Target-specific Binding Research Articles

Engineered polyspecific antibodies: A new frontier in the field of immunotherapeutics.

The 21st-century beginning remarked with the huge success of monospecific MAbs, however, in the last couple of years, polyspecific MAbs (PsAbs) have been an interesting topic and show promise of being biobetter than monospecific MAbs. Polyspecificity, in which a single antibody serves multiple specific target binding, has been hypothesized to contribute to the development of a highly effective antibody repertoire for immune defence. This polyspecific MAb trend represents an explosion that is gripping the whole pharmaceutical industry. This review is concerned with the current development and quality enforcement of PsAbs. All provided literature on monospecific MAbs and polyspecific MAbs (PsAbs) were searched using various electronic databases such as PubMed, Google Scholar, Web of Science, Elsevier, Springer, ACS, Google Patent and books via the keywords Antibody engineering, Polyspecific antibody, Conventional antibody, non-conventional antibody, and Single domain antibody. In the literature, there are more than 100 different formats to construct PsAb by quadroma technology, chemical conjugation and genetic engineering. Till March 2023, nine PsAb have been approved around the world, and around 330 are in advanced developmental stages, showing the dominancy of PsAb in the growing health sector. Recent advancements in protein engineering techniques and the fusion of non-conventional antibodies have made it possible to create complex PsAbs that demonstrate higher stability and enhanced potency. This marks the most significant achievement for cancer immunotherapy, in which PsAbs have immense promise. It is worth mentioning that seven out of the nine PsAbs have been approved as anti-cancer therapy. As PsAbs continue to acquire prominence, they could pave the way for the development of novel immunotherapies for multiple diseases.

Specializing Carbon Nanozyme Active Sites for Sensitive Alkaline Phosphatase Activity Metal-Free Detection.

As biological enzymes regulate metabolic processes, alkaline phosphatase (ALP) is a critical diagnostic indicator associated with many diseases. To accurately measure the enzyme activity, nanozymactic materials can offer sensitive strategies for ALP detection. However, nanozymes often lack specific target binding sites, and the presence of common co-components, e. g., metal ions, may cause false-positive or false-negative results in enzyme activity determination. Herein, we developed a colorimetric assay for ALP detection using metal-free nanozymatic carbon dots (CDs). The ALP hydrolysis of pyrophosphate ions (PPi) to phosphate ions (Pi) induces a "turn-on" response based on the nanozyme activity. This PPi-induced inhibition mechanism is extensively studied via the Michaelis-Menten model, revealing that PPi acts as a noncompetitive inhibitor for CDs at a binding site distinct from the common nanozyme active site. With superior responses to ALP substrates, a highly sensitive and selective method is established for sensing ALP activity with a linear range of 0.010-0.200 U/L and a detection limit of 0.009 U/L. This finding explores the recognition and binding behavior of nanozymes, allowing for precise and reliable measurements even in complex samples, and represents a significant breakthrough for nanozyme-based assays in biological analysis.

Using anti-PD-L1 antibody conjugated gold nanoshelled poly (Lactic-co-glycolic acid) nanocapsules loaded with doxorubicin: A theranostic agent for ultrasound imaging and photothermal/chemo combination therapy of triple negative breast cancer.

