Cell Surface Binding Research Articles

Initial Outcome of Subconjunctival Bevacizumab for Corneal Neovascularization - a Case Series

Introduction : Corneal neovascularization (CNV) has a variety of causes and threatens corneal clarity, thus optimal visual acuity. Conventional medical management includes topical steroids and matrix metalloproteinase inhibitors like doxycycline. Anti-vascular endothelial growth factor (anti-VEGF) agents have demonstrated promise but remain off-label for this indication.
 Case Illustration : We present three patients with corneal neovascularization varied in etiology who underwent subconjunctival injection of Bevacizumab . Case 1: a-female-69 years old came with blurred vision after had Stephen Johnson Syndrome which affect her cornea. The examination revealed opacification of cornea accompanied by neovascularization at 5 until 9 o’clock position up to edge of pupil in the left. Case 2: a-male-47 years old presented corneal haze with neovascularization almostin all corneal region on background of fungal keratitis. Case 3: a-male-63 years old who underwent penetrating keratoplasty of indication corneal lekoma following an episode of fungal keratitis. There was opacity in all corneal region and neovascularization in 360 degree of limbus. All of these patients showed regression of neovessel after first injection of bevacizumab.
 Discussion : Corneal neovascularization is a condition caused by disruption homeostasis in which proangiogenic balanced by antiangiogenic, thereby maintaining avascularity. Bevacizumab as VEGF-A-binding proteins could prevent VEGF-A from binding cell surface receptors.
 Conclusion : Subconjunctival injection of Bevacizumab can be considered as a treatment for corneal neovascularization. It has shown promising effects in recess neovascularization in cornea regardlessthe etiology.

Bacterial surface display of human lectins in Escherichia coli.

Lectin-glycan interactions sustain fundamental biological processes involved in development and disease. Owing to their unique sugar-binding properties, lectins have great potential in glycobiology and biomedicine. However, their relatively low affinities and broad specificities pose a significant challenge when used as analytical reagents. New approaches for expression and engineering of lectins are in demand to overcome current limitations. Herein, we report the application of bacterial display for the expression of human galectin-3 and mannose-binding lectin in Escherichia coli. The analysis of the cell surface expression and binding activity of the surface-displayed lectins, including point and deletion mutants, in combination with molecular dynamics simulation, demonstrate the robustness and suitability of this approach. Furthermore, the display of functional mannose-binding lectin in the bacterial surface proved the feasibility of this method for disulfide bond-containing lectins. This work establishes for the first time bacterial display as an efficient means for the expression and engineering of human lectins, thereby increasing the available toolbox for glycobiology research.

Development of a [89Zr]Zr-labeled Human Antibody using a Novel Phage-displayed Human scFv Library.

Tax-interacting protein 1 (TIP1) is a cancer-specific radiation-inducible cell surface antigen that plays a role in cancer progression and resistance to therapy. This study aimed to develop a novel anti-TIP1 human antibody for noninvasive PET imaging in patients with cancer. A phage-displayed single-chain variable fragment (scFv) library was created from healthy donors' blood. High-affinity anti-TIP1 scFvs were selected from the library and engineered to human IgG1. Purified Abs were characterized by size exclusion chromatography high-performance liquid chromatography (SEC-HPLC), native mass spectrometry (native MS), ELISA, BIAcore, and flow cytometry. The labeling of positron emitter [89Zr]Zr to the lead Ab, L111, was optimized using deferoxamine (DFO) chelator. The stability of [89Zr]Zr-DFO-L111 was assessed in human serum. Small animal PET studies were performed in lung cancer tumor models (A549 and H460). We obtained 95% pure L111 by SEC-HPLC. Native MS confirmed the intact mass and glycosylation pattern of L111. Conjugation of three molar equivalents of DFO led to the optimal DFO-to-L111 ratio of 1.05. Radiochemical purity of 99.9% and specific activity of 0.37 MBq/μg was obtained for [89Zr]Zr-DFO-L111. [89Zr]Zr-DFO-L111 was stable in human serum over 7 days. The immunoreactive fraction in cell surface binding studies was 96%. In PET, preinjection with 4 mg/kg cold L111 before [89Zr]Zr-DFO-L111 (7.4 MBq; 20 μg) significantly (P < 0.01) enhanced the tumor-to-muscle standard uptake values (SUVmax) ratios on day 5 compared with day 2 postinjection. L111 Ab targets lung cancer cells in vitro and in vivo. [89Zr]Zr-DFO-L111 is a human antibody that will be evaluated in the first in-human study of safety and PET imaging.

Development of therapeutic monoclonal antibodies against DKK1 peptide-HLA-A2 complex to treat human cancers

BackgroundTargeted immunotherapy with monoclonal antibodies (mAbs) is an effective and safe method for the treatment of malignancies. Development of mAbs with improved cytotoxicity, targeting new and known tumor-associated antigens, therefore...

