Intracellular expression of Ab fragments has been efficiently used to inactivate therapeutic targets, oncogene products, and to induce viral resistance in plants. Ab fragments expressed in the appropriate cell compartment may also help to elucidate the functions of a protein of interest. We report in this study the successful targeting of the protein tyrosine kinase Syk in the RBL-2H3 rat basophilic leukemia cell line. We isolated from a phage display library human single-chain variable fragments (scFv) directed against the portion of Syk containing the Src homology 2 domains and the linker region that separates them. Among them, two scFv named G4G11 and G4E4 exhibited the best binding to Syk in vivo in a yeast two-hybrid selection system. Stable transfectants of RBL-2H3 cells expressing cytosolic G4G11 and G4E4 were established. Immunoprecipitation experiments showed that intracellular G4G11 and G4E4 bind to Syk, but do not inhibit the activation of Syk following FcepsilonRI aggregation, ...
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Antibodies that recognize antigens restricted to leukemia, lymphoma, and normal hematopoietic cells represent a unique opportunity to develop therapeutics, because they have the potential for relatively selective treatment of these diseases. Antibodies that recognize the CD19 antigen found on normal and malignant B cells, but not on stem cells, have been used to develop immunoconjugates. However, these conjugates are large and might be suboptimal in tumor penetration when compared to molecules using smaller single chain Fv (scFv) antibody fragments. scFv has the advantage of being a molecularly engineered homogeneous molecule. In this report, we demonstrate the cloning, expression, and binding of three anti-CD19 antibodies as scFvs. All three scFvs were successfully cloned and expressed. FVS191, derived from cell line B43, and FVS192, derived from SJ25C1, were properly refolded and bound CD19 antigen in FACS competition assays. These anti-CD19 scFv should be useful in the further development of ...
Antibody fragments selected from large combinatorial libraries have numerous applications in diagnosis and therapy. Most existing antibody repertoires are derived from human immunoglobulin genes. Genes from other species can, however, also be used. Because of the way in which gene conversion introduces diversity, the naïve antibody repertoire of the chicken can easily be accessed using only two sets of primers. With in vitro diagnostic applications in mind, we have constructed a large library of recombinant filamentous bacteriophages displaying single chain antibody fragments derived from combinatorial pairings of chicken variable heavy and light chains. Synthetically randomised complementarity determining regions are included in some of the heavy chains. Single chain antibody fragments that recognise haptens, proteins and virus particles were selected from this repertoire. Affinities of three different antibody fragments were determined using surface plasmon resonance. Two were in the low nanomolar
Antibodies are applied in both basic research and diagnostics, and represent an increasingly important class of therapeutics. Monoclonal antibodies is the largest and fastest growing class of protein pharmaceuticals [1]. In the discovery and development of these antibodies, antibody fragments such as the antigen binding fragment (Fab), the single-chain variable fragment (scFv), and the single variable domains (VH and VL, collectively sdAb) are often employed [2]. The present recombinant antibody discovery platforms, such as ribosome and phage display [3], enable easy screening and selection of antibody fragments against virtually any antigen [4]. Based on the initial screening or selection, a number of candidate antibodies are obtained [3]. Often, these recombinant antibody candidates can be expressed in E. coli. However, although the field of recombinant protein expression in E. coli is developed and expanded [5, 6], the codon usage and folding dynamics of some recombinant antibody clones are ...
Serum half-life of IgG is controlled by the neonatal Fc receptor (FcRn) that interacts with the IgG Fc region and may be increased or decreased as a function of altered FcRn binding. Preclinical evaluations of modified IgGs are frequently carried out in mice, but such IgGs may bind differently to mouse and human FcRn (mFcRn and hFcRn). Here, we report a detailed characterization of a matched set of mouse-human chimeric T84.66 scFv-Fc variants with specificity for the tumor carcinoembryonic antigen and mutations in the FcRn-binding site. Binding to soluble mFcRn and hFcRn was measured using in vitro assays, and the results were compared with blood clearance in vivo in normal (mFcRn bearing) and hFcRn transgenic mice. All variants bound better to mFcRn than to hFcRn. The loss of affinity varied among the mutants, however, and also the hierarchy of binding differed depending on the receptor. The mutations had no major impact on binding to the classical Fcγ receptors. Importantly, the trend of blood
Background: Aborted myocardial infarction defined by , 2 fold elevation in biomarkers occurs in 13-14% of STEMI patients (pts). Its relationship with other metrics of infarct size and LV function defined by MRI is unclear.. Methods: Of the 5745 pts enrolled in APEX-AMI (STEMI pts undergoing primary PCI , 6hrs after symptom onset) 73 were part of a substudy including MRIs 3-5 days after randomization.. Results: Aborted MI (ST elevation/LBBB on presenting ECG with subsequent peak CK/CKMB,2 X ULN) was observed in 11% (437/3938) overall and in 19% (14/73) pts in the MRI study. Aborted MI pts tended to be older (median 62 (59-73) yrs vs. 57 (46-67); p=0.081) and have complete ST-elevation resolution 30 minutes post-PCI (≥70% resolution: 64% versus 32% p=0.076) than other MI patients. MRI revealed that aborted MI pts had smaller infarct size (4.7 vs. 14.9 LV%, p50% wall thickness) p=0.01. (See Figure). Conclusions: In APEX-AMI, 11% of all pts had an aborted MI by biomarker evaluation, which was ...
