Single-chain Fv Antibody Research Articles

Affinity-matured antibody with a disulfide bond in H-CDR3 loop

Affinity maturation increases antigen-binding affinity and specificity of antibodies by somatic hypermutation. Various monoclonal antibodies against (4-hydroxy-3-nitrophenyl)acetyl (NP) were obtained during affinity maturation. Among them, highly matured anti-NP antibodies, such as E11 and E3, possess Cys96H and Cys100H in the complementarity-determining region 3 of the heavy chain, which would form a disulfide bond. In this study, we evaluated the effects of disulfide bonds on antigen binding by generating single-chain Fv (scFv) antibodies of E11 and its mutants, E11_C96KH/C100EH and E11_C96KH/C100QH, and determined their antigen-binding thermodynamics and kinetics. The binding affinities of the Cys mutants were lower than that of E11 scFv, indicating that the disulfide bond contributed to antigen binding, especially for stable complex formation. This was also supported by the decreased affinity of E11 scFv in the presence of a reducing agent. The crystal structures of NP-free and NP-bound E11 scFvs were determined at high resolution, showing the existence of a disulfide bond between Cys96H and Cys100H, and the antigen recognition mechanism, which could be compared with those of other anti-NP antibodies, such as germline-type N1G9 and matured-type C6, as reported previously. These structures could explain the molecular basis of changes in antigen-binding affinity and thermal stability in the absence or presence of antigens. Small-angle X-ray scattering further showed a local conformational change in E11 scFv upon antigen binding in solution.

Evaluation of the cyclic single-chain Fv antibody derived from nivolumab by biophysical analyses and in vitro cell-based bioassay.

Single-chain Fv (scFv) is a recombinant small antibody in which a polypeptide linker connects the variable regions of the light chain (VL) and the heavy chain (VH). The practical use of scFv, however, has been prevented by its tendency to aggregate due to interchain VL-VH interactions. We recently developed a cyclic scFv whose N-terminus and C-terminus were connected by protein ligation techniques. Biophysical comparisons between cyclic and linear scFv have been conducted, but cell biological evaluations remain unexplored. Here we studied the properties of cyclic and linear scFv derived from nivolumab. Biophysical studies revealed that the thermal stability was not changed but that the antigen-binding activity was approximately 3-fold higher as a result of circularization. A cell-based PD-1/PD-L1 interaction inhibitory assay revealed that the biological activity of scFv was markedly higher in the circularized form. In addition, biophysical analysis of scFv proteins incubated in the presence of serum revealed that circularization suppressed the decrease in antigen-binding activity. It could be assumed that circularization of scFv improved stability in the presence of serum, which in turn would suggest the applicability of cyclic scFv as a biopharmaceutical format.

Analysis of the Structural Dynamics of Proteins in the Ligand-Unbound and -Bound States by Diffracted X-ray Tracking.

Although many protein structures have been determined at atomic resolution, the majority of them are static and represent only the most stable or averaged structures in solution. When a protein binds to its ligand, it usually undergoes fluctuation and changes its conformation. One attractive method for obtaining an accurate view of proteins in solution, which is required for applications such as the rational design of proteins and structure-based drug design, is diffracted X-ray tracking (DXT). DXT can detect the protein structural dynamics on a timeline via gold nanocrystals attached to the protein. Here, the structure dynamics of single-chain Fv antibodies, helix bundle-forming de novo designed proteins, and DNA-binding proteins in both ligand-unbound and ligand-bound states were analyzed using the DXT method. The resultant mean square angular displacements (MSD) curves in both the tilting and twisting directions clearly demonstrated that structural fluctuations were suppressed upon ligand binding, and the binding energies determined using the angular diffusion coefficients from the MSD agreed well with the binding thermodynamics determined using isothermal titration calorimetry. In addition, the size of gold nanocrystals is discussed, which is one of the technical concerns of DXT.