Triple negative breast cancer (TNBC) has the worst prognosis of all breast cancers, and it is difficult to progress through traditional chemotherapy. Therefore, the treatment of TNBC urgently requires agents with effective diagnostic and therapeutic capabilities. In this study, we obtained programmed death-ligand 1 (PD-L1) antibody conjugated gold nanoshelled poly(lactic-co-glycolic acid) (PLGA) nanocapsules (NCs) encapsulating doxorubicin (DOX) (DOX@PLGA@Au-PD-L1 NCs). PLGA NCs encapsulating DOX were prepared by a modified single-emulsion oil-in-water (O/W) solvent evaporation method, and gold nanoshells were formed on the surface by gold seed growth method, which were coupled with PD-L1 antibodies by carbodiimide method. The fabricated DOX@PLGA@Au-PD-L1 NCs exhibited promising contrast enhancement in vitro ultrasound imaging. Furthermore, DOX encapsulated in NCs displayed good pH-responsive and photo-triggered drug release properties. After irradiating 200 μg/mL NCs solution with a laser for 10 min, the solution temperature increased by nearly 23°C, indicating that the NCs had good photothermal conversion ability. The targeting experiments confirmed that the NCs had specific target binding ability to TNBC cells overexpressing PD-L1 molecules. Cell experiments exhibited that the agent significantly reduced the survival rate of TNBC cells through photochemotherapy combination therapy. As a multifunctional diagnostic agent, DOX@PLGA@Au-PD-L1 NCs could be used for ultrasound targeted contrast imaging and photochemotherapy combination therapy of TNBC cells, providing a promising idea for early diagnosis and treatment of TNBC.

TiO2-guanine as a new amalgamation compoundfor fabrication of a disposable biosensor with high sensitivity and rapid detection of H1N1 swine flu virus.

A TiO2-guanine nanocomposite (TG NC)-based electrochemical biosensor was immobilized with hemagglutinin (HA) gene specific probe with 5' NH2 group on screen-printed gold electrode (probe(ss)DNA-TG-SPGE). The modified biosensor was examined for H1N1 swine flu virus. TG NCs along with precursors were characterized spectroscopically and morphologically by employing several approaches. Electrochemical investigations were performed with the help of cyclic voltammetric (CV) and electrochemical impedance spectroscopy (EIS) in 0.1 M phosphate buffer saline (PBS; pH 7.4) with 1 µM methylene blue (MB) redox indicator. For better detection of single-stranded virus DNA, the modified electrode was optimized at various concentrations, pH, and scan rates. The modified biosensor showed high sensitivity (40.32 μA/ng.cm2), low LOD (0.00024 ng/6 µL), and broad linear range 0.0002-20 ng/6µL with coefficient of determination ofR2=0.9981 for H1N1 virus detection. The HA gene-modified biosensor presented decent stability and specificity against different infectious pathogens including H3N2 virus and human DNA with negative response. Furthermore, the modified biosensor also responded well for real sample target DNA detection with a recovery of >96%. The simply designed HA gene-modified biosensor transduces decreased current response towards target-specific (ss)DNA binding and could be used as a rapid detection tool for H1N1 swine flu virus diagnosis.

Polyaniline and polystyrene sulfonic acid modified on conductive biochar nanoparticle with strong signal enhancement for trace atrazine detection

A label and reagent free and cost effective immunosensing system was newly developed for detection of trace leveled atrazine in water. This was constructed by synthesis of polyaniline (PA) and polystyrene sulfonic acid (PSS) modified biochar (BC) nanoparticle composite (PA-PSS-BC) and coating them on screen printed electrode as conductive and electrochemically active layer, and covalently immobilizing anti-atrazine antibody on electrode for specific target binding. The low cost biochar from sugarcane was highly conductive and favorable for attaching PA and PSS due to large surface area and sp2 carbon structure. Crediting to dopant PSS, PA-PSS-BC achieved significant enhancement of redox current signal even in phosphate buffered saline of neutral pH compared to that of without PSS. And redox current of PA was extremely stable with negligible signal change even after twenty cycles repeated reactions owning to protection by BC. These essentially ensured sensing platform being sensitively performed in neutral solution with exemption of adding external redox reagent. As a result, fairly low concentrations of atrazine could be detected within 5 min, with limit of detection being 0.407 ng/L in a dynamic detection range varying from 1 to 100 ng/L. Moreover, the immunosensor exhibited reasonable signal recoveries for detecting trace leveled atrazine in real water samples, high selectivity, and good stability with dry and cold storage.

Profiling the Biophysical Developability Properties of Common IgG1 Fc Effector Silencing Variants.