Chimaeric toxins have considerable therapeutic potential to treat various malignancies. We have previously used the fungal ribonucleolytic toxin restrictocin to make chimaeric toxins in which the ligand was fused at either the N-terminus or the C-terminus of the toxin. Chimaeric toxins containing ligand at the C-terminus of restrictocin were shown to be more active than those having ligand at the N-terminus of the toxin. Here we describe the further engineering of restrictocin-based chimaeric toxins, anti-TFR(scFv)-restrictocin and restrictocin-anti-TFR(scFv), containing restrictocin and a single chain fragment variable (scFv) of a monoclonal antibody directed at the human transferrin receptor (TFR), to enhance their cell-killing activity. To promote the independent folding of the two proteins in the chimaeric toxin, a linear flexible peptide, Gly-Gly-Gly-Gly-Ser, was inserted between the toxin and the ligand to generate restrictocin-linker-anti-TFR(scFv) and anti-TFR(scFv)-linker-restrictocin. ...
Methods The authors chose type II collagen (CII) as a target as it is uniquely present in cartilage. In the arthritic joint, CII is damaged by reactive oxidant species (ROS) generated in the inflammation process. The authors used ROS-modified CII to select a human single chain fragment variable (scFv) specific to ROS-CII.. In order to target therapeutic proteins to the inflamed joints, the authors have fused anti-ROS-CII scFv to anti-inflammatory proteins via an MMP cleavage site linker. MMPs are up-regulated in arthritis, and therefore when the fusion protein is localised to inflamed areas by the scFv, the therapeutic is liberated, and is free to engage its target.. ...
In the present study, we describe the synthesis and characterization of new generation of cancer-targeting magnetic nanoprobes: superparamagnetic iron oxide nanoparticles (SPIONs) coated with polyethylene glycol (PEG) shell functionalized with recombinant anti-HER2 single chain fragment variable (scFv) of Trastuzumab antibody. An anti-HER2 scFv with terminal cysteine (scFv 4D5-Cys) has been rationally engineered in order to favor its orientation- and site-directed covalent conjugation to the polymeric surface of PEGylated SPIONs. Optimization of scFv and nanoparticles production allowed to obtain well-characterized SPIONs-PEG-scFv nanoparticles carrying ∼7 fragments per nanoparticle, having a hydrodynamic diameter of ca. 86 nm and nearly neutral surface. The nanoprobes-scFv capability to recognize the HER2 protein has been confirmed by enzyme-linked immunosorbent assay (ELISA). Compared to non-targeted PEGylated SPIONs, the SPIONs-PEG-scFv nanoprobes showed an enhanced binding to HER2-overexpressing
Breast cancer is a major cause of cancer mortality, second only to lung cancer as a cause of cancer death in women. The five-year survival rate for localized breast cancer has increased from 80 percent in the 1950s to 98 percent today. However, the mortality rate in the most advanced forms remains unsatisfactory. Indeed, the extensive use of mammography within screening programs has led to cancers being detected earlier, when early treatments may be more effective. A greater understanding of the molecular biology and genetic expression of breast cancer has therefore led to new pre-surgical and post-surgical treatments, including hormone modulators and monoclonal antibodies. Many of these agents have led to decreased mortality and disease recurrence.. F16 is a human recombinant antibody fragment in the scFv (single chain Fragment variable) format that is directed against tenascin C, an angiogenesis marker common to most solid tumors independent of the tumor type. ScFv(F16) selectively localizes ...