Structure, dynamics and transferability of the metal-dependent polyhistidine tetramerization motif TetrHis for single-chain Fv antibodies

The polyhistidine (6XHis) motif is one of the most ubiquitous protein purification tags. The 6XHis motif enables the binding of tagged proteins to various metals, which can be advantageously used for purification with immobilized metal affinity chromatography. Despite its popularity, protein structures encompassing metal-bound 6XHis are rare. Here, we obtained a 2.5 Å resolution crystal structure of a single chain Fv antibody (scFv) bearing a C-terminal sortase motif, 6XHis and TwinStrep tags (LPETGHHHHHHWSHPQFEK[G3S]3WSHPQFEK). The structure, obtained in the presence of cobalt, reveals a unique tetramerization motif (TetrHis) stabilized by 8 Co2+ ions. The TetrHis motif contains four 6 residues-long β-strands, and each metal center coordinates 3 to 5 residues, including all 6XHis histidines. By combining dynamic light scattering, small angle x-ray scattering and molecular dynamics simulations, We investigated the influence of Co2+ on the conformational dynamics of scFv 2A2, observing an open/close equilibrium of the monomer and the formation of cobalt-stabilized tetramers. By using a similar scFv design, we demonstrate the transferability of the tetramerization property. This novel metal-dependent tetramerization motif might be used as a fiducial marker for cryoelectron microscopy of scFv complexes, or even provide a starting point for designing metal-loaded biomaterials.

Development and characterization of a latex turbidimetric immunoassay using rabbit anti-CRP single-chain Fv antibodies

In this study, we developed and demonstrated a latex turbidimetric immunoassay (LTIA) using latex beads immobilized with rabbit monoclonal single-chain variable fragments (scFvs) selected from an scFv-displayed phage library. Sixty-five different anti-c-reactive protein (anti-CRP) scFv clones were identified after biopanning selection using antigen-coupled multi-lamellar vesicles. By ranking antigen-binding clones using the apparent dissociation rate constant (appkoff) as a sorting index, scFv clones with a dissociation constant (KD free) ranging from 4.07 × 10−9 M to 1.21 × 10−11 M were isolated. Among them, three candidates (R2–6, R2–45, and R3–2) were produced in the culture supernatant at concentrations of 50 mg/L or higher in flask culture and maintained at considerably high antigen-binding activity in immobilized state on the CM5 sensor chip surface.All the scFv-immobilized latexes (scFv-Ltxs) prepared were well-dispersed in 50 mM MOPS at pH 7.0, without additives for dispersion, and their antigen-dependent aggregation was sufficiently detectable. The reactivity of scFv-Ltx to antigen differed among the scFv clones, in particular, R2–45 scFv-Ltx detected the CRP with the highest signal. Furthermore, the reactivity of scFv-Ltx varied significantly with salt concentration, scFv immobilization density, and the type of blocking protein. Particularly, antigen-dependent latex aggregation improved significantly in all rabbit scFv clones when scFv-Ltx was blocked with horse muscle myoglobin compared with conventional bovine serum albumin; while their baseline signals in the absence of antigen were fully stable. Under optimal conditions, R2–45 scFv-Ltx exhibited greater aggregation signals with antigen concentrations higher than those produced by conventional polyclonal antibody-immobilized latex for CRP detection in LTIA. The methodology for rabbit scFv isolation, immobilization, and antigen-dependent latex aggregation demonstrated in the present study can be applicable to scFv-based LTIA for various target antigens.

Molecular Dynamics-Based Design and Biophysical Evaluation of Thermostable Single-Chain Fv Antibody Mutants Derived from Pharmaceutical Antibodies.

Antibody drugs are denatured under physical stress, e.g., friction, heat, and freezing, which triggers formation of aggregates and resultant allergic reactions. Design of a stable antibody is thus critical for the development of antibody drugs. Here, we obtained a thermostable single-chain Fv (scFv) antibody clone by rigidifying the flexible region. We first conducted a short molecular dynamics (MD) simulation (3 runs of 50 ns) to search for weak spots in the scFv antibody, i.e., flexible regions located outside the CDR (complementarity determining region) and the interface between the heavy-chain and light-chain variable regions. We then designed a thermostable mutant and evaluated it by means of a short MD simulation (3 runs of 50 ns) based on reductions in the root-mean-square fluctuation (RMSF) values and formation of new hydrophilic interactions around the weak spot. Finally, we designed the VL-R66G mutant by applying our strategy to scFv derived from trastuzumab. Trastuzumab scFv variants were prepared by using an Escherichia coli expression system, and the melting temperature-measured as a thermostability index-was 5 °C higher than that of the wild-type trastuzumab scFv, while the antigen-binding affinity was unchanged. Our strategy required few computational resources, and would be applicable to antibody drug discovery.

