytotoxic activity against prostate cancer cells in vitro. The humanized/de-immunized version of our murine anti-PSMA single-chain antibody fragment (scFv) D7, termed hD7-1(VL-VH), was ligated to the 40-kDa toxin domain of Pseudomonas aeruginosa exotoxin A (PE40), and to the deimmunized 24-kDa toxin domains PE24 or PE24mut. The immunotoxins designated as hD7-1(VL-VH)-PE40, hD7-1(VL-VH)-PE24 and hD7-1(VL-VH)-PE24mut were bacterially expressed and purified by affinity chromatography. Binding and cytotoxicity were examined by flow cytometry and viability assay, respectively. All immunotoxins revealed strong binding to prostate cancer cells expressing PSMA and specific cytotoxicity, with half-maximal inhibitory concentration values in the picomolar range. We successfully created powerful anti-PSMA immunotoxins with reduced immunogenicity for further clinical development and application against advanced prostate cancer.

Abstract B004: Enhanced efficacy of immune modulators with albumin binding domains

Abstract Numerous approaches to improve the biologic half-life and tumor targeting of small therapeutic proteins (e.g., interleukins), peptides, and fragment-based antibodies (scFv) have been described. The attractiveness of these smaller biologic formats includes improved tumor penetration and the ability to engineer a single molecule that can modulate several tumor targets. However, the limitation of these smaller biologic-based therapeutics (less than 150kd) is their short half-life and lack of tumor targeting. Here we describe a unique (24Kd) single-chain fragment antibody (ScFv) that binds to serum albumin across a range of pHs and that retains binding to, and recycling by, the neonatal Fc-receptor (FcRn), resulting in a significant extension of the biologic half-life of three different therapeutic proteins that were molecularly linked to this albumin binding domain (ABD) fragment antibody. A second advantage to having an ABD construct is enhanced tumor targeting. Numerous studies have shown that albumin-mediated delivery of therapeutic proteins and small molecules results in tumor accumulation that was higher than that produced by PEGylation, Thus, fusion with an ABD is a promising approach for improving the therapeutic/safety window of small recombinant therapeutic proteins (e.g., interleukins, receptor ligands) or enhancing the targeting of receptor-directed scFv therapeutic proteins. Using the XOMA phage library, we discovered an scFv ABD that has high binding affinity to mouse, human, and cynomolgus serum albumin. We demonstrate that fusion constructs of this scFv ABD with several different small therapeutic proteins (recombinant interleukin proteins and scFvs targeting relevant immune-oncology receptors) demonstrated improved serum half-life and greater efficacy compared to the naked proteins without the ABD present. We have further characterized our scFV-ABD and have demonstrated that (1) biologic activity is retained when the therapeutic protein is attached via the N- or C-terminus, suggesting utility for delivering more than one therapeutic protein; and (2) half-life in mouse serum in vivo was extended from minutes to hours/days for three different recombinant proteins with MWs of 10-80Kd. Additionally, we have demonstrated markedly superior reductions in tumor growth and improved overall survival with our ABD constructs vs free recombinant protein in an established B6F10 melanoma model. Moreover, superior efficacy was observed at lower doses and with a single dose of the ABD constructs vs free recombinant protein. The versatility of this ABD construct to attached combinations of various types of recombinant immune stimulators and/or scFv to immune checkpoint inhibitors can result in significantly enhanced clinical efficacy. Citation Format: John Cini. Enhanced efficacy of immune modulators with albumin binding domains [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B004.

Array-In-Well Epitope Mapping of Phage-Displayed Antibodies.

Novel affinity reagents, such as single chain (scFv) antibody fragments, can be generated by isolating them from recombinant protein libraries using phage display selection. A successful selection process against a target protein can produce a number of binder candidates among which the desired binders are identified by screening and characterization of individual clones. Obtaining information on the binding properties, such as the binding epitope, already during the screening step helps to choose the most useful candidates for further development at early phase saving time and resources. To this end, we describe here an Array-in-Well-based screening procedure to perform activity testing and epitope mapping for filamentous phage-displayed scFvs in an integrated manner with a single assay.

Enhanced delivery of siRNA to triple negative breast cancer cells in vitro and in vivo through functionalizing lipid-coated calcium phosphate nanoparticles with dual target ligands.