Therapeutic antibodies represent the most significant modality in biologics, with around 150 approved drugs on the market. In addition to specific target binding mediated by the variable fragments (Fvs) of the heavy and light chains, antibodies possess effector functions through binding of the constant region (Fc) to Fcγ receptors (FcγR), which allow immune cells to attack and kill target cells using a variety of mechanisms. However, for some applications, including T-cell-engaging bispecifics, this effector function is typically undesired. Mutations within the lower hinge and the second constant domain (CH2) of IgG1 that comprise the FcγR binding interface reduce or eliminate effector function ("Fc silencing") while retaining binding to the neonatal Fc receptor (FcRn), important for normal antibody pharmacokinetics (PKs). Comprehensive profiling of biophysical developability properties would benefit the choice of constant region variants for development. Here, we produce a large panel of representative mutations previously described in the literature and in many cases in clinical or approved molecules, generate select combinations thereof, and characterize their binding and biophysical properties. We find that some commonly used CH2 mutations, including D265A and P331S, are effective in reducing binding to FcγR but significantly reduce stability, promoting aggregation, particularly under acidic conditions commonly employed in manufacturing. We highlight mutation sets that are particularly effective for eliminating Fc effector function with the retention of WT-like stability, including L234A, L235A, and S267K (LALA-S267K), L234A, L235E, and S267K (LALE-S267K), L234A, L235A, and P329A (LALA-P329A), and L234A, L235E, and P329G (LALE-P329G).

Structure-guided engineering of aptamers to enhanced structural stability and application performance in alleviating β-lactoglobulin allergenicity

Aptamers are synthetic single-strand oligonucleotide-based ligands with great application potential due to their intriguing properties of specific and high affinity target binding and inhibition. However, the excessive flexibility of aptamer conformation will lead to instability and diversity of the folded structure. It remains a great challenge to keep high binding performance in real-world applications. Herein, we rational engineered a structurally enhanced aptamer (Cb-#T4M8) through hairpin structural enhancement guided by structural information combined with cyclization strategies, that possesses excellent binding affinity (5.1 ± 1.2 nM) against β-lactoglobulin (β-Lg), as well as highest stability. Notably, molecular simulation and competitive experimental analysis revealed that Cb-#T4M8 shares the same binding epitopes on β-Lg with IgE. Using Cb-#T4M8 as an inhibitor, Cb-#T4M8 was successfully demonstrated to interfere with IgE binding to β-Lg in vitro experiments. Furthermore, The application of Cb-#T4M8 in allergy models of cells and mice significantly reduced the allergic response induced by β-Lg. Overall, our study not only provided a reference for the engineering of aptamers to obtain a good application in the real world but also proposed a new direction for developing treatment against food allergy.

Abstract A13: Development of LILRB4 biparatopic synthetic T-cell receptor and antigen receptor (STAR)-T cells for the treatment of acute myeloid leukemia (AML)