Structural, functional, and physiological properties of anti-(4-hydroxy-3-nitrophenyl)acetyl antibodies during the course of affinity maturation.

Structural and functional analyses of antibodies in the affinity maturation pathway can help us understand the molecular mechanisms of protein recognition. Using one of the haptens, (4-hydroxy-3-nitrophenyl)acetyl (NP), various monoclonal antibodies have been obtained, either at the early or late stage of immunization. The variable regions of monoclonal antibodies and their site-directed mutants can also be obtained as single-chain Fv (scFv) antibodies. The change in antigen-binding affinity and avidity of matured-type antibodies from germline-type antibodies could be evaluated based on binding kinetics and thermodynamics, proposing the antigen recognition mode. Crystal structures of a germline-type antibody, N1G9, and a matured-type antibody, C6, in complex with NP were determined, revealing different antigen-binding mode at atomic resolution. Notably, the Tyr to Gly mutation at the 95th residue of the heavy chain is critical for changing the configuration of complementarity determining region 3, which is involved in antigen binding. Furthermore, thermal stability analyses of scFv antibodies have revealed trade-off between antigen-binding affinity and thermal stability in the antigen-unbound state. To increase affinity, the stability of the variable region may be decreased, possibly due to protein architecture. The high stability of germline-type antibodies and the low stability of matured-type antibodies, which increase upon antigen binding, can be explained by the stability of antibodies required at the respective stages of immunization.

Evaluation of multi-specificity of antibody G2 using its single-chain Fv and its covalently linked antigen peptides

The antibody G2 specifically binds to four peptides with different amino acid sequences: Pep18mer, Pep8, Pep395, and PepH4P6. To elucidate the multi-specificity of G2, we generated a G2 single-chain Fv (scFv) antibody and analyzed its binding thermodynamics and kinetics to antigen peptides. Our results clearly showed that the recognition of PepH4P6 was similar to that of Pep18mer, to which G2 could obtain binding ability through the deletion of Pro95 at light chain on the affinity maturation process. The covalent linking of peptides could increase the thermal stability of G2 scFv due to intramolecular antigen binding. In the effects of respective peptides, the increased thermal stability of G2 scFv linked to Pep8 was significant, possibly due to the rapid dissociation. Binding experiments of G2 scFv linked to peptides to other peptides showed decreased association rates relative to those of antigen-free G2 scFv while the dissociation rates were almost unchanged.

Anti-mesothelin CAR-T immunotherapy in patients with ovarian cancer.

Recently, chimeric antigen receptor T cell (CAR-T) therapy has received increasing attention as an adoptive cellular immunotherapy that targets tumors. However, numerous challenges remain for the effective use of CAR-T to treat solid tumors, including ovarian cancer, which is an aggressive and metastatic cancer with a poor therapeutic response. We screened for an effective anti-MSLN single-chain Fv antibody with comparable binding activity and non-off-target properties using human phage display library. A second-generation of anti-MSLN CAR was designed and generated. We demonstrated the efficacy of our anti-MSLN CAR-T cells for ovarian cancer treatment in an in vitro experiment to kill ovarian tumor cell lines. The anti-MSLN CAR-T cells impeded MSLN-positive tumor growth concomitant with a significant increase in cytokine levels compared with the control. Then, we demonstrated the efficacy of anti-MSLN CAR-T cells in an in vivo experiment against ovarian cancer cell-derived xenografts. Furthermore, we herein report three cases with ovarian cancer who were treated with autologous anti-MSLN CAR-T cells and evaluate the safety and effectiveness of adoptive cell therapy. In this investigator-initiated clinical trials, no patients experienced cytokine release syndrome or neurological symptoms over 2 grads. Disease stabilized in two patients, with progression-free survival times of 5.8 and 4.6months. Transient CAR expression was detected in patient blood after infusion each time. The tumor partially subsided, and the patient's condition was relieved. In conclusion, this work proves the efficacy of the anti-MSLN CAR-T treatment strategy in ovarian cancer and provides preliminary data for the development of further clinical trials.