The conjugation of ligands to nanoparticle platforms for the target delivery of therapeutic agents to the tumor tissue is one of the promising anti-cancer strategies. However, conventional nanoparticle platforms are not so effective in terms of the selectivity and transfection efficiency. In this study, we designed and developed a dual-target drug/gene delivery system based on lipid-coated calcium phosphate (LCP) nanoparticles (NPs) for significantly enhanced siRNA cellular uptake and transfection efficiency. LCP NPs loaded with therapeutic siRNA were conjugated with a controlled number of folic acid and/or EGFR-specific single chain fragment antibody (ABX-EGF scFv). The uptake of ABX-EGF scFv-modified (LCP-scFv) and folic acid-modified LCP NPs (LCP-FA) by human breast tumor cells (MDA-MB-468) was significantly higher with an optimal ligand density on each NP surface (LCP-125scFv and LCP-100FA). Co-conjugation with sub-optimal dual ligands (50 FA and 75 ABX-EGF scFv) per LCP NP (LCP-50FA-75scFv) further enhanced the cellular uptake. More significantly, much more NPs were delivered to the MDA-MB-468 tumor tissue in the nude mouse model when LCP-50FA-75scFv NPs were used. Therefore, the new dual-ligand LCP NPs may be a valuable targeting system for human breast cancer diagnosis and therapy.

Improvement of solubility and refolding of an anti-human epidermal growth factor receptor 2 single-chain antibody fragment inclusion bodies.

Single chain variable fragment antibodies (scFvs) have attracted many attentions due to their small size, faster bio-distribution and better penetration in to the target tissues, and ease of expression in Escherichia coli. Although, scFv expression in E. coli usually leads to formation of inclusion bodies (IBs). The aim of this research was to improve solubilizing and refolding conditions for IBs of scFv version of pertuzumab (anti-human epidermal growth factor receptor 2 (HER2) antibody). After protein overexpression in E. coli BL21 (DE3), bacterial cells were lysed and IBs were extracted via repeated washing and centrifugation. The effect of different types, concentrations, pHs, and additive of denaturing agents on IBs solubility were evaluated. More than 40 refolding additives were screened and combinations of 10 of the best additives were check out using Plackett-Burman design to choose three refolding additives with the most positive effect on refolding of the scFv. Response surface methodology (RSM) was used to optimize the concentration of adopted additives. The most efficient buffer to solubilize IBs was a buffer containing 6 M urea with 6 mM beta mercaptoethanol, pH 11. The optimum concentration of three buffer additives for refolding of the scFv was 23 mM tricine, 0.55 mM arginine, and 14.3 mM imidazole. The bioactivity of the refolded scFv was confirmed by immunohistochemical staining of breast cancer tissue, a specific binding based method. The systematic optimization of refolding buffer developed in the present work will contribute to improve the refolding of other scFv fragments.

Purification and Characterization of Antibodies in Single-Chain Format against the E6 Oncoprotein of Human Papillomavirus Type 16

In Human Papillomaviruses- (HPV-) associated carcinogenesis, continuous expression of the E6 oncoprotein supports its value as a potential target for the development of diagnostics and therapeutics for HPV cancer. We previously reported that the I7 single-chain antibody fragment (scFv) specific for HPV16 E6, expressed as an intrabody by retroviral system, could inhibit significantly the growth of cervical cancer cells in vitro and was even able to reduce tumor development in experimental HPV-related cancer models. Nevertheless, for the development of therapeutic tools to be employed in humans, it is important to achieve maximum safety guarantee, which can be provided by the protein format. In the current study, two anti-16E6 scFvs derived from I7 were expressed in E. coli and purified in soluble form by affinity chromatography. Specificity, sensitivity and stability in physiologic environment of the purified scFvs were demonstrated by binding studies using recombinant 16E6 as an antigen. The scFvs functionality was confirmed by immunofluorescence in cervical cancer cells, where the scFvs were able to recognize the nuclear E6. Furthermore, an antiproliferative activity of the scFvI7nuc delivered in protein format to HPV16-positive cell lines was observed. Our results demonstrate that functional anti-16E6 scFvs can be produced in E. coli, suggesting that such purified antibodies could be used in the diagnosis and treatment of HPV-induced malignancies.

