Efficient generation of recombinant anti-HER2 scFv with high yield and purity using a simple method.

We developed a method to produce a soluble form of a single-chain fragment variable (scFv) targeting human epithelial growth factor receptor 2 (HER2) in Escherichia coli. By optimizing the orientations of the variable heavy (VH) and variable light (VL) domains and the His-tag, we identified the HL-His type antibody with the highest HER2-binding activity. Purification of HL-His yielded 40.7 mg from a 1 L culture, achieving >99% purity. The limit of detection was determined to be 2.9 ng, demonstrating high production yield, purity, and sensitivity. Moreover, we successfully labeled HER2+ cell lines with fluorescent dye-conjugated scFv, resulting in a significantly higher observed signal-to-background ratio, compared to that of HER2- cell lines. This highlights the potential of these fluorescent scFvs as valuable probes for HER2+ breast cancer diagnostics. Notably, the process for the complete scFv production was streamlined and required only 4-5 days. Additionally, the product maintained its activity after freeze storage, allowing for large-scale production and a wide range of practical applications.

Evaluation of PDL1 positive cancer cell-specific binding activity of recombinant anti-PDL1 scFv.

Programmed cell death-ligand 1 (PDL1) is a transmembrane protein that is characterized as an immune regulatory molecule. We recently developed a recombinant single-chain fragment of variable domain (scFv) against PDL1, which showed high binding efficiency to purified recombinant PDL1 protein. However, at that time, proof-of-concept data for the effect of scFv using PDL1-expressing cells was lacking. In this study, we conducted two kinds of cell-based immunoassays, western blotting and enzyme-linked immunosorbent assay, using anti-PDL1 scFv. The results indicate that scFv can selectively and sensitively detect PDL1 from PDL1 positive human cancer cell lines. Our findings suggest that scFv could be used as a potential PDL1 inhibitor agent and probe for cell-based immunoassays to detect PDL1.

Fluorogenic enzyme-linked immunosorbent assay with a dual color variation.

The development of accurate and high-throughput biomarker detection tools is crucial for the diagnosis, monitoring, and treatment of various diseases. In this study, a sensitive fluorogenic enzyme-linked immunosorbent assay (FELISA) using Amplex Red or QuantaBlu fluorescent substrate was developed for the detection of tumor necrosis factor alpha and programmed cell death-ligand 1. The limit of detection of FELISA was in the nanogram order and multiple samples were conveniently assayed within 20 h using FELISA, demonstrating its applicability as a powerful immunoassay tool. FELISA can be widely used for rapid and accurate TNFα and PDL1 detection and applied to various fluorogenic immunoassays against other antigens of interest.

Discovery of Orally Bioavailable Phthalazinone Analogues as an ENPP1 Inhibitor for STING-Mediated Cancer Immunotherapy.

A lack of the T cell-inflamed tumor microenvironment limits the efficacy of immune checkpoint inhibitors (ICIs). Activation of stimulator of interferon genes (STING)-mediated innate immunity has emerged as a novel therapeutic approach in cancer therapy. 2',3'-Cyclic GMP-AMP (cGAMP) is a natural STING agonist; however, cGAMP is subjected to endogenous degradation by ecto-nucleotide pyrophosphatase phosphodiesterase 1 (ENPP1). To improve the ICI response rate, we developed 29f, a novel ENPP1 inhibitor with phthalazin-1(2H)-one as the core scaffold. 29f inhibited the cGAMP hydrolysis by ENPP1 in vitro (IC50 = 68 nM) and enhanced the STING-mediated type I interferon response in both immune and tumor cells. 29f demonstrated excellent metabolic stability and bioavailability (F = 65%). Orally administered 29f promoted tumor growth inhibition in a CT26 syngeneic model and increased the anti-PD-L1 response. Furthermore, 29f-induced immunological memory prevented the tumor relapse against tumor rechallenge, suggesting the promising therapeutic potential of 29f.

Generation of a Recombinant scFv against Deoxycholic Acid and Its Conversion to a Quenchbody for One-Step Immunoassay.