Strategies for selection and identification of rabbit single-chain Fv antibodies as ligand in affinity chromatography

We developed affinity chromatographic resins that immobilized rabbit single-chain Fv antibodies (scFvs). By biopanning using antigen-coupled multilamellar vesicles (Ag-MLVs), 152 types of original scFv clones that specifically bind to human IgG were isolated and identified. Apparent dissociation rate constants, appkoff, of six different candidates were less than 10-3 s-1 and their dissociation constants, KDs, were ranged from 5.56× 10-10 to 4.04× 10-8M. Consequently, the clones, R1-27, R2-18, and R3-26 were further investigated for use in affinity purification of human IgG. Both the clones, R1-27 and R3-26 maintained more than 40% of antigen-binding activities on the surface of affinity resins. Especially, R3-26 had a relatively high alkaline resistance. The direct separation of human IgG from 10% FBS-D-MEM by use of the column with R1-27 achieved 97.2% purity, while the column with R3-26 showed almost 100% recovery. The affinity resins at the densities between 4.32 and 15.19mg-scFv/cm3 exhibited maximum binding amount of human IgG, while the highest ligand utilization was achieved by use of the resin at approximately 9mg-scFv/cm3. The resin exhibited 7.69mg/cm3 of equilibrium binding capacity (EBC) in affinity chromatography. It was expected that the EBC of affinity resins was strongly dependent on the specific surface area as well as the pore volume of the base resin. Therefore, the strategies to develop affinity ligands will be beneficial for development of on-demand affinity columns with higher affinity/selectivity, chemical resistance, while optimization of pore size and pore volume for scFv-coupled resins will further improve the EBC.

Shelf-Life Extension of Fc-Fused Single Chain Fragment Variable Antibodies by Lyophilization.

Generation of sequence defined antibodies from universal libraries by phage display has been established over the past three decades as a robust method to cope with the increasing market demand in therapy, diagnostics and research. For applications requiring the bivalent antigen binding and an Fc part for detection, phage display generated single chain Fv (scFv) antibody fragments can rapidly be genetically fused to the Fc moiety of an IgG for the production in eukaryotic cells of antibodies with IgG-like properties. In contrast to conversion of scFv into IgG format, the conversion to scFv-Fc requires only a single cloning step, and provides significantly higher yields in transient cell culture production than IgG. ScFv-Fcs can be effective as neutralizing antibodies in vivo against a panel of pathogens and toxins. However, different scFv fragments are more heterologous in respect of stability than Fab fragments. While some scFv fragments can be made extremely stable, this may change due to few mutations, and is not predictable from the sequence of a newly selected antibody. To mitigate the necessity to assess the stability for every scFv-Fc antibody, we developed a generic lyophilization protocol to improve their shelf life. We compared long-term stability and binding activity of phage display-derived antibodies in the scFv-Fc and IgG format, either stored in liquid or lyophilized state. Conversion of scFv-Fcs into the full IgG format reduced protein degradation and aggregation, but in some cases compromised binding activity. Comparably to IgG conversion, lyophilization of scFv-Fc resulted in the preservation of the antibodies’ initial properties after storage, without any drop in affinity for any of the tested antibody clones.

Shotgun scanning glycomutagenesis: A simple and efficient strategy for constructing and characterizing neoglycoproteins