Abstract Background: Acute myeloid leukemia (AML) is one of the most common hematopoietic malignancies. Adoptive cellular immunotherapy like chimeric antigen receptor (CAR) T therapy has demonstrated remarkable antitumor activity against B cell malignancies. However, CAR-T therapy exhibited very limited efficacy in treating AML due to the heterogeneity of AML cells and their similarity to normal hematopoietic cells and lack of leukemic cell-specific targets. LILRB4 (also known as ILT3, CD85k) is an immunosuppressive receptor that is highly expressed on monocyte AML blasts (FAB M4 and M5 AML subtypes), which makes it an ideal therapeutic target for monocytic AML. Here we generated LILRB4-specific nanobody-based STAR-T (Synthetic T-Cell Receptor and Antigen Receptor-T) cells, aiming to develop a new treatment strategy to improve outcomes for monocytic AML. Methods: LILRB4-specific nanobodies were obtained from alpacas immunized with the resombinant LILRB4 extracellular domain. These nanobodies were then fused to IgG1 Fc, expressed and purified from CHO cells and used for measuring the affinity, epitope binning, target binding specificity by SPR, BLI and flow cytometry. The LILRB4 STAR was designed as a double-chain TCR-based receptor with nanobodies fused to the N-terminus of the modified mouse TCR-Cα or TCR-Cβ, the two chains were linked with Furin-P2A sequence. Based on STAR structure, LILRB4 single epitopic STAR was generated by fusing an nanobody to the TCR-Cβ, whereas LILRB4 biparatopic STAR contained two nanobodies fused to the TCR-Cα and TCR-Cβ separately. In vitro assays and xenograft models were used to assess the potential of LILRB4 STAR-T cells for elimination of leukemic disease. Results and Conclusions: Multiple STAR-T cells were generated using a lentiviral-based vector and tested for their tumor lysis activity. Among multiple nanobodies tested, two nanobodies that displayed high antigen binding affinity and specificity were identified for further studies. They recognized two distinctive epitopes of LILRB4, and neither of them had competitive binding with APOE or FN1, known ligands of LILRB4. We generated both single paratopic and bi-paratopic STAR constructs and engineered the corresponding STAR-T cells. In vitro and in vivo cytotoxicity assays demonstrated that all LILRB4 STAR-T cells had high cytotoxic activity against LILRB4+ AML cell lines. In addition, LILRB4 biparatopic STAR-T cells exhibited the highest anti-leukemic activity and the best expansion in vivo. Therefore, the LILRB4 STAR-T cells described here may be further developed for the treatment of AML and represent a new immune cell therapy strategy for LILRB4+ AML patients. Citation Format: Wei Rui, Lei Lei, Zezhong Zhang, Chunyan Wu, Yu Xia, Yingying Liu, Xiaojing Pang, Ruifang Du, Lan Wang, Dengyue Sheng, Jing Guo, Yanyan Zhang, Xueqiang Zhao, James Pan. Development of LILRB4 biparatopic synthetic T-cell receptor and antigen receptor (STAR)-T cells for the treatment of acute myeloid leukemia (AML) [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A13.

Efficient evolution of human antibodies from general protein language models

Natural evolution must explore a vast landscape of possible sequences for desirable yet rare mutations, suggesting that learning from natural evolutionary strategies could guide artificial evolution. Here we report that general protein language models can efficiently evolve human antibodies by suggesting mutations that are evolutionarily plausible, despite providing the model with no information about the target antigen, binding specificity or protein structure. We performed language-model-guided affinity maturation of seven antibodies, screening 20 or fewer variants of each antibody across only two rounds of laboratory evolution, and improved the binding affinities of four clinically relevant, highly mature antibodies up to sevenfold and three unmatured antibodies up to 160-fold, with many designs also demonstrating favorable thermostability and viral neutralization activity against Ebola and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudoviruses. The same models that improve antibody binding also guide efficient evolution across diverse protein families and selection pressures, including antibiotic resistance and enzyme activity, suggesting that these results generalize to many settings.

Imaging angiogenesis in an intracerebrally induced model of brain macrometastasis using αv β3 -targeted iron oxide microparticles.

Brain metastasis is responsible for a large proportion of cancer mortality, and there are currently no effective treatments. Moreover, the impact of treatments, particularly antiangiogenic therapeutics, is difficult to ascertain using current magnetic resonance imaging (MRI) methods. Imaging of the angiogenic vasculature has been successfully carried out in solid tumours using microparticles of iron oxide (MPIO) conjugated to a Arg-Gly-Asp peptide (RGD) targeting integrin αv β3 . The aim of this study was to determine whether RGD-MPIO could be used to identify angiogenic blood vessels in brain metastases in vivo. A mouse model of intracerebrally implanted brain macrometastasis was established through intracerebral injection of 4T1-GFP cells. T2 *-weighted imaging was used to visualise MPIO-induced hypointense voxels in vivo, and Prussian blue staining was used to visualise MPIO and endogenous iron histologically ex vivo. The RGD-MPIO showed target-specific binding in vivo, but the sensitivity of the agent for visualising angiogenic vessels per se was reduced by the presence of endogenous iron-laden macrophages in larger metastases, resulting in pre-existing hypointense areas within the tumour. Further, our data suggest that peptide-targeted MPIO, but not antibody-targeted MPIO, are taken up by perivascular macrophages within the macrometastatic microenvironment, resulting in additional nonspecific contrast. While pre-MPIO imaging will circumvent the issues surrounding pre-existing hypointensities and enable detection of specific contrast, our preliminary findings suggest that the use of antibodies rather than peptides as the targeting ligand may represent a preferable route forward for new angiogenesis-targeted molecular MRI agents.