The cellular heterogeneity seen in tumors, with subpopulations of cells capable of resisting different treatments, renders single-treatment regimens generally ineffective. Accordingly, there is a great need to increase the repertoire of drug treatments from which combinations may be selected to efficiently target sets of pathological processes, while suppressing the emergence of resistance mutations. In this regard, members of the TGF- signaling pathway may furnish new, valuable therapeutic targets. In the present work, we developed in situ proximity ligation assays (isPLA) to monitor the state of the TGF- signaling pathway. Moreover, we extended the range of suitable affinity reagents for this analysis by developing a set of in-vitro-derived human antibody fragments (single chain fragment variable, scFv) that bind SMAD2 (Mothers against decapentaplegic 2), 3, 4, and 7 using phage display. These four proteins are all intracellular mediators of TGF- signaling. We also developed an scFv specific ...
Organophosphorus pesticides (OPs), a kind of important agricultural production material, have been playing an important role in pest control and food production increase. But more and more attention has been paid on the serious environmental problems because of the excess use of OPs. The quick and accurate detection of residues, especially the multi-residues of OPs is always a research focus. In recent years, a great deal of research and exploration has been made on the multi-analyte immunoassay of pesticides based on the spe-cific recognition between antigen and antibody. This article reviewed the recent advances on the mul-ti-residue determination of organophosphorus insecticides, including the progresses of mixed antibodies, antibodies against multi-antigenic determinants, antibodies against common moiety and single chain fragment variable (ScFv) antibodies, and discussed the existed problems and future research.
Alberini, Isabella, Del Tordello, Elena, Fasolo, Alba, Temperton, Nigel J., Galli, Grazia, Gentile, Chiara, Montomoli, Emanuele, Hilbert, Anne K., Banzhoff, Angelika, Del Giudice, Giuseppe, Donnelly, John J., Rappuoli, Rino and Capecchi, Barbara (2009) Pseudoparticle neutralization is a reliable assay to measure immunity and cross-reactivity to H5N1 influenza viruses. Vaccine, 27 (43). pp. 5998-6003. ISSN 0264-410X (doi:10.1016/j.vaccine.2009.07.079) Ascione, Alessandro, Capecchi, Barbara, Campitelli, Laura, Imperiale, Valentina, Flegoa, Michela, Zamboni, Silvia, Gellini, Mara, Alberini, Isabella, Pittiglio, Eliana, Donatelli, Isabella, Temperton, Nigel J. and Cianfriglia, Maurizio (2009) Human monoclonal antibodies in single chain fragment variable format with potent neutralization activity against influenza virus H5N1. Antiviral Research, 83 (3). pp. 238-244. ISSN 0166-3542 (doi:10.1016/j.antiviral.2009.05.005) Oha, Sawyin, Selleck, Paul, Temperton, Nigel J., Chan, Paul K.S., Capecchi, ...
The V3 loop in the envelope (Env) of HIV-1 is one of the major targets of neutralizing antibodies. However, this antigen is hidden inside the Env trimer in most isolates and is fully exposed only during CD4-gp120 interaction. Thus, primary HIV-1 isolates are relatively resistant to anti-V3 antibodies because IgG is too large to access the V3 loop. To overcome this obstacle, we constructed single-chain variable fragments (scFvs) from anti-V3 monoclonal antibodies 0.5γ, 5G2, and 16G6. Enhanced neutralization by 0.5γ and 5G2 scFvs was observed in strains resistant to their IgG counterparts. Neutralization coverage by 0.5γ scFv reached up to 90% of the tested viruses (tier 2 and 3 classes). The temperature-regulated neutralization assay revealed that extensive cross-neutralization of 0.5γ scFv can be explained by post-attachment neutralization. Neutralization assay involving viruses carrying an inter-subunit disulfide bond (SOS virus) showed that the neutralization-susceptible timeframe after ...
TY - JOUR. T1 - Properties of a single-chain antibody containing different linker peptides. AU - Alfthan, Kaija. AU - Takkinen, Kristiina. AU - Sizmann, Dorothea. AU - Söderlund, Hans. AU - Teeri, Tuula. N1 - Project code: BEL2026. PY - 1995. Y1 - 1995. N2 - Single-chain antibodies were constructed using six different linker peptides to join the VH and VL domains of an anti-2-phenyloxazolone (Ox) antibody. Four of the linker peptides originated from the interdomain linker region of the fungal cellulase CBHI and consisted of 28, 11, six and two amino acid residues. The two other linker peptides used were the (GGGGS)3 linker with 15 amino acid residues and a modified IgG2b hinge peptide with 22 residues. Proteolytic stability and Ox binding properties of the six different scFv derivatives produced in Escherichia coli were investigated and compared with those of the corresponding Fv fragment containing no joining peptide between the V domains. The hapten binding properties of different antibody ...