As a common protein modification, asparagine-linked (N-linked) glycosylation has the capacity to greatly influence the biological and biophysical properties of proteins. However, the routine use of glycosylation as a strategy for engineering proteins with advantageous properties is limited by our inability to construct and screen large collections of glycoproteins for cataloguing the consequences of glycan installation. To address this challenge, we describe a combinatorial strategy termed shotgun scanning glycomutagenesis in which DNA libraries encoding all possible glycosylation site variants of a given protein are constructed and subsequently expressed in glycosylation-competent bacteria, thereby enabling rapid determination of glycosylatable sites in the protein. The resulting neoglycoproteins can be readily subjected to available high-throughput assays, making it possible to systematically investigate the structural and functional consequences of glycan conjugation along a protein backbone. The utility of this approach was demonstrated with three different acceptor proteins, namely bacterial immunity protein Im7, bovine pancreatic ribonuclease A, and human anti-HER2 single-chain Fv antibody, all of which were found to tolerate N-glycan attachment at a large number of positions and with relatively high efficiency. The stability and activity of many glycovariants was measurably altered by N-linked glycans in a manner that critically depended on the precise location of the modification. Structural models suggested that affinity was improved by creating novel interfacial contacts with a glycan at the periphery of a protein-protein interface. Importantly, we anticipate that our glycomutagenesis workflow should provide access to unexplored regions of glycoprotein structural space and to custom-made neoglycoproteins with desirable properties.

Anti-EGFR Single-Chain Fv Antibody Fragment Displayed on the Surface of Ferritin H-Chain Protein Nanoparticle for Asthma Therapy.

Epidermal growth factor receptor (EGFR)-dependent signaling contributes to the pathophysiology of asthma. However, these findings have not been translated into a clinical application. We recently generated ferritin H-chain protein (FTH1)-based nanoparticles with an anti-EGFR single-chain Fv (anti-EGFR scFv) on the surface of FTH1, namely, anti-EGFR scFv-FTH1/FTH1 nanoparticles. In the present study, we found that these nanoparticles could specifically bind to EGFR-expressing cells, leading to downregulation of EGFR and mucin 5AC (MUC5AC) protein expression and growth suppression of House Dust Mite (HDM)-stimulated human bronchial epithelial 16HBE and lipopolysaccharides (LPS)-activated murine macrophage-like RAW264.7 cells. In vivo, intraperitoneal administration of anti-EGFR scFv-FTH1/FTH1 nanoparticles, but not FTH1 nanoparticles, alleviated the major pathological symptoms including airway hyperresponsiveness, airway inflammation, goblet cell hyperplasia, mucus hyperproduction, and increased release of Th2 cytokines in an allergen ovalbumin (OVA)-induced asthma mouse model. Importantly, during the dosing period these nanoparticles were safe for both heathy and asthmatic mice, and more effective in controlling airway inflammation than cetuximab, an EGFR monoclonal antibody. Altogether, our studies provide insights into the control of airway inflammation for treatment of asthma by targeting EGFR. The similar strategy can be used to fabricate scFv-based recombinant protein nanoparticles for other clinical applications.

Stabilization of single-chain Fv antibody production using recombinant Escherichia coli by DO-stat fed-batch culture employing yeast extract-enriched feeding medium

The stabilized production of anti-CRP single-chain Fv (scFv) antibody using recombinant E. coli by prolonged dissolved oxygen (DO)-stat fed-batch culture was demonstrated, focusing on the supply of nitrogen sources during fed-batch operation. The serious degradation of anti-CRP scFv by intracellular proteases was observed when the fermentation time was prolonged from 50 h to 100 h, which was presumably induced by the insufficient supply of free amino acids. To evaluate the effects of amino acid supply on the proteolysis of anti-CRP scFv, fed-batch experiments using yeast extract (YE)-enriched feeding medium, which had various blend ratios of glucose and YE (G/YE), were performed. By increasing the YE content in the feeding medium, the expression of intracellular proteases was significantly suppressed; consequently, the proteolysis of the scFv production was successfully prevented in 100 h fed-batch culture. The desired G/YE ratio to avoid proteolysis was 1.0 g-glucose/g-YE. The stabilization of scFv production accordingly resulted in the enhanced scFv production from 3.0 g/L in 50 h fed-batch culture to 4.0–5.2 g/L in 100 h fed-batch culture with more than 97% solubility which corresponded to 94.9% of increase in total scFv production by one fed-batch operation.