Development of a rapid detection method for deoxycholic acid (DCA) is crucial for its diagnosis in the early stages of inflammation and cancer. In this study, we expressed a soluble recombinant anti-DCA single-chain variable fragment (scFv) in Escherichia coli. To convert scFv into a Quenchbody (Q-body), we labeled scFv using commercially available maleimide-linked fluorophores. The TAMRA-C5-maleimide-conjugated Q-body showed the highest response within a few minutes of DCA addition, indicating its applicability as a wash-free immunoassay probe for onsite DCA detection.

Production and engineering of nanobody-based quenchbody sensors for detecting recombinant human growth hormone and its isoforms.

Due to athletes' misuse of recombinant human growth hormone (rhGH) for performance improvement, the World Anti-Doping Agency has designated rhGH as a prohibited substance. This study focuses on the development and improvement of a simple and fast rhGH detection method using a fluorescence-incorporated antibody sensor "Quenchbody (Q-body)" that activates upon antigen binding. Camelid-derived nanobodies were used to produce stable Q-bodies that withstand high temperatures and pH levels. Notably, pituitary human growth hormone (phGH) comprises two major isoforms, namely 22 and 20 kDa GH, which exist in a specific ratio, and the rhGH variant shares the same sequence as the 22 kDa GH isoform. Therefore, we aimed to discriminate rhGH abuse by analyzing its specific isoform ratio. Two nanobodies, NbPit (recognizing phGH) and NbRec (preferentially recognizing 22 kDa rhGH), were used to develop the Q-bodies. Nanobody production in Escherichia coli involved the utilization of a vector containing 6xHis-tag, and Q-bodies were obtained using a maleimide-thiol reaction between the N-terminal of the cysteine tag and a fluorescent dye. The addition of tryptophan residue through antibody engineering resulted in increased fluorescence intensity (FI) (from 2.58-fold to 3.04-fold). The limit of detection (LOD) was determined using a fluorescence response, with TAMRA-labeled NbRec successfully detecting 6.38 ng/ml of 22 kDa rhGH while unable to detect 20 kDa GH. However, ATTO520-labeled NbPit detected 7.00 ng/ml of 20 kDa GH and 2.20 ng/ml 22 kDa rhGH. Q-bodies successfully detected changes in the GH concentration ratio from 10 to 40 ng/ml in human serum within 10 min without requiring specialized equipment and kits. Overall, these findings have potential applications in the field of anti-doping measures and can contribute to improved monitoring and enforcement of rhGH misuse, ultimately enhancing fairness and integrity in competitive sports.

Synthesis and biological evaluation of N-(3-fluorobenzyl)-4-(1-(methyl-d3)-1H-indazol-5-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-amine as a novel, potent ALK5 receptor inhibitor.

Specific inhibition of ALK5 provides a novel method for controlling the development of cancers and fibrotic diseases. In this work, a novel series of N-(3-fluorobenzyl)-4-(1-(methyl-d3)-1H-indazol-5-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-amine (11), a potential clinical candidate, was synthesized by strategic incorporation of deuterium at potential metabolic soft spots and identified as ALK5 inhibitors. This compound has a low potential for CYP-mediated drug-drug interactions as a CYP450 inhibitor (IC50 = >10 µM) and showed potent inhibitory effects in cellular assay (IC50 = 3.5 ± 0.4 nM). The pharmacokinetic evaluation of 11 in mice demonstrated moderate clearance (29.0 mL/min/kg) and also revealed high oral bioavailability in mice (F = 67.6%).

Generation of a novel monoclonal antibody against inflammatory biomarker S100A8 using hybridoma technology.

OBJECTIVES: S100A8 is highly expressed in several inflammatory and oncological conditions. To address the current lack of a reliable and sensitive detection method for S100A8, we generated a monoclonal antibody with a high binding affinity to human S100A8 to enable early disease diagnosis. RESULTS: A soluble recombinant S100A8 protein with a high yield and purity was produced using Escherichia coli. Next, mice were immunized with recombinant S100A8 to obtain anti-human S100A8 monoclonal antibodies using hybridoma technology. Lastly, the high binding activity of the antibody was confirmed and its sequence was identified. CONCLUSIONS: This method, including the production of antigens and antibodies, will be useful for the generation of hybridoma cell lines that produce anti-S100A8 monoclonal antibodies. Moreover, the sequence information of the antibody can be used to develop a recombinant antibody for use in various research and clinical applications.

A rapid ELISA for the detection of matrix metallopeptidase 9 using a recombinant Fab-type antibody.

