Application of Recombinant Human scFv Antibody as a Powerful Tool to Monitor Nitrogen Fixing Biofertilizer in Rice and Legume

Human scFv antibody generated from phage display technology was successfully used for the generation of specific recombinant antibodies: yiN92-1e10 and yiDOA9-162 for the detection of Bradyrhizobium strains SUTN9-2 and DOA9, respectively.

Expression, purification and characterisation of a human anti-CDK4 single-chain variable fragment antibody

Background Cyclin-dependent kinase 4 (CDK4) when hyperactivated drives development and maintenance of most tumour types, thus prompting its use as an essential cancer treatment target and a diagnostic tool. Target-binding molecules, such as single-chain variable fragment (scFv) antibodies, hold tremendous potential for use in a wide range of cancer diagnostic and therapeutic applications. Results A human anti-CDK4 scFv antibody (AK2) derived from a human phage display library was expressed in soluble form in Escherichia coli and shown to be secreted into the culture supernatant. Next, soluble AK2 within culture supernatant was successfully purified using affinity chromatography then was shown, using enzyme-linked immunosorbent assays, to bind to recombinant human CDK4 with high affinity and specificity. Further analyses of AK2 interactions with intracellular components demonstrated that AK2 recognised and interacted specifically with endogenous CDK4 and thus could be useful for detection of CDK4 within tumour cells. Conclusions A novel anti-CDK4 scFv antibody that can recognise and interact specifically with recombinant human CDK4 and endogenous CDK4 in tumour cells was expressed and purified successfully. These results suggest that the anti-CDK4 scFv antibody may serve as a new and promising tool for achieving CDK4-targeted diagnosis, prognosis and treatment of numerous types of cancers.

Bispecific antibody-mediated redirection of NKG2D-CAR natural killer cells facilitates dual targeting and enhances antitumor activity

BackgroundNatural killer group 2D (NKG2D) is an activating receptor of natural killer (NK) cells and other lymphocytes that mediates lysis of malignant cells through recognition of stress-induced ligands such as MICA and MICB. Such ligands are broadly expressed by cancer cells of various origins and serve as targets for adoptive immunotherapy with effector cells endogenously expressing NKG2D or carrying an NKG2D-based chimeric antigen receptor (CAR). However, shedding or downregulation of NKG2D ligands (NKG2DL) can prevent NKG2D activation, resulting in escape of cancer cells from NKG2D-dependent immune surveillance.MethodsTo enable tumor-specific targeting of NKG2D-expressing effector cells independent of membrane-anchored NKG2DLs, we generated a homodimeric recombinant antibody which harbors an N-terminal single-chain fragment variable (scFv) antibody domain for binding to NKG2D, linked via a human IgG4 Fc region to a second C-terminal scFv antibody domain for recognition of the tumor-associated antigen ErbB2 (HER2). The ability of this molecule, termed NKAB-ErbB2, to redirect NKG2D-expressing effector cells to ErbB2-positive tumor cells of different origins was investigated using peripheral blood mononuclear cells, ex vivo expanded NK cells, and NK and T cells engineered with an NKG2D-based chimeric receptor.ResultsOn its own, bispecific NKAB-ErbB2 increased lysis of ErbB2-positive breast carcinoma cells by peripheral blood-derived NK cells endogenously expressing NKG2D more effectively than an ErbB2-specific IgG1 mini-antibody able to induce antibody-dependent cell-mediated cytotoxicity via activation of CD16. Furthermore, NKAB-ErbB2 synergized with NK-92 cells or primary T cells engineered to express an NKG2D-CD3ζ chimeric antigen receptor (NKAR), leading to targeted cell killing and greatly enhanced antitumor activity, which remained unaffected by soluble MICA known as an inhibitor of NKG2D-mediated natural cytotoxicity. In an immunocompetent mouse glioblastoma model mimicking low or absent NKG2DL expression, the combination of NKAR-NK-92 cells and NKAB-ErbB2 effectively suppressed outgrowth of ErbB2-positive tumors, resulting in treatment-induced endogenous antitumor immunity and cures in the majority of animals.ConclusionsOur results demonstrate that combining an NKAB antibody with effector cells expressing an activating NKAR receptor represents a powerful and versatile approach to simultaneously enhance tumor antigen-specific as well as NKG2D-CAR and natural NKG2D-mediated cytotoxicity, which may be particularly useful to target tumors with heterogeneous target antigen expression.