Site-Specific Modification of Single-Chain Antibody Fragments for Bioconjugation and Vascular Immunotargeting.

The conjugation of antibodies to drugs and drug carriers improves delivery to target tissues. Widespread implementation and effective translation of this pharmacologic strategy awaits the development of affinity ligands capable of a defined degree of modification and highly efficient bioconjugation without loss of affinity. To date, such ligands are lacking for the targeting of therapeutics to vascular endothelial cells. To enable site-specific, click-chemistry conjugation to therapeutic cargo, we used the bacterial transpeptidase, sortase A, to attach short azidolysine containing peptides to three endothelial-specific single chain antibody fragments (scFv). While direct fusion of a recognition motif (sortag) to the scFv C-terminus generally resulted in low levels of sortase-mediated modification, improved reaction efficiency was observed for one protein, in which two amino acids had been introduced during cloning. This prompted insertion of a short, semi-rigid linker between scFv and sortag. The linker significantly enhanced modification of all three proteins, to the extent that unmodified scFv could no longer be detected. As proof of principle, purified, azide-modified scFv was conjugated to the antioxidant enzyme, catalase, resulting in robust endothelial targeting of functional cargo in vitro and in vivo.

Pacific Biosciences Sequencing and IMGT/HighV-QUEST Analysis of Full-Length Single Chain Fragment Variable from an In Vivo Selected Phage-Display Combinatorial Library.

Phage-display selection of immunoglobulin (IG) or antibody single chain Fragment variable (scFv) from combinatorial libraries is widely used for identifying new antibodies for novel targets. Next-generation sequencing (NGS) has recently emerged as a new method for the high throughput characterization of IG and T cell receptor (TR) immune repertoires both in vivo and in vitro. However, challenges remain for the NGS sequencing of scFv from combinatorial libraries owing to the scFv length (>800 bp) and the presence of two variable domains [variable heavy (VH) and variable light (VL) for IG] associated by a peptide linker in a single chain. Here, we show that single-molecule real-time (SMRT) sequencing with the Pacific Biosciences RS II platform allows for the generation of full-length scFv reads obtained from an in vivo selection of scFv-phages in an animal model of atherosclerosis. We first amplified the DNA of the phagemid inserts from scFv-phages eluted from an aortic section at the third round of the in vivo selection. From this amplified DNA, 450,558 reads were obtained from 15 SMRT cells. Highly accurate circular consensus sequences from these reads were generated, filtered by quality and then analyzed by IMGT/HighV-QUEST with the functionality for scFv. Full-length scFv were identified and characterized in 348,659 reads. Full-length scFv sequencing is an absolute requirement for analyzing the associated VH and VL domains enriched during the in vivo panning rounds. In order to further validate the ability of SMRT sequencing to provide high quality, full-length scFv sequences, we tracked the reads of an scFv-phage clone P3 previously identified by biological assays and Sanger sequencing. Sixty P3 reads showed 100% identity with the full-length scFv of 767 bp, 53 of them covering the whole insert of 977 bp, which encompassed the primer sequences. The remaining seven reads were identical over a shortened length of 939 bp that excludes the vicinity of primers at both ends. Interestingly these reads were obtained from each of the 15 SMRT cells. Thus, the SMRT sequencing method and the IMGT/HighV-QUEST functionality for scFv provides a straightforward protocol for characterization of full-length scFv from combinatorial phage libraries.

Evolution of the magic bullet: Single chain antibody fragments for the targeted delivery of immunomodulatory proteins.

Immunocytokines are fusion proteins that combine the specific antigen binding capacities of an antibody or derivative thereof and the potent bioactivity of a cytokine partner. These novel biopharmaceuticals have been directed to various targets of oncological as well as non-oncological origin and a handful of promising constructs are currently advancing in the clinical trial pipeline. Several factors such as the choice of a disease specific antigen, the antibody format and the modulatory nature of the payload are crucial, not only for therapeutic efficacy and safety but also for the commercial success of such a product. In this review, we provide an overview of the basic principles and obstacles in immunocytokine design with a specific focus on single chain antibody fragment-based constructs that employ interleukins as the immunoactive component. Impact statement Selective activation of the immune system in a variety of malignancies represents an attractive approach when existing strategies have failed to provide adequate treatment options. Immunocytokines as a novel class of bifunctional protein therapeutics have emerged recently and generated promising results in preclinical and clinical studies. In order to harness their full potential, multiple different aspects have to be taken into consideration. Several key points of these fusion constructs are discussed here and should provide an outline for the development of novel products based on an overview of selected formats.