Matrix metalloproteinase 9 (MMP9) contributes to several aspects of inflammation and cancer pathology, including invasion, metastasis, and angiogenesis. In this study, we expressed a recombinant fragment antigen-binding (Fab)-type anti-MMP9 antibody in Escherichia coli with high purity within five days and confirmed the nanomolar order of antigen-binding efficiency of the recombinant Fab. Moreover, we optimized the experimental time for performing enzyme-linked immunosorbent assay (ELISA), and decreased the reaction time from the conventional 20.5 h to 3.5 h. The rapid and sensitive MMP9 detection system developed in this study can be applied to a range of applications, including the diagnosis of diseases with MMP9 overexpression including inflammatory and cancer-related diseases.

Identification of novel pyrrolopyrimidine and pyrrolopyridine derivatives as potent ENPP1 inhibitors.

In an effort to discover novel scaffolds of non-nucleotide-derived Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) inhibitors to stimulate the Stimulator of Interferon Genes (STING) pathway, we designed and synthesised pyrrolopyrimidine and pyrrolopyridine derivatives and performed structure-activity relationship (SAR) study. We found 18p possessed high potency (IC50 = 25.0 nM) against ENPP1, and activated STING pathway in a concentration dependent manner. Also, in response to STING pathway activation, cytokines such as IFN-ß and IP-10 were induced by 18p in a concentration dependent manner. Finally, we discovered that 18p causes inhibition of tumour growth in 4T1 syngeneic mouse model. This study provides new insight into the designing of novel ENPP1 inhibitors and warrants further development of small molecule immune modulators for cancer immunotherapy.

Discovery of 3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one and 3,4-dihydropyrido[2,3-d]pyrimidin-2(1H)-one derivatives as novel ENPP1 inhibitors.

Ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) negatively regulates the anti-cancer Stimulator of Interferon Genes (STING) pathway. We discovered that 3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one and 3,4-dihydropyrido[2,3-d]pyrimidin-2(1H)-one derivatives possessed inhibitory activities on ENPP1. A structure-activity relationship (SAR) study led to the identification of 46 and 23 as potent ENPP1 inhibitors. Also, compounds 46 and 23 possessed high microsomal stabilities in human, rat, and mouse liver microsome. Additionally, CYPs (1A2, 2C9, 2C19, 2D6, and 3A4) were not inhibited by 46 and 23. Molecular dynamics simulations provided an insight of binding modes between ENPP1 and compounds (46 and 23).

Soluble Expression of a Neo2/15-Conjugated Single Chain Fv against PD-L1 in Escherichia coli.

Immunocytokines, antibody-cytokine fusion proteins, have the potential to improve the therapeutic index of cytokines by delivering the cytokine to the site of localized tumor cells using antibodies. In this study, we produced a recombinant anti-programmed death-ligand 1 (PD-L1) scFv, an antibody fragment against PD-L1 combined with a Neo2/15, which is an engineered interleukin with superior function using an E. coli expression system. We expressed the fusion protein in a soluble form and purified it, resulting in high yield and purity. The high PD-L1-binding efficiency of the fusion protein was confirmed via enzyme-linked immunosorbent assay, suggesting the application of this immunocytokine as a cancer-related therapeutic agent.

Identification of highly selective type II kinase inhibitors with chiral peptidomimetic tails.

Identification of highly selective type II kinase inhibitors is described. Two different chiral peptidomimetic scaffolds were introduced on the tail region of non-selective type II kinase inhibitor GNF-7 to enhance the selectivity. Kinome-wide selectivity profiling analysis showed that type II kinase inhibitor 7a potently inhibited Lck kinase with great selectivity (IC50 of 23.0 nM). It was found that 7a and its derivatives possessed high selectivity for Lck over even structurally conserved all Src family kinases. We also observed that 7a inhibited Lck activation in Jurkat T cells. Moreover, 7a was found to alleviate clinical symptoms in DSS-induced colitis mice. This study provides a novel insight into the design of selective type II kinase inhibitors by adopting chiral peptidomimetic moieties on the tail region.

Development of a CHO cell line for stable production of recombinant antibodies against human MMP9.