Gene therapy can be defined as the introduction of nucleic acids into cells with the purpose of altering the course of a medical condition or disease. In many clinical settings, efficient and successful gene therapy relies on the delivery of genes to specific cells in the human body. Such specific delivery can only be achieved through the design of vehicles/vectors that are able to recognize the target cell � targeting. Adeno-associated virus type 2, a non-pathogenic human virus, has received an increased amount of attention as a vector for gene therapy since it was first cloned into a bacterial plasmid in 1982. Although the first attempt to construct an AAV-2 targeting vector made use of a single chain antibody fragment, it was the insertion of small peptide ligands into the AAV-2 capsid that marked the beginning of AAV-2 vector targeting. Sequence alignment of AAV-2 with CPV led to the discovery of amino acid position 587. Several reports have shown that small peptide ligands, once inserted ...
The primary endpoint of death or MI at 30 days occurred in 15.2% of the pexelizumab group and 16.3% of the placebo group (relative risk reduction [RRR] 6.7%, p=0.201). Among the components of the composite, MI occurred in 12.6% of the pexelizumab group and 13.3% of the placebo group, (p=0.311) and death in 3.8% and 4.6%, respectively (RRR 17%, p=0.177). Results of the primary endpoint were similar in the different risk factor subgroups. Serious adverse events did not differ by treatment group (31.2% for pexelizumab and 32.5% for placebo). Sepsis occurred less often in the pexelizumab group (2.1% for 3.1%).. ...
JPT Peptide Technologies is a DIN ISO 9001:2015 certified and GCLP compliant integrated provider of innovative peptide based catalog products and custom services.
The continuously growing natural killer (NK) cell line NK-92 is highly cytotoxic against malignant cells of various origin without affecting normal human cells. Based on this selectivity, the potential of NK-92 cells for adoptive therapy is currently being investigated in phase I clinical studies. To further enhance the antitumoral activity of NK-92 cells and expand the range of tumor entities suitable for NK-92-based therapies, here by transduction with retroviral vectors we have generated genetically modified NK-92 cells expressing chimeric antigen receptors specific either for the tumor-associated ErbB2 (HER2/neu) antigen or the human Epithelial Cell Adhesion Molecule (Ep-CAM). Both antigens are overexpressed by many tumors of epithelial origin. The chimeric antigen receptors consist of either the ErbB2 specific scFv(FRP5) antibody fragment or the Ep-CAM specific scFv(MOC31), a flexible hinge region derived from CD8, and transmembrane and intracellular regions of the CD3 zeta chain. ...
Recombinant antibody (scFv-Fc) production services include: molecular construction of recombinant scFv-Fc antibodies; transient production of recombinant antibodies; purification of recombinant antibodies.
Single chain variable domain (Fv) fragments (scFv) are powerful tools in research and clinical settings, owing to better pharmacokinetic properties compared to the parent monoclonal antibodies and the relative ease of producing them in large quantities, at low cost. Though they offer several advantages, they suffer from lower binding affinity and rapid clearance from circulation, which limits their therapeutic potential. However, these fragments can be genetically modified to enhance desirable properties, such as multivalency, high target retention and slower blood clearance, and as such, a variety of scFv formats have been generated. ScFvs can be administered by systemic injection for diagnostic and therapeutic purposes. They can be expressed in vivo through viral vectors in instances where large infection rates and sustenance of high levels of the antibody is required. ScFvs have found applications as tools for in vivo loss-of-function studies and inactivation of specific protein domains, diagnostic
Single chain variable domain (Fv) fragments (scFv) are powerful tools in research and clinical settings, owing to better pharmacokinetic properties compared to the parent monoclonal antibodies and the relative ease of producing them in large quantities, at low cost. Though they offer several advantages, they suffer from lower binding affinity and rapid clearance from circulation, which limits their therapeutic potential. However, these fragments can be genetically modified to enhance desirable properties, such as multivalency, high target retention and slower blood clearance, and as such, a variety of scFv formats have been generated. ScFvs can be administered by systemic injection for diagnostic and therapeutic purposes. They can be expressed in vivo through viral vectors in instances where large infection rates and sustenance of high levels of the antibody is required. ScFvs have found applications as tools for in vivo loss-of-function studies and inactivation of specific protein domains, diagnostic
The overall goal of this thesis was the development of fast immunological methods for the detection of mycotoxins in crops and of polycyclic aromatic hydrocarbons (PAHs) in water intended for human consumption. The main focus of the qualitative and quantitative determination of mycotoxins was on the development of a new detection method. Therefore a chemiluminescence microarray for the detection of aflatoxins and ochratoxin A in crops was successfully produced and validated. For the detection of PAHs in drinking water the emphasis was on the development of new affine antibodies. In addition to highly affine monoclonal antibodies, new recombinant antibodies (single chain variable fragments) were developed for the first time. « ...