Single-chain Fv antibody covalently linked to antigen peptides and its structural evaluation

The monoclonal antibody G2 specifically recognizes different peptides. The single-chain Fv (scFv) antibodies of G2 covalently linked to antigen peptides, Pep18mer and Pep395, via a flexible linker were expressed in Escherichia coli in the insoluble fraction, and were solubilized using guanidine HCl, followed by refolding. We analyzed the folding thermodynamics of the refolded proteins, purified as monomers using size-exclusion chromatography (SEC). The results of the differential scanning calorimetry (DSC) showed that the thermal stabilities of antigen peptide-linked G2 scFvs were higher than those of antigen-free G2 scFv in the absence or presence of antigen peptides. The folding thermodynamics further indicated how the antigen-antibody affinity affect the intramolecular interactions. The combination of SEC and DSC experiments could confirm the folding correctness of antigen peptide-linked G2 scFvs and could be applied for “structural screening” of refolded proteins in the case that the “functional screening” like antigen binding is difficult to apply. The present method to covalently link the peptide would contribute to the stable complex structure, and would be widely applied to other antibodies recognizing peptide antigens.

Autoinduction as Means for Optimization of the Heterologous Expression of Recombinant Single-Chain Fv (scFv) Antibodies.

The monoclonal antibodies and the recombinant antibody fragments are widely used in the biotechnology studies and in medicine as a powerful therapeutic and diagnostic tool. The most commonly used recombinant antibody fragments are single-chain fragment variable (scFv) because of their small size and minimal immunogenicity while still retaining high-affinity antigen binding. A wide range of expression systems such as bacterial and eukaryotic cell systems enable the sufficient production of scFv antibodies. However, their stable expression in soluble form and correct protein folding are often insufficient. In the present study, we present the autoinduction as a key element of the optimized scheme for heterologous expression of human monoclonal scFv antibodies (clones A1 and A12) in Escherichia coli HB2151, which resulted in two-fold increase of the total protein yield in 24h.

Molecular recognition of a single-chain Fv antibody specific for GA-pyridine, an advanced glycation end-product (AGE), elucidated using biophysical techniques and synthetic antigen analogues.

Advanced glycation end-products (AGEs) are a heterogeneous group of compounds formed by non-enzymatic reaction between reducing-sugar and Arg/Lys in proteins and are involved in various diabetic complications. GA-pyridine is derived from glycolaldehyde and is one of the most cytotoxic AGEs. Here, we established a single-chain Fv (scFv) antibody against GA-pyridine, 73MuL9-scFv, and examined the details of its specificity and antigen recognition by using various techniques involving biophysics, chemical biology and structural biology. We also synthesized several compounds that differ slightly in regard to the position and number of GA-pyridine substituent groups, and revealed that GA-pyridine was specifically bound to 73MuL9-scFv. Thermodynamic analysis revealed that the association of GA-pyridine to 73MuL9-scFv was an exothermic and enthalpy driven reaction, and thus that the antigen recognition involved multiple specific interactions. Crystallographic analysis of the Fv fragment of 73MuL9-scFv revealed that several CH-π and hydrogen bond interactions took place between the Fv-fragment and GA-pyridine, which was consistent with the results of thermodynamic analysis. Further studies using 73MuL9-scFv as a tool to clarify the relevance of GA-pyridine to diabetic complications are warranted.

The Relevance of CD20 Targeting of Oncolytic Measles Virus in the Treatment of Adult Acute Lymphoblastic Leukemia