Potential of Animal Excreta as a Source of Probiotic Lactic Acid Bacteria for Aflatoxin B1 Detoxification by the Surface Binding Mechanism

Aflatoxins (AFs) are the most potent and ubiquitously found mycotoxins, capable of causing contamination in agricultural products. Aflatoxin B1 (AFB1) is the most toxic and primarily produced Aflatoxin and will be a real threat to the safety of food and feeds. The current study searched for the potential of Lactic acid bacteria (LAB) isolated from animal excreta for AFB1 mitigation. Three LAB out of 56 isolates were found to exhibit more than 50% sorbent action with AFB1 in phosphate-buffered saline (PBS) and were identified as Lactococcuslactis strain CF_6 (OP183481) (65.38%), Lacticaseibacillus casei strain CW_3 (OP183482)(52.63%) and Lactobacillus acidophilus strain CE_4 (OP183483)(63.13%). More than 60% of the total AFB1 removal was observed in 2 hr of incubation, and maximum sorbent action was found at a pH 6-7 range at 37oC for 24 hours. In the Scanning Electron Microscope (SEM) analysis, heat-killed cells showed a significant increase in cell surface binding area, which improved the surface binding for all isolates except L. casei strain CW_3; however, it proves that LAB surface binding is strain-specific rather than heat treatment. Moreover, the rise in AFB1 concentration improved the rate of the sorbent action but did not observe any substantial changes in total AFB1 detoxification. So, it is concluded that the animal excreta may be a versatile source of probiotic LAB for AFB1 detoxification by surface binding.

The Role of Chlamydia pneumoniae in the Aetiology of Autoimmune Diseases.

Introduction The most prevalent chronic human autoimmune disorder worldwide is rheumatoid arthritis. Synovial samples from acute-phase patients are polymerase chain reaction-positive for Chlamydia pneumoniae (C. pneumoniae)DNA and express chlamydial hsp60. Furthermore, anti-cyclic citrullinated peptide (anti-CCP) antibodies promote apoptosis of mature human Saos-2 osteoblasts via cell surface binding to citrullinated heat shock protein 60 (HSP60). Hence, we hypothesised that C. pneumoniae infection is associated with anti-CCP antibodies. Methods C. pneumoniae IgA and anti-CCP antibody levels were determined in 26 healthy subjects in this cross-sectional study. Serum C. pneumoniae IgA antibody levels were assessed using an enzyme-linked immunosorbent assay. Serum anti-CCP antibody levels were assessed using fluoroenzymeimmunoassay. Results There was a highly significant positive correlation between the two sets of antibodies (rs = 0.621; P = 0.0007). Linear regression analysis showed that this correlation was not the result of age or sex. Discussion A biologically plausible mechanism is put forward for these results, involving HSP60 acting as an endogenous ligand for toll-like receptor 4 (TLR4) and the interaction of TLR4 with lipopolysaccharides, which occur in the outer membrane of the C. pneumoniae elementary body. Pronounced pro-inflammatory mediator secretion then takes place. The release of Ca2+ ions may then activate local peptidylarginine deiminases, leading to the formation of CCPs and thus the reported finding. Confirmation of these results may have potential clinical implications in terms of diagnosis, including pre-symptomatic diagnosis, and treatment.

Role of peptide-cell surface interactions in cosmetic peptide application.

Cosmetic peptides have gained popularity in a wide range of skincare products due to their good biocompatibility, effective anti-oxidative properties, and anti-aging effects. However, low binding between peptides and the cell surface limits the efficacy of functional peptides. In this study, we designed two novel targeting peptide motifs to enhance the interaction between cosmetic peptides and the cell surface, thereby improving their performance for skin health. To achieve this, we optimized the well-known peptide tripeptide-1 (GHK) by separately grafting the integrin αvβ3-binding motif RGD and the chondroitin sulfate (CS)-binding motif sOtx2 onto it, forming two chimeric targeting peptides, RGD-GHK and sOtx2-GHK. Comparative analysis showed that both RGD-GHK and sOtx2-GHK exhibited superior anti-oxidative and anti-apoptotic effects compared to the non-targeting peptide, GHK. Furthermore, RGD-GHK demonstrated exceptional anti-aging activity, and its potential for promoting wound healing and repairing the skin barrier was evaluated in vitro using cells and skin models. In vitro permeation and in vivo adsorption testing confirmed that RGD-GHK achieved a high local concentration in the skin layer, initiating peptide effects and facilitating in vivo wound healing, while maintaining excellent biocompatibility. The enhancement of signaling cosmetic peptides can be attributed to the specific interaction between the binding motif and cell surface components. Consequently, this targeting peptide holds promising potential as a novel functional peptide for application in cosmetics.

A Cell Surface-Binding Antibody Atlas Nominates a MUC18-Directed Antibody-Drug Conjugate for Targeting Melanoma.

Discovery of melanoma-targeting antibodies using a proteome-scale array and use of a cutting-edge linker-payload system led to development of a MUC18-targeting antibody-exatecan conjugate with clinical potential for treating major melanoma subtypes.