Introduction: ScFv(FRP5)-ETA is a recombinant antibody toxin with binding specificity for ErbB2 (HER2). It consists of an N-terminal single-chain antibody fragment (scFv), genetically linked to truncated Pseudomonas exotoxin A (ETA). Potent antitumoral activity of scFv(FRP5)-ETA against ErbB2-overexpressing tumor cells was previously demonstrated in vitro and in animal models. Here we report the first systemic application of scFv(FRP5)-ETA in human cancer patients. Methods: We have performed a phase I dose-finding study, with the objective to assess the maximum tolerated dose and the dose-limiting toxicity of intravenously injected scFv(FRP5)-ETA. Eighteen patients suffering from ErbB2-expressing metastatic breast cancers, prostate cancers, head and neck cancer, non small cell lung cancer, or transitional cell carcinoma were treated. Dose levels of 2, 4, 10, 12.5, and 20 μg/kg scFv(FRP5)-ETA were administered as five daily infusions each for two consecutive weeks. Results: No hematologic, ...
A soluble human single-chain T cell receptor (TCR) having the structure: Vα2-L-Vβ or Vβ-L-Vα2, wherein L is a linker peptide that links Vβ with Vα, Vβ is a TCR variable β region, and Vα2 is a TCR variable α region of the family 2 is provided. The provided scTCR is useful for many purposes, including the treatment of cancer, viral diseases and autoimmune diseases.
The discovery of an HIV-1 cure remains a medical challenge because the virus rebounds quickly after the cessation of combination antiretroviral therapy (cART). Here, we investigate the potential of an engineered tandem bispecific broadly neutralizing antibody (bs-bnAb) as an innovative product for HIV-1 prophylactic and therapeutic interventions. We discovered that by preserving 2 single-chain variable fragment (scFv) binding domains of each parental bnAb, a single gene-encoded tandem bs-bnAb, BiIA-SG, displayed substantially improved breadth and potency. BiIA-SG neutralized all 124 HIV-1-pseudotyped viruses tested, including global subtypes/recombinant forms, transmitted/founder viruses, variants not susceptible to parental bnAbs and to many other bnAbs with an average IC50 value of 0.073 μg/ml (range , 0.001-1.03 μg/ml). In humanized mice, an injection of BiIA-SG conferred sterile protection when administered prior to challenges with diverse live HIV-1 stains. Moreover, whereas BiIA-SG ...
The adhesion-molecule group of integrins share the property to change conformation, on cell activation, thereby exposing their receptor ligand-binding pocket.2 This provides the unique possibility to design agents that specifically block only the activated receptor. We designed a strategy to develop human single-chain antibodies specific for integrins in the activated conformation that could be used for the therapy of the many diseases in which integrins play a major role, such as inflammation, cancer, and thrombosis.18 The platelet integrin GPIIb/IIIa is of pivotal importance for coagulation and thrombosis. Its conformation-unspecific blockade has been one of the major advances in antithrombotic therapy of recent years, although unexpected limitations and side effects, in particular with orally applicable blockers, have damped the original enthusiasm. We have taken advantage of the existence of different conformational states of GPIIb/IIIa to develop an alternative, unique strategy that targets ...
In this study of diverse antibody libraries, the authors demonstrate that by targeting different residues of the same antibody gene, it is possible to create sublibraries with distinct profiles in terms of the number and affinities of the library clones against different-sized antigens. Apparent affinities for anti-streptavidin clones were measured on the BLItz system using Streptavidin biosensors.