The Edmonston-B, vaccine strain of measles virus (MVNSe) has oncolytic activity in numerous preclinical tumour models with safety and some efficacy being demonstrated in phase 1 clinical trials for multiple myeloma and cutaneous non-Hodgkin lymphoma.Adult acute lymphoblastic leukemia (ALL) is an aggressive malignancy where long term survival is achieved in only 50% of patients. The GRAALL study showed that anti-CD20 monoclonal antibody, rituximab, improved outcome in ALL and the results of the UKALL14 trial are eagerly anticipated in this regard. To determine whether targeting MV to the CD20 antigen can recapitulate some of these benefits we used a MV expressing a single chain Fv antibody against CD20 as a C-terminal extension of the H glycoprotein. We also used targeted MV ‘blinded’ to the native MV receptors previously cloned by site-directed mutagenesis. The mechanisms of MV oncolysis remain incompletely understood and our experiments were particularly designed to help clarify the role of the innate immune system.First, we retrovirally transduced CD20 negative B-precursor ALL cell line NALM6 cells to express human CD20. The cells were flow sorted to give ‘high’ and ‘low’ expressing populations based on mean fluorescence intensity. Viral replication was measured by TCID50 at consecutive 24 hourly time-points and MV-N mRNA/GADPH by RQ-PCR showing similar growth curves and MV-N mRNA levels for the targeted and parent virus. Oncolytic activity was measured by trypan blue exclusion at 24, 48, 72 and 96 hours post infection (hpi). Infection of CD20 negative NALM6 cells by MVHaCD20 virus showed similar levels of killing to the parent virus MVNSe, and there was significantly greater levels of killing by MVHaCD20 of the CD20 positive cells when compared to MVNSe (p =0.03). Since it is also possible for the MVHaCD20 to enter cells via native MV receptors CD46 and CD150 (SLAM) we investigated the effect of ‘blinding’ the virus to these receptors. As expected, there was a small, non-significant reduction in efficacy in the CD46 blinded CD20 virus, MVHaCD20CD46blind, but no difference with the CD150 blinded virus, MVHaCD20SLAMblind, as CD150 is not expressed on NALM6 cells.Our previous work has shown that neutrophils are important effectors in mediating MV oncolysis. To further investigate specific mechanisms, we carried out a FACS-based neutrophil phagocytosis assay in which target cells were labelled with PKH67 and the percentage phagocytosis determined by the frequency of dual expressing CD15+/PKH67+ cells relative to the total number of CD15+ cells. These assays demonstrated that in the presence of anti-MV antibody a similar level of phagocytosis was seen 24 hpi with MVNSe, at a multiplicity of infection of 1.0, to uninfected CD20 expressing NALM6 cells in the presence of rituximab (53% c/w 58%). The percentage phagocytosis strongly correlated with the anti-MV IgG concentration from individual donors' serum (Pearson's correlation, r =0.937; p <0.001) The level of phagocytosis was significantly increased when complement was active (student t-test, p =0.008). There was no increase in cell death as indicated by DAPI staining.To further investigate the role of complement we carried out complement cytotoxicity (CDC) assays at 2hpi and 24hpi. As CD46 is a negative regulator to complement the CD46 blind viruses were included in the comparison. The results showed CDC to be a minor mechanism at 24hpi causing 5-10% cell death which was less effective than rituximab which resulted in 13-17% cell death. There was no additional effect with our ‘blinded’ viruses.In conclusion, CD20 targeted MV has increased oncolytic efficacy in-vitro compared to the native MV when infecting CD20 expressing NALM6 cells. Our results suggest that ADCP is a mechanism of MV oncolysis however a recent study in chronic lymphocytic leukemia (Valgardsdottir et al Blood 2017) has indicated that this effect is ‘trogocytosis’ whereby the neutrophils take up small amounts of the cell membrane rather than engulfing the entire leukemic cell. In our study complement was implicated both in increasing the efficacy of this mechanism and also in CDC. Although this requires further exploration in ALL, it remains that neutrophils appear to be an important effector cell of MV oncolysis in the presence of anti-MV antibody. Overall our data suggest that targeting MV to CD20 could have some efficacy in the treatment of ALL. DisclosuresFielding:Baxalta: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

Generation of a Half-Life Extended Anti-CD19 BiTE® Antibody Construct Compatible with Once-Weekly Dosing for Treatment of CD19-Positive Malignancies