BACKGROUND: Human matrix metalloproteinase 9 (hMMP9) is a biomarker in several diseases, including cancer, and the need for developing detectors and inhibitors of hMMP9 is increasing. As an antibody against hMMP9 can be selectively bound to hMMP9, the use of anti-MMP9 antibody presents new possibilities to address hMMP9-related diseases. In this study, we aimed to establish a stable Chinese hamster ovary (CHO) cell line for the stable production of antibodies against hMMP9. RESULTS: Weconstructed recombinant anti-hMMP9 antibody fragment-expressing genes and transfected these to CHO cells. We chose a single clone, and successfully produced a full-sized antibody against hMMP9 with high purity, sensitivity, and reproducibility. Subsequently, we confirmed the antigen-binding efficiency of the antibody. CONCLUSIONS: We developed a novel recombinant anti-hMMP9 antibody via a CHO cell-based mammalian expression system, which has a high potential to be used in a broad range of medical and industrial areas.

Production of a Soluble Recombinant Antibody Fragment against MMP9 Using Escherichia coli.

Matrix metalloproteinase 9 (MMP9) is involved in several aspects of the pathology of cancer, including invasion, metastasis, and angiogenesis. In this study, we expressed a recombinant scFv-type anti-MMP9 antibody in soluble form using Escherichia coli, purified it, and confirmed its antigen-binding ability. The convenient, rapid, inexpressive system used in this study for producing recombinant antibody fragments needs only five days, and thus can be used for the efficient production of scFv against MMP9, which can be used in a range of applications and industrial fields, including diagnosis and treatment of inflammatory and cancer-related diseases.

Diagnostic ability of salivary matrix metalloproteinase-9 lateral flow test point-of-care test for periodontitis.

AIM: This cross-sectional study aimed to examine the diagnostic ability of salivary matrix metalloproteinase (MMP)-9 lateral flow test (LFT) point-of-care (POC) kit and develop an algorithm for diagnosis of periodontitis. MATERIALS AND METHODS: Through Seoul National Dental Hospital, 137 participants (46 LFT negatives, 91 LFT positives) were recruited. For salivary diagnostics, 150 µl of the unstimulated saliva was applied to LFT-POC kit. To make a diagnosis of periodontitis, stage II-IV in modified new international classification system was used. Covariates encompassing age, sex, smoking and obesity were evaluated through face-to-face interview. Enzyme-linked immunosorbent assay was used for quantification of salivary MMP-9. To develop a diagnostic algorithm, multivariable logistic regression analysis was used. Receiver operating characteristic curve was applied for evaluating diagnostic ability. RESULTS: Diagnostic ability of salivary MMP-9 LFT-POC test was 0.82 (sensitivity of 0.92, specificity of 0.72) in total participants. Diagnostic algorithm using POC test resulted in a response equation, that is algorithm score = -3.675 + 2.877*LFT + 0.034*age + 0.121*sex + 0.372*smoking + 0.192*obesity. Diagnostic ability of the algorithm was 0.88 (sensitivity of 0.92, specificity of 0.85) with cut-off score of 0.589. CONCLUSIONS: Salivary MMP-9 LFT-POC kit showed appropriate diagnostic ability for periodontitis and would be an efficient tool for screening of periodontitis.

PM Q-probe: A fluorescent binding protein that converts many antibodies to a fluorescent biosensor.

Quenchbody (Q-body) is a fluorescent biosensor in which a fluorescent dye is tagged near the antigen binding site of an antibody. The fluorescence of the dye is quenched by the tryptophan residues present in the variable region of the antibody, and is recovered when the antigen binds. Q-bodies have been prepared using recombinant DNA technology by introducing one or more tag sequence(s) at either the N-terminal of the Fab or the single chain variable region fragment of the antibody, and labeling the tag with a fluorescent dye. However, preparation of recombinant antibody fragments is time-consuming and the performance of the Q-body is unpredictable. Here we report an antibody-binding quenching probe made from protein M from Mycoplasma genitalium that can transform the IgG antibody into an immunosensor. By using bacterially expressed and purified protein M and labeling the C-terminal cysteine-containing tag, we prepared a TAMRA-labeled PM Q-probe. When the Q-probe was incubated with Fab or IgG recognizing the bone Gla protein, the fluorescence of the probe was quenched and subsequently recovered by the adding of antigens in a dose-dependent manner. We also succeeded in detecting several small biomarkers with nanomolar sensitivity, including thyroxine extracted from human serum. The clone found to be suitable for the detection of cortisol was confirmed to work as a recombinant Q-body as well, which also worked in 50% human serum. The results suggest that the Q-probe can quickly convert an IgG to a biosensor, which will be useful in rapid diagnosis of small biomarkers.