This trial assessed the efficacy and tolerability of pexelizumab in patients undergoing coronary artery bypass grafting (CABG) with cardiopulmonary bypass
Principal Investigator:SUDA Yasuo, Project Period (FY):2011-04-01 - 2014-03-31, Research Category:Grant-in-Aid for Scientific Research (B), Section:一般, Research Field:Tumor diagnosis
Impaired apoptosis contributes to cancer development and resistance towards chemotherapy, since apoptosis normally eliminates cells with damaged DNA or increased malignant potential. The increased resistance towards cell death often limits therapeutic options in the clinic and is one major problemin current tumor therapy. Different approaches, which have been described so far intend to lower the apoptotic threshold in order to eliminate chemoresistant cancer cells. In the first part of this thesis the anti-tumor potential of the bispecific 4625 oligonucleotide was investigated in combination with chemotherapeutic drugs in vitro and in vivo. The second part describes the anti tumor activity of the recombinant Ep-CAM specific scFv immunotoxin 4D5MOC-B-ETA in vitro and in nude mice. Bcl-2 and Bcl-xL are inhibitors of apoptosis frequently overexpressed in malignant tumor cells. Downregulation of either Bcl-2 or Bcl-xL lowers the apoptotic threshold and tumor cells undergo apoptosis. The 4625 ...
Impaired apoptosis contributes to cancer development and resistance towards chemotherapy, since apoptosis normally eliminates cells with damaged DNA or increased malignant potential. The increased resistance towards cell death often limits therapeutic options in the clinic and is one major problemin current tumor therapy. Different approaches, which have been described so far intend to lower the apoptotic threshold in order to eliminate chemoresistant cancer cells. In the first part of this thesis the anti-tumor potential of the bispecific 4625 oligonucleotide was investigated in combination with chemotherapeutic drugs in vitro and in vivo. The second part describes the anti tumor activity of the recombinant Ep-CAM specific scFv immunotoxin 4D5MOC-B-ETA in vitro and in nude mice. Bcl-2 and Bcl-xL are inhibitors of apoptosis frequently overexpressed in malignant tumor cells. Downregulation of either Bcl-2 or Bcl-xL lowers the apoptotic threshold and tumor cells undergo apoptosis. The 4625 ...
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Effective neutralization of different strains of HIV virus is shown by neutralizing antibodies against HIV-1 envelope glycoproteins. It has been demonstrated that gp41 is involved in virus mediated membrane fusion which results in HIV-entry into target cells. Recombinant single chain antibodies (scFvs) with high specificity and high affinity properties have been identified as useful agents in anti-viral targeted therapy. In this study we selected specific scFvs against a conserved neutralizing epitope of gp41. Four rounds of panning were performed to select the specific clones. The reactivity of the selected scFvs against the corresponding epitope was tested in ELISA. Results demonstrated that the specific clones were selected with the frequencies of 65% and 30%. The ELISA evaluation demonstrated significant higher OD of scFvs in reaction with the corresponding epitope than the negative control. Specific scFvs against conserved neutralizing epitope of gp41 of HIV has the potential to be ...
Recombinant Antibody Network (RAN) is an international tri-center consortium that develops renewable and highly validated recombinant antibodies.
Recombinant Antibody Network (RAN) is an international tri-center consortium that develops renewable and highly validated recombinant antibodies.
Fast and cost-effective production of recombinant antibodies in up to gram scale by our expert team using Icosagens patented QMCF technology
We and others have reported that fusion proteins comprising CD20-targeting MAbs and IFNα are more effective against NHL compared with combinations of MAb and IFNα in xenograft and syngeneic mouse models, indicating that MAb-IFNα could overcome the toxicity and pharmacokinetic limitations associated with IFNα (10, 16). Although CD20 is an attractive candidate for targeted MAb-IFNα therapy of B-cell lymphoma, its expression is largely limited to malignancies of this lineage, with some individuals exhibiting low antigen density. Here, we report the first bispecific immunocytokine, 20-C2-2b, which specifically targets IFNα2b to both CD20 and HLA-DR, thus potentially expanding the hematopoietic tumor types amenable to this immunocytokine therapy.. Anti-HLA-DR MAbs efficiently induce apoptosis, which is mediated by direct signaling without the requirement of additional crosslinking, and are also potent inducers of ADCC and CDC (11, 15, 17). Where ADCC may enhance therapeutic potential, CDC is ...