An atherosclerotic plaque-targeted single-chain antibody for MR/NIR-II imaging of atherosclerosis and anti-atherosclerosis therapy

Background Oxidation-specific epitopes (OSEs) are rich in atherosclerotic plaques. Innate and adaptive immune responses to OSEs play an important role in atherosclerosis. The purpose of this study was to develop novel human single-chain variable fragment (scFv) antibody specific to OSEs to image and inhibit atherosclerosis. Results Here, we screened a novel scFv antibody, named as ASA6, from phage-displayed human scFv library. ASA6 can bind to oxidized LDL (Ox-LDL) and atherosclerotic plaques. Meanwhile, ASA6 can also inhibit the uptake of Ox-LDL into macrophage to reduce macrophage apoptosis. The atherosclerotic lesion area of ApoE−/− mice administrated with ASA6 antibody was significantly reduced. Transcriptome analysis reveals the anti-atherosclerosis effect of ASA6 is related to the regulation of fatty acid metabolism and inhibition of M1 macrophage polarization. Moreover, we conjugated ASA6 antibody to NaNdF4@NaGdF4 nanoparticles for noninvasive imaging of atherosclerotic plaques by magnetic resonance (MR) and near-infrared window II (NIR-II) imaging. Conclusions Together, these data demonstrate the potential of ASA6 antibody in targeted therapy and noninvasive imaging for atherosclerosis. Graphic abstract

CRISPR/Cas9-Mediated Gene Drive to Prevent the Replication of Dengue Virus in the Mosquito Vectors to Reduce the Impact of Dengue Epidemic in Bangladesh

Dengue is a flavivirus transmitted by Aedes aegypti, leading to mosquito-borne illness causing significant morbidity and mortality each year. A majority of dengue cases around the world are caused by four serotypes-dengue virus (DENV) 1-4. The recent outbreak has broken all the previous record of infections with 101, 354 dengue cases, which has increased the urgency of finding an effective way to reduce the level of infection. CRISPR-Cas9 mediated gene drive is a novel technology that can be used to reduce the transmission by Aedes mosquitoes. Ae. aegypti can be engineered to express anti-DENV, 1C19-based, single-chain variable fragment (scFv) antibody, to provide protection against all four DENV serotypes by neutralizing them. Anti-DENV scFv-antibody transgene can be incorporated into a clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR associated (Cas); i.e., the CRISPR/Cas9-based gene drive system gene for effective suppression of dengue infection. CRISPR/Cas9-based gene drive system allows it to pass down this phenotype across the wild population in the urban area. However, the incautious release of gene drive in the environment can swipe away the entire population. This technology can greatly impact on the environment, creating an imbalance in the ecosystem if not applied carefully. Rigorous studies and mass level cooperation are needed among the scientists, local authorities and the government to make the informed decisions on the outcome of this technology.

Construction and directional evolution of anti-enrofloxacin ScFv antibody for the immunoassay of fluoroquinolones

A recombinant anti-enrofloxacin single-chain antibody (scFv) was produced for the detection of enrofloxacin. An immunized mouse phage display scFv library with a capacity of 2.35×109 CFU/mL was constructed and used for anti-enrofloxacin scFv screening. After four rounds of bio-panning, 10 positives were isolated and identified successfully. The highest positive scFv was expressed in E. coli BL21. Then, its recognition mechanisms were studied using the molecular docking method. The result showed the amino acid residues Leu121 were the key residues for the binding of ScFv to ENR. Based on the results of virtual mutation, the ScFv antibody was evolved by directional mutagenesis of contact amino acid residue Leu121 to Asn. After the expression and purification, an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) based on the parental and mutant ScFv were established for enrofloxacin respectively. The IC50 value of the assay established with the ScFv mutant was 1.63 ng/mL, while the parental ScFv was 21.08 ng/mL, this result showed highly increased affinity with up to 12.9-folds improved sensitivity. The mean recovery for ENR ranged from 71.80% to 117.35% with 10.46% relative standard deviation between the intra-assay and the inter-assay. The results indicate that we have obtained a highly sensitive anti-ENR scFv by the phage library construction and directional evolution, and the scFv-based IC-ELISA is suitable for the detection of ENR residue in animal derived edible tissues and milk.