Abstract 3185: Development of LILRB4 biparatopic synthetic T-cell receptor and antigen receptor (STAR)-T cells for the treatment of acute myeloid leukemia (AML)

Abstract Background: Acute myeloid leukemia (AML) is one of the most common hematopoietic malignancies. Adoptive cellular immunotherapy like chimeric antigen receptor (CAR) T therapy has demonstrated remarkable antitumor activity against B cell malignancies. However, CAR-T therapy exhibited very limited efficacy in treating AML due to the heterogeneity of AML cells and their similarity to normal hematopoietic cells and lack of leukemic cell-specific targets.LILRB4 (also known as ILT3, CD85k) is an immunosuppressive receptor that is highly expressed on monocyte AML blasts (FAB M4 and M5 AML subtypes), which makes it an ideal therapeutic target for monocytic AML. Here we generated LILRB4-specific nanobody-based STAR-T (Synthetic T-Cell Receptor and Antigen Receptor-T) cells, aiming to develop a new treatment strategy to improve outcomes for monocytic AML. Methods: LILRB4-specific nanobodies were obtained from alpacas immunized with the recombinant LILRB4 extracellular domain. These nanobodies were then fused to IgG1 Fc, expressed and purified from CHO cells and used for measuring the affinity, epitope binning, target binding specificity by SPR, BLI and flow cytometry. The LILRB4 STAR was designed as a double-chain TCR-based receptor with nanobodies fused to the N-terminus of the modified mouse TCR-Cα or TCR-Cβ, the two chains were linked with Furin-P2A sequence. Based on STAR structure, LILRB4 single epitopic STAR was generated by fusing an nanobody to the TCR-Cβ,whereas LILRB4 biparatopic STAR contained two nanobodies fused to the TCR-Cα and TCR-Cβ separately. In vitro assays and xenograft models were used to assess the potential of LILRB4 STAR-T cells for elimination of leukemic disease. Results and Conclusions: Multiple STAR-T cells were generated using a lentiviral-based vector and tested for their tumor lysis activity. Among multiple nanobodies tested, two nanobodies that displayed high antigen binding affinity and specificity were identified for further studies. They recognized two distinctive epitopes of LILRB4, and neither of them had competitive binding with APOE or FN1, known ligands of LILRB4. We generated both single paratopic and bi-paratopic STAR constructs and engineered the corresponding STAR-T cells. In vitro and in vivo cytotoxicity assays demonstrated that all LILRB4 STAR-T cells had high cytotoxic activity against LILRB4+ AML cell lines. In addition, LILRB4 biparatopic STAR-T cells exhibited the highest anti-leukemic activity and the best expansion in vivo. Therefore, the LILRB4 STAR-T cells described here may be further developed for the treatment of AML and represent a new immune cell therapy strategy for LILRB4+ AML patients. Citation Format: Wei Rui, Lei Lei, Zezhong Zhang, Chunyan Wu, Yu Xia, Yingying Liu, Xiaojing Pang, Ruifang Du, Lan Wang, Dengyue Sheng, Jing Guo, Yanyan Zhang, Xueqiang Zhao, James Pan. Development of LILRB4 biparatopic synthetic T-cell receptor and antigen receptor (STAR)-T cells for the treatment of acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3185.