Transgenic mosquitoes resistant to malaria parasites are being developed to test the hypothesis that they may be used to control disease transmission. We have developed an effector portion of an antiparasite gene that can be used to test malaria resistance in transgenic mosquitoes. Mouse monoclonal antibodies that recognize the circumsporozoite protein of Plasmodium gallinaceum can block sporozoite invasion of Aedes aegypti salivary glands. An anti-circumsporozoite monoclonal antibody, N2H6D5, whose corresponding heavy- and light-chain gene variable regions were engineered as a single-chain antibody construct, binds to P. gallinaceum sporozoites and prevents infection of Ae. aegypti salivary glands when expressed from a Sindbis virus. Mean intensities of sporozoite infections of salivary glands in mosquitoes expressing N2scFv were reduced as much as 99.9% when compared to controls.
CEA is a tumor-associated antigen abundantly expressed on several cancer types, including those naturally refractory to chemotherapy. The selection and characterization of human anti-CEA single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer monoclonal antibodies designed for optimal blood clearance and tumor penetration. The human MA39 scFv, selected for its ability to recognize a CEA epitope expressed on human colon carcinomas, was first isolated from a large semi-synthetic ETH-2 antibody phage library, panned on human purified CEA protein. Subsequently, by in vitro mutagenesis of a gene encoding for the scFv MA39, a new library was established, and new scFv antibodies with improved affinity towards the CEA cognate epitope were selected and characterized. The scFv MA39 antibody was affinity-maturated by in vitro mutagenesis and the new scFv clone, E8, was isolated, typed for CEA family member recognition and its CEACAM1, 3 and 5 shared epitope characterized
TY - JOUR. T1 - Intramuscular delivery of a single chain antibody gene prevents brain Aβ deposition and cognitive impairment in a mouse model of Alzheimers disease. AU - Wang, Yan Jiang. AU - Gao, Chang. AU - Yang, Miao. AU - Liu, Xiao-Hong. AU - Sun, Annie. AU - Pollard, Anthony. AU - Dong, X. AU - Wu, Xiao-Bing. AU - Zhong, Jin Hua. AU - Zhou, Hua-Dong. AU - Zhou, Xin-Fu. PY - 2010/11. Y1 - 2010/11. N2 - Anti-beta-amyloid (Aβ) immunotherapy is effective in removing brain Aβ, but has shown to be associated with detrimental effects. We have demonstrated that Adeno-associated virus (AAV)-mediated delivery of an anti-Aβ single chain antibody (scFv) gene was effective in clearing brain Aβ without eliciting any inflammatory side effects in old APPSwe/PS1dE9 transgenic mice. In the present study, we tested the efficacy and safety of intramuscular delivery of the scFv gene in preventing brain Aβ deposition. The scFv gene was intramuscularly delivered to APPSwe/PS1dE9 transgenic mice at 3months ...
Celiac disease (CD) is a chronic, small intestinal inflammatory disease mediated by dietary gluten and related prolamins. The only current therapeutic option is maintenance of a strict life-long gluten-free diet, which implies substantial burden for CD patients. Different treatment regimes might be feasible, including masking of toxic celiac peptides with blocking antibodies or fragments thereof. The objective of this study was therefore to select and produce a recombinant avian single-chain fragment variable (scFv) directed against peptic-tryptic digested gliadin (PT-Gliadin) and related celiac toxic entities. Gluten-free raised chicken of same age were immunized with PT-Gliadin. Chicken splenic lymphocytes, selected with antigen-coated magnetic beads, served as RNA source for the generation of cDNA. Chicken VH and VL genes were amplified from the cDNA by PCR to generate full-length scFv constructs consisting of VH and VL fragments joined by a linker sequence. ScFv constructs were ligated in a
article{c58957be-5df1-4efc-b433-f02e50dc2d3f, abstract = {CD40 plays a central regulatory role in the immune system and antibodies able to modulate CD40 signalling may consequently have a potential in immunotherapy, in particular for treatment of lymphomas and autoimmune disease like multiple sclerosis. As a first step to achieve this goal, we describe the selection and characterization of a novel set of fully human anti-CD40 antibody fragments (scFv) from a phage display library (n-CoDeR). In order to determine their biological potential, these antibody fragments have been analysed for their ability to promote B-cell activation, rescue from apoptosis and to block the CD40-CD40 ligand (CD40L) interaction. The selected cohort of human scFv could be subcategorized, each expressing a distinct functional signature. Thus scFv were generated that induced B-cell proliferation, rescued B cells from apoptosis and blocked the CD40-CD40L interaction to different extents. In particular, one of the scFv ...