Complementarity determining regions and frameworks contribute to the disulfide bond independent folding of intrinsically stable scFv.

CyDisCo is a system facilitating disulfide bond formation in recombinant proteins in the cytoplasm of Escherichia coli. Previously we screened for soluble expression of single chain antibody fragments (scFv) in the cytoplasm of E. coli in the presence and absence of CyDisCo, with >90% being solubly expressed. Two scFv, those derived from natalizumab and trastuzumab, were solubly produced in high amounts even in the absence of folding catalysts i.e. disulfide bond formation is not critical for their folding.Here we investigate the contribution of the framework and the complementarity determining regions (CDRs) of scFv to the disulfide-independence of folding. We swapped CDRs between four scFv that have different properties, including two scFv that can efficiently fold independently from disulfide bonds and two more disulfide-dependent scFv. To confirm disulfide-independence we generated cysteine to alanine mutants of the disulfide-independent scFv. All of the scFv were tested for soluble expression in the cytoplasm of E. coli in the presence and absence of the oxidative folding catalysts Erv1p and PDI.Eight of the hybrid scFv were solubly produced in the presence of CyDisCo, while seven were solubly produced in the absence of CyDisCo, though the yields were often much lower when CyDisCo was absent. Soluble expression was also observed for scFv natalizumab and trastuzumab containing no cysteines. We compared yields, thermal stability and secondary structure of solubly produced scFv and undertook binding studies by western blotting, dot blotting or surface plasmon resonance of those produced in good yields. Our results indicate that both the CDRs and the framework contribute to the disulfide-dependence of soluble production of scFv, with the CDRs having the largest effect. In addition, there was no correlation between thermal stability and disulfide-dependence of folding and only a weak correlation between the yield of protein and the thermal stability of the protein.

A streamlined cloning workflow minimising the time-to-strain pipeline for Pichia pastoris

Although recent advances in E. coli self-assembly have greatly simplified cloning, these have not yet been harnessed for the high-throughput generation of expression strains in the early research and discovery phases of biopharmaceutical production. Here, we have refined the technique and incorporated it into a streamlined workflow for the generation of Pichia pastoris expression strains, reducing the timeline by a third and removing the reliance on DNA editing enzymes, which often require troubleshooting and increase costs. We have validated the workflow by cloning 24 human proteins of biopharmaceutical value, either as direct therapeutics or as research targets, which span a continuous range in size and GC content. This includes demonstrating the applicability of the workflow to three-part assemblies for a monoclonal antibody and its single-chain antibody fragments derivatives. This workflow should enable future research into recombinant protein production by P. pastoris and a synthetic biology approach to this industrial host.

Generation of High-Sensitivity Monoclonal Antibodies Specific for Homoserine Lactones.

A number of bacteria use a class of chemical compounds called acyl-homoserine lactones (AHLs) as quorum sensing (QS) signals to coordinate their behavior at the population level, including pathogens like Pseudomonas aeruginosa. Blocking QS using antibodies is an attractive strategy for infection control as this process takes a central role in P. aeruginosa infections. Here the methods involved in the generation of high sensitivity anti-QS monoclonal antibodies from an immunized sheep phage display antibody library are described. A panel of AHL compounds conjugated to carrier proteins are used for sheep immunization and a phage display antibody library is constructed using the immune repertoire of sheep as a source of antibody genes. High sensitivity single chain antibody fragments (scFv) are isolated from the library using "smart selection strategies" and reformatted into single chain antibodies (scAbs). The resultant monoclonal antibodies: (1) recognize HSL compounds at low nanomolar concentrations; (2) have the potential to reduce virulence gene expression in P. aeruginosa; and (3) offer protection in a nematode model of infection.