CD19 is an immunoglobulin family member expressed ubiquitously on B cell lineage cells, from the pre-B cell to the mature B cell stage, until downregulation during terminal differentiation into plasma cells. CD19 is important for B cell activation, maturation and proliferation, and is widely expressed on the majority of B cell neoplasms.The T cell engaging bispecific antibody construct (BiTE®) blinatumomab, which targets CD19, is approved for the treatment of relapsed or refractory B cell precursor acute lymphoblastic leukemia in adults and children in the US and has shown great promise for treating other CD19-positive hematological malignancies. The short serum half-life of blinatumomab necessitates administration by continuous IV infusion to maintain therapeutic serum concentrations. To extend the half-life and allow for a more convenient administration, a next generation BiTE® antibody construct designated CD19 HLE BiTE® has been generated. The CD19 HLE BiTE® comprises a new CD19- and CD3-targeting tandem single-chain Fv antibody fused to an Fc domain.The biological properties of CD19 HLE BiTE® were evaluated in vitro and in vivo . CD19 HLE BiTE® binds with high affinity to both human and non-human primate (NHP) CD19 as well as CD3. In vitro experiments demonstrated that CD19 HLE BiTE® can recruit and activate human and NHP T cells, mediating redirected lysis of CD19-expressing cells at half-maximal effective concentrations in the low picomolar range. The fusion to an Fc domain resulted in a serum half-life of 210 hours following a single intravenous administration of a 5 µg/kg dose in NHP. In a repeat-dose study CD19 HLE BiTE® caused robust depletion of circulating B cells, B lineage bone marrow cells, and peripheral lymphoid organ B cells in NHPs, whilst no overt signs of toxicity were observed in clinical and laboratory examinations of the animals.These results demonstrate that CD19 HLE BiTE® effectively eliminated CD19-positive cells in vitro and in vivo, and may be suitable for once weekly dosing in patients with CD19-positive malignancies. DisclosuresLorenczewski:Amgen: Employment, Equity Ownership. Friedrich:Amgen Research (Munich) GmbH: Employment, Equity Ownership. Kischel:AMGEN: Employment. Dahlhoff:Amgen Research (Munich): Employment, Equity Ownership. Anlahr:Amgen: Employment, Equity Ownership. Balazs:Amgen Inc.: Employment, Equity Ownership. Rock:Amgen, Inc.: Employment, Equity Ownership. Boyle:Amgen: Employment. Goldstein:Amgen: Employment. Coxon:Amgen, Inc.: Employment. Chapman-Arvedson:Amgen, Inc.: Employment, Equity Ownership.

NanoLuc luciferase as a suitable fusion partner of recombinant antibody fragments for developing sensitive luminescent immunoassays

Enzyme-linked immunosorbent assays (ELISAs) are essential for monitoring various biomarkers. Competitive and noncompetitive (sandwich) assay formats are used to determine hapten and macromolecule levels, respectively. Both formats require more sensitive detection of reporter enzymes for greater assay sensitivities. We previously reported the utility of wild-type Gaussia luciferase (wtGLuc) as a fusion partner with antibody single-chain Fv fragments (scFvs) for developing sensitive luminescent ELISAs. Here, we evaluated utility of NanoLuc luciferase (NLuc), a recently developed luciferase, as fusion partner with scFvs from the view of comparison with wtGLuc and a mutant of alkaline phosphatase (ALP’). Thyroxine (T4) and T4-labeled albumin were chosen as model haptenic and macromolecular antigens, respectively. An in-house-prepared anti-T4 scFv was fused with NLuc, wtGLuc, or ALP’. The scFv−NLuc fusion protein showed 47-fold and 29-fold lower limit of detection [LOD; 59 zmol (per assay)] than the wtGLuc- and ALP’-fusions, respectively. In a competitive T4 ELISA, the NLuc-fusion showed 9.3- and 6.3-fold lower LOD, (0.67 pg) than the wtGLuc- and ALP’-fusions, respectively, with a higher specificity in clinical applications. A typical colorimetric ELISA using a peroxidase-labeled second antibody showed 70-fold higher LOD than NLuc-based ELISA. Another advantage of the NLuc-fusion was shown in the sandwich assays; the LOD of T4-labeled albumin (5.0 fmol) was >6-fold lower than that of the other luminescent ELISAs. In an additional sandwich assay developed to count bacteriophage particles, NLuc enabled more sensitive determination than wtGLuc, whereas ALP’ showed nearly equivalent performance. Its slowest alteration rate for light intensity after starting the enzyme reaction should enable robust batch-by-batch assay operations. Thus, we concluded that scFv−NLuc fusions serve as suitable probes in various types of immunoassays and may facilitate higher sensitivities with practical specificities.