Based on years of experience in phage display technology, Creative Biolabs now is able to provide services for the construction and screening of immune antibody library.. With years of research and development in the past decades, phage display has been a powerful technology to display millions or even billions of different peptides or proteins. It is now a common choice for the studies of protein-protein, protein-peptide and protein-DNA interactions. Among all the applications of phage display technology, one of the most successful ones is the isolation of monoclonal antibodies utilizing large capacity phage antibody libraries.. Thanks to the rapid development of this technology, it has become a reliable tool for the production of monoclonal antibody with high specificity and affinity. "Compared with conventional hybridoma technology, the generation of immune antibody libraries is not limited by the requirement of fusion partners. This expands the possibility to develop monoclonal antibodies ...
Sialic acids are a family of acidic monosaccharides that typically reside as terminal moieties on N- and O-linked glycans. These sugars are actively involved in a plethora of biological phenomena, ranging from cell-cell adhesion and recognition, intracellular signalling events, pathogen attack, viral infection and inflammatory disease. In addition, many cancer-related antigens contain terminal sialic acids or altered sialylation patterns. The identification of sialic acid-specific antibodies is important in the fields of basic research and diagnostics. Therefore the primary objective of this thesis was the generation and characterisation of recombinant antisialic acid antibodies.A single-chain antibody fragment (scFv) library was constructed from a chicken that was immunised with a novel synthetic sialic acid protein-conjugate (Neu5Gchumanserum albumin). The scFv library was biopanned using a second novel sialoneoglycoconjugate(Neu5Gc-bovine serum albumin). Anti-sialic scFvs were isolated ...
Heparan sulfate (HS) binds and modulates the transport and activity of a large repertoire of regulatory proteins. The HS phage display antibodies are powerful tools for the analysis of native HS structure in situ; however, their epitopes are not well defined. Analysis of the binding specificities of a set of HS antibodies by competitive binding assays with well defined chemically modified heparins demonstrates that O-sulfates are essential for binding; however, increasing sulfation does not necessarily correlate with increased antibody reactivity. IC50 values for competition with double modified heparins were not predictable from IC50 values with corresponding singly modified heparins. Binding assays and immunohistochemistry revealed that individual antibodies recognize distinct epitopes and that these are not single linear sequences but families of structurally similar motifs in which subtle variations in sulfation and conformation modify the affinity of interaction. Modeling of the antibodies ...
1A14: COMPLEX BETWEEN NC10 ANTI-INFLUENZA VIRUS NEURAMINIDASE SINGLE CHAIN ANTIBODY WITH A 5 RESIDUE LINKER AND INFLUENZA VIRUS NEURAMINIDASE
Bispecific antibodies are a versatile class of targeted therapeutics designed to bind two different sites, which can be located on a single antigen or on two antigens. Although bispecific antibodies were conceptualized ~60 years ago, various challenges associated with protein engineering, stability and manufacturing delayed their wide-spread development. However, as of 2020, numerous validated platforms, i.e., those that have produced bispecific clinical candidates, are readily available (1). Using these platforms, the commercial clinical pipeline has grown to over 100 bispecific antibodies, ranging from tandem single-chain variable fragments (scFv) to full-length immunoglobulins with dual variable domains. Substantial growth in the pipeline has occurred only relatively recently, though. During the early 2010s, bispecific antibodies comprised less than 10% of the total number of antibody therapeutics entering clinical study per year, but this number rose to 25% by 2018. Reflecting the general ...
Chimeric antigen receptors (CARs) have been developed as a promising immunotherapy for ALL. This technology uses a single chain variable fragment (scFv) designed to recognize the cell surface marker CD19 as a method of treating ALL. CD19 is a molecule found on all B-cells and can be used as a means of distinguishing the potentially malignant B-cell population. In this therapy, mice are immunized with the CD19 antigen and produce anti-CD19 antibodies. Hybridomas developed from mouse spleen cells fused to a myeloma cell line can be developed as a source for the cDNA encoding the CD19 specific antibody.[51] The cDNA is sequenced and the sequence encoding the variable heavy and variable light chains of these antibodies are cloned together using a small peptide linker. This resulting sequence encodes the scFv. This can be cloned into a transgene, encoding what will become the endodomain of the CAR. Varying arrangements of subunits serve as the endodomain, but they generally consist of the hinge ...