Using display technologies to identify macrocyclic peptide antibiotics

Antibiotic resistant bacterial infections are now a leading cause of global mortality. While drug resistance continues to spread, the clinical antibiotic pipeline has become bare. This discord has focused attention on developing new strategies for antimicrobial discovery. Natural macrocyclic peptide-based products have provided novel antibiotics and antibiotic scaffolds targeting several essential bacterial cell envelope processes, but discovery of such natural products remains a slow and inefficient process. Synthetic strategies employing peptide display technologies can quickly screen large libraries of macrocyclic sequences for specific target binding and general antibacterial potential providing alternative approaches for new antibiotic discovery. Here we review cell envelope processes that can be targeted with macrocyclic peptide therapeutics, outline important macrocyclic peptide display technologies, and discuss future strategies for both library design and screening.

Selection of phage-displayed antibodies with high affinity and specificity by electrophoresis in microfluidic devices.

A method development aimed for high-throughput and automated antibody screening holds great potential for areas ranging from fundamental molecular interactions to the discovery of novel disease markers, therapeutic targets and monoclonal antibody engineering. Surface display techniques enable efficient manipulation of large molecular libraries in small volumes. Specifically, phage display appeared as a powerful technology for selecting peptides and proteins with enhanced, target-specific binding affinities. Here, we present a phage-selection microfluidic device wherein electrophoresis was performed under two orthogonal electric fields through an agarose gel functionalized with the respective antigen. This microdevice was capable of screening and sorting in a single round high affinity phage-displayed antibodies against virus glycoproteins, including Human Immunodeficiency Virus-1 (HIV-1) glycoprotein (gp120) or the Ebola Virus glycoprotein (EBOV-GP). Phages were differentially and laterally swept depending on their antigen affinity; the high affinity phages were recovered at channels proximal to the application site, whereas low affinity phages migrated distal after electrophoresis. These experiments proved that the microfluidic device specifically designed for phage-selection is rapid, sensitive and effective. Therefore, this is an efficient and cost-effective method that allowed highly controlled assay conditions for isolating and sorting high affinity ligands displayed in phages. This article is protected by copyright. All rights reserved.

OregonFluor enables quantitative intracellular paired agent imaging to assess drug target availability in live cells and tissues.

Non-destructive fluorophore diffusion across cell membranes to provide an unbiased fluorescence intensity readout is critical for quantitative imaging applications in live cells and tissues. Commercially available small-molecule fluorophores have been engineered for biological compatibility, imparting high water solubility by modifying rhodamine and cyanine dye scaffolds with multiple sulfonate groups. The resulting net negative charge, however, often renders these fluorophores cell-membrane-impermeant. Here we report the design and development of our biologically compatible, water-soluble and cell-membrane-permeable fluorophores, termed OregonFluor (ORFluor). By adapting previously established ratiometric imaging methodology using bio-affinity agents, it is now possible to use small-molecule ORFluor-labelled therapeutic inhibitors to quantitatively visualize their intracellular distribution and protein target-specific binding, providing a chemical toolkit for quantifying drug target availability in live cells and tissues.

89Zr-immuno-PET using the anti-LAG-3 tracer [89Zr]Zr-BI 754111: demonstrating target specific binding in NSCLC and HNSCC