Neoleukin-2 enhances anti-tumour immunity downstream of peptide vaccination targeted by an anti-MHC class II VHH.

Cancer-specific mutations can lead to peptides of unique sequence presented on MHC class I to CD8 T cells. These neoantigens can be potent tumour-rejection antigens, appear to be the driving force behind responsiveness to anti-CTLA-4 and anti-PD1/L1-based therapies and have been used to develop personalized vaccines. The platform for delivering neoantigen-based vaccines has varied, and further optimization of both platform and adjuvant will be necessary to achieve scalable vaccine products that are therapeutically effective at a reasonable cost. Here, we developed a platform for testing potential CD8 T cell tumour vaccine candidates. We used a high-affinity alpaca-derived VHH against MHC class II to deliver peptides to professional antigen-presenting cells. We show in vitro and in vivo that peptides derived from the model antigen ovalbumin are better able to activate naive ovalbumin-specific CD8 T cells when conjugated to an MHC class II-specific VHH when compared with an irrelevant control VHH. We then used the VHH-peptide platform to evaluate a panel of candidate neoantigens in vivo in a mouse model of pancreatic cancer. None of the candidate neoantigens tested led to protection from tumour challenge; however, we were able to show vaccine-induced CD8 T cell responses to a melanoma self-antigen that was augmented by combination therapy with the synthetic cytokine mimetic Neo2/15.

Detection and destruction of HER2-positive cancer cells by Ultra Quenchbody-siRNA complex.

Ultra Quenchbody (UQ-body) is a biosensor that utilizes the quenching behavior of the fluorescent dye linked to the antibody V region. When the corresponding antigen is bound to the UQ-body, the fluorescence is restored and allows the detection of target molecules easily and sensitively. In this paper, we constructed UQ-bodies to sensitively detect the human epidermal growth factor receptor 2 (HER2) cancer marker in solution or on cancer cells, which was further used to kill the cancer cells. A synthetic Fab fragment of anti-HER2 antibody Fab37 with many Trp residues at hypervariable region was prepared and labeled with fluorescent dyes to obtain the UQ-bodies. The UQ-body could detect HER2 in solution at concentrations as low as 20 pM with an EC50 of 0.3 nM with a fourfold response. Fluorescence imaging of HER2-positive cells was successfully performed without any washing steps. To deliver small interfering RNA (siRNA) to cancer cells, a modified UQ-body with C-terminal 9R sequence was also prepared. HER2-positive cancer cells were effectively killed by polo-like kinase 1 siRNA intracellularly delivered by the UQ-body-9R. The novel approach employing siRNA-empowered UQ-body could detect and image the HER2 antigen easily and sensitively, and effectively kill the HER2-positive cancer cells.

Transnuclear mice reveal Peyer's patch iNKT cells that regulate B-cell class switching to IgG1.

Tissue-resident iNKT cells maintain tissue homeostasis and peripheral surveillance against pathogens; however, studying these cells is challenging due to their low abundance and poor recovery from tissues. We here show that iNKT transnuclear mice, generated by somatic cell nuclear transfer, have increased tissue resident iNKT cells. We examined expression of PLZF, T-bet, and RORγt, as well as cytokine/chemokine profiles, and found that both monoclonal and polyclonal iNKT cells differentiated into functional subsets that faithfully replicated those seen in wild-type mice. We detected iNKT cells from tissues in which they are rare, including adipose, lung, skin-draining lymph nodes, and a previously undescribed population in Peyer's patches (PP). PP-NKT cells produce the majority of the IL-4 in Peyer's patches and provide indirect help for B-cell class switching to IgG1 in both transnuclear and wild-type mice. Oral vaccination with α-galactosylceramide shows enhanced fecal IgG1 titers in iNKT cell-sufficient mice. Transcriptional profiling reveals a unique signature of PP-NKT cells, characterized by tissue residency. We thus define PP-NKT as potentially important for surveillance for mucosal pathogens.