TRIAD: a transposition-based approach for gene mutagenesis by random short in-frame insertions and deletions for directed protein evolution

Insertions and deletions (InDels) are among the most frequent changes observed in natural protein evolution, yet their potential has hardly been harnessed in directed evolution experiments. Here we describe the standard protocol for TRIAD (Transposition-based Random Insertion And Deletion mutagenesis), a simple and efficient Mu transposon mutagenesis approach for generating libraries of single InDel variants with one, two or three triplet nucleotide insertions or deletions. This method has recently been employed in three published examples of InDel-based directed evolution of proteins, including a phosphotriesterase, a scFv antibody and an ancestral luciferase.

Development of a single-chain fragment variable fused-mutant HALT-1 recombinant immunotoxin against G12V mutated KRAS colorectal cancer cells

Background KRAS oncogenes harboring codon G12 and G13 substitutions are considered gatekeeper mutations which drive oncogenesis in many cancers. To date, there are still no target-specific vaccines or drugs available against this genotype, thus reinforcing the need towards the development of targeted therapies such as immunotoxins. Methods This study aims to develop a recombinant anti-mKRAS scFv-fused mutant Hydra actinoporin-like-toxin-1 (mHALT-1) immunotoxin that is capable of recognizing and eradicating codon-12 mutated k-ras antigen abnormal cells. One G13D peptide mimotope (164-D) and one G12V peptide mimotope (68-V) were designed to elicit antigen specific IgG titres against mutated K-ras antigens in immunised Balb/c mice. The RNA was extracted from splenocytes following ELISA confirmation on post-immunized mice sera and was reverse transcribed into cDNA. The scFv combinatorial library was constructed from cDNA repertoire of variable regions of heavy chain (VH) and light chain (VL) fusions connected by a flexible glycine-serine linker, using splicing by overlap extension PCR (SOE-PCR). Anti-mKRAS G12V and G13D scFvs were cloned in pCANTAB5E phagemid and superinfected with helper phage. After few rounds of bio-panning, a specific mKRAS G12V and G13D scFv antibody against G12V and G13D control mimotope was identified and confirmed using ELISA without any cross-reactivity with other mimotopes or controls. Subsequently, the anti-mKRAS scFv was fused to mHALT-1 using SOE-PCR and cloned in pET22b vector. Expressed recombinant immunotoxins were analyzed for their effects on cell proliferation by the MTT assay and targeted specificity by cell-based ELISA on KRAS-positive and KRAS-negative cancer cells. Results The VH and VL genes from spleen RNA of mice immunized with 164-D and 68-V were amplified and randomly linked together, using SOE-PCR producing band sizes about 750 bp. Anti-mKRAS G12V and G13D scFvs were constructed in phagemid pCANTAB5E vectors with a library containing 3.4 × 106 and 2.9 × 106 individual clones, respectively. After three rounds of bio-panning, the anti-mKRAS G12V-34 scFv antibody against G12V control mimotope was identified and confirmed without any cross-reactivity with other controls using ELISA. Anti-mKRAS G12V-34 scFv fragment was fused to mHALT-1 toxin and cloned in pET22b vector with expression as inclusion bodies in E. coli BL21(DE3) (molecular weight of ~46.8 kDa). After successful solubilization and refolding, the mHALT-1-scFv immunotoxin exhibited cytotoxic effects on SW-480 colorectal cancer cells with IC50 of 25.39 μg/mL, with minimal cytotoxicity effect on NHDF cells. Discussion These results suggested that the development of such immunotoxins is potentially useful as an immunotherapeutic application against KRAS-positive malignancies.