PurposeAlthough lymphocyte activation gene-3 (LAG-3) directed therapies demonstrate promising clinical anti-cancer activity, only a subset of patients seems to benefit and predictive biomarkers are lacking. Here, we explored the potential use of the anti-LAG-3 antibody tracer [89Zr]Zr-BI 754111 as a predictive imaging biomarker and investigated its target specific uptake as well as the correlation of its tumor uptake and the tumor immune infiltration.MethodsPatients with head and neck (N = 2) or lung cancer (N = 4) were included in an imaging substudy of a phase 1 trial with BI 754091 (anti-PD-1) and BI 754111 (anti-LAG-3). After baseline tumor biopsy and [18F]FDG-PET, patients were given 240 mg of BI 754091, followed 8 days later by administration of [89Zr]Zr-BI 754111 (37 MBq, 4 mg). PET scans were performed 2 h, 96 h, and 144 h post-injection. To investigate target specificity, a second tracer administration was given two weeks later, this time with pre-administration of 40 (N = 3) or 600 mg (N = 3) unlabeled BI 754111, followed by PET scans at 96 h and 144 h post-injection. Tumor immune cell infiltration was assessed by immunohistochemistry and RNA sequencing.ResultsTracer uptake in tumors was clearly visible at the 4-mg mass dose (tumor-to-plasma ratio 1.63 [IQR 0.37-2.89]) and could be saturated by increasing mass doses (44 mg: 0.67 [IQR 0.50–0.85]; 604 mg: 0.56 [IQR 0.42–0.75]), demonstrating target specificity. Tumor uptake correlated to immune cell-derived RNA signatures.Conclusions[89Zr]Zr-BI-754111 PET imaging shows favorable technical and biological characteristics for developing a potential predictive imaging biomarker for LAG-3-directed therapies.Trial registrationClinicalTrials.gov, NCT03780725. Registered 19 December 2018

TCP-1, a novel peptide to diagnose early colon cancer

A nine cyclic peptide (TCP-1) showed excellent specificity for colon cancer. TCP-1 binds with human tumor tissues at early stages and mice tumor with diameters of 1-4 mm, suggesting that TCP-1 may be used for early diagnosis of colon cancer. The mechanism of the targeted binding of TCP-1 to colon cancer was also studied using immunoprecipitation, LC-MS and bioinformatics. After screening and identifying of the possible binding target proteins of TCP-1, keratin, type II cytoskeletal 5 was speculated to be the specific binding target protein of TCP-1 in human tumor tissue. Pharmacokinetics studies were conducted to investigate the target-mediated drug disposition of the new tumor-specific peptide by LC-MS/MS. The tissue distribution study showed that TCP-1 was found only in colon tumors (the target site) in tumor mice did not bind to any other tissues. Conjugating TCP-1 to tumor markedly increased its removal rate from blood circulation but mildly extended its staying time in vivo. In tumor mice, a lower AUC of TCP-1 (reduced by almost 35%) and 2-fold higher clearance were found compared to that of normal mice. The proposed metabolic pathway of TCP-1 in the kidney was also determined using LC-MSn-IT-TOF. The high specificity and low toxicity of the peptide may be caused by its extremely tight binding to the targets. Potential applications for future clinical use, including MRI and PET/CT were also explored, and this research may promote the development of colon cancer diagnostic technology research and provide new ideas and technical routes for tumor diagnostic technology.

Integrated Signal Amplification on a Fiber Optic SPR Sensor Using Duplexed Aptamers.

Throughout the past decades, fiber optic surface plasmon resonance (FO-SPR)-based biosensors have proven to be powerful tools for both the characterization of biomolecular interactions and target detection. However, as FO-SPR signals are generally related to the mass that binds to the sensor surface, multistep processes and external reagents are often required to obtain significant signals for low molecular weight targets. This increases the time, cost, and complexity of the respective bioassays and hinders continuous measurements. To overcome these requirements, in this work, cis-duplexed aptamers (DAs) were implemented on FO-SPR sensors, which underwent a conformational change upon target binding. This induced a spatial redistribution of gold nanoparticles (AuNPs) upon specific target binding and resulted in an amplified and concentration-dependent signal. Importantly, the AuNPs were covalently conjugated to the sensor, so the principle does not rely on multistep processes or external reagents. To implement this concept, first, the thickness of the gold fiber coating was adapted to match the resonance conditions of the surface plasmons present on the FO-SPR sensors with those on the AuNPs. As a result, the signal obtained due to the spatial redistribution of the AuNPs was amplified by a factor of 3 compared to the most commonly used thickness. Subsequently, the cis-DAs were successfully implemented on the FO-SPR sensors, and it was demonstrated that the DA-based FO-SPR sensors could specifically and quantitatively detect an ssDNA target with a detection limit of 230 nM. Furthermore, the redistribution of the AuNPs was proven to be reversible, which is an important prerequisite for continuous measurements. Altogether, the established DA-based FO-SPR bioassay holds much promise for the detection of low molecular weight targets in the future and opens up possibilities for FO-SPR-based continuous biosensing.

Однодоменное антитело для связывания консервативного эпитопа рецептор-связывающего домена белка Spike коронавируса SARS-CoV-2

Several COVID-19 vaccines have been developed in the last three years using various tecnhiques. Multiple virus-neutralizing antibodies against SARS-CoV-2 have been also obtained to combat the pandemic. However, the use of these medications for prevention and potential treatment faces significant challenges due to the emergence of new mutant virus variants, both more contagious and escaping neutralization by the immune system, that is why it is necessary to continuously renew the vaccines and develop new therapeutic antibodies. The study was aimed to use the technology of generating single-domain antibodies (nanobodies) to target the surface spike (S) protein RBD conserved epitope of the broad spectrum of SARS-CoV-2 variants. Recombinant proteins that corresponded to RBDs of three important SARS-СoV-2 strains and the full-length S protein (Wuhan) were used as antigens for immunization of a camel in order to induce production of appropriate antibodies and/or as immobilized proteins for further cross selection of the nanobody clones with pre-set specificity by the phage display. A nanobody capable of effectively recognizing the conservative region in the S protein RBDs of the broad spectrum of pandemic SARS-CoV-2 variants, including Omicron, was selected from the generated immune library. Along with conventional use in immunoassays and diagnosis, the generated nanobody can be potentially used as a module for target-specific binding used to trap coronavirus in human upper airways during the development of novel combination antiviral drugs.

Anti-AMPA Receptor Autoantibodies Reduce Excitatory Currents in Rat Hippocampal Neurons.

The GluR3 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) has been identified as a target for autoantibodies (Aabs) in autoimmune encephalopathy and other diseases. Recent studies have proposed mechanisms by which these Aabs act, but their exact role in neuronal excitability is yet to be established. Patient Aabs have been shown to bind to specific regions within the GluR3 subunit. GLUR3B peptides were designed based on described (ELISA) immunogenic epitopes for Aabs and an immunisation strategy was used to generate novel anti-AMPAR Aabs. Target-specific binding and specificity of affinity-purified anti-AMPAR Aabs was confirmed using enzyme-linked immunosorbent assay, immunocytochemistry and Western blot. Functional anti-AMPAR Aab effects were determined on excitatory postsynaptic currents (EPSCs) from primary hippocampal neurons using whole-cell patch-clamp electrophysiology. Acute (10 or 30 min) or longer-term (24 h) application of anti-AMPAR Aabs caused a significant reduction in the mean frequency of spontaneous and miniature EPSCs in hippocampal neurons. Our data demonstrate that anti-AMPAR Aabs targeting peptides linked to auto-immune diseases mediate inhibitory effects on neuronal excitability at the synaptic level, such effects may lead to disruption of the excitatory/inhibitory balance at a network level.

Receptor-ligand non-equilibrium kinetics (RLNEK) 1.0: An integrated Trackmate laminar flow chamber analysis

Although parallel plate flow chamber assays are widely performed, extraction of kinetic parameters is limited to specialized labs with mathematical expertise and customized video-microscopy tracking tools. The recent development of Trackmate has increased researcher accessibility to tracking particles in video-microscopy experiments; however, there is a lack of tools that analyze this tracking information. We report a software tool, compatible with Trackmate, that extracts Receptor Ligand Non-Equilibrium Kinetic (RLNEK) parameters from video-microscopy data. This software should be of particular interest to the community of researchers and scientists interrogating the target-specific binding and release of immune cells.