Development and characterization of phage display-derived anti-toxin antibodies neutralizing TcdA and TcdB of Clostridioides difficile.

TcdA and TcdB are known as the major virulence attributes of Clostridioides difficile. Hence, neutralizing the TcdA and TcdB activities can be considered as an efficient therapeutic approach against C. difficile infection (CDI). In this work, we utilized phage display technique to select single-chain fragment variable (scFv) fragments as recombinant antibodies displayed on the surface of phages, which specifically target native TcdA, or TcdB (nTcdA and nTcdB), and their recombinant C-terminal combined repetitive oligopeptide (CROP) domains (rTcdA and rTcdB). After three rounds of biopanning, abundance of phage clones displaying high reactivity with TcdA or TcdB was quantified through enzyme-linked immunosorbent assay (ELISA). Furthermore, selected scFvs were characterized by cell viability and neutralization assays. The gene expression of immunological markers, IL-8 and TNF-α, was examined in treated Caco-2 cells by RT-qPCR. The epitopes of neutralizing scFvs were also identified by molecular docking. Totally, 18 scFv antibodies (seven for TcdA and 11 for TcdB) were identified by ELISA. Among selected scFvs, two clones for TcdA (rA-C2, A-C9) and three clones for TcdB (rB-B4, B-F5, B-F11) exhibited the highest neutralizing activity in Caco-2 and Vero cells. Moreover, the cocktail of anti-TcdA and anti-TcdB antibodies notably decreased the mRNA expression of TNF-α and IL-8 in Caco-2 cells. Molecular docking revealed that the interaction between scFv and toxin was mostly restricted to CROP domain of TcdA or TcdB. Our results collectively provided more insights for the development of neutralizing scFvs against C. difficile toxins using phage display. Further research is needed to meticulously evaluate the potential of scFvs as an alternative treatment for CDI using animal models and clinical trials.IMPORTANCETargeting the major toxins of Clostridioides difficile by neutralizing antibodies is a novel therapeutic approach for CDI. Here, we report a panel of new anti-TcdA (rA-C2, A-C9) and anti-TcdB (rB-B4, B-F5, and B-F11) recombinant antibody fragments (scFvs) isolated from Tomlinson I and J libraries using phage display technique. These scFv antibodies were capable of neutralizing their respective toxin and showed promise as potential therapeutics against TcdA and TcdB of C. difficile in different in vitro models. In addition, in silico analysis showed that at least two neutralization mechanisms, including inhibiting cell surface binding of toxins and inhibiting toxin internalization can be proposed for the isolated scFvs in this work. These findings provide more insights for the applicability of specific scFvs toward C. difficile toxins at in vitro level. However, further research is required to evaluate the potential application of these scFvs as therapeutic agents for CDI treatment in clinical setting.

Design of a mucin-selective protease for targeted degradation of cancer-associated mucins.

Targeted protein degradation is an emerging strategy for the elimination of classically undruggable proteins. Here, to expand the landscape of targetable substrates, we designed degraders that achieve substrate selectivity via recognition of a discrete peptide and glycan motif and achieve cell-type selectivity via antigen-driven cell-surface binding. We applied this approach to mucins, O-glycosylated proteins that drive cancer progression through biophysical and immunological mechanisms. Engineering of a bacterial mucin-selective protease yielded a variant for fusion to a cancer antigen-binding nanobody. The resulting conjugate selectively degraded mucins on cancer cells, promoted cell death in culture models of mucin-driven growth and survival, and reduced tumor growth in mouse models of breast cancer progression. This work establishes a blueprint for the development of biologics that degrade specific protein glycoforms on target cells.

Exploring the chemical space for potential inhibitors against cell surface binding protein of Mpox virus using molecular fingerprint based screening approach

Mpox virus is the latest member of the Poxviridae family of which small pox virus is a member. Monekypox virus has led to thousands of infections across the globe. Poxvirus gains entry into the cell making use of glycosaminoglycans like chondroitin sulphate and heparan sulphate. The interaction of the Mpox virus protein E8L also called cell surface binding protein is crucial for host cell attachment, membrane fusion and viral entry into the host cell leading to establishment of infection thus making this protein a very attractive therapeutic target. In this study we have tried to utilize the chondroitin sulphate binding groove present in the protein and identify molecules which are structurally similar to chondroitin sulphate. These molecules can thus occupy the same pocket but with a better binding affinity than chondroitin sulphate in order to outcompete the latter molecule from binding to the E8L protein and thus prevent it from performing its function. This study may pave the way for development of highly efficient therapeutics against the Mpox virus and further curb its infective potential. Communicated by Ramaswamy H. Sarma

γ-Bungarotoxin impairs the vascular endothelial barrier function by inhibiting integrin α5

γ-bungarotoxin (γ-BGT) is an RGD motif-containing protein, derived from the venom of Bungarus multicinctus, leading to acute death in mice. These RGD motif-containing proteins from snake venom belonging to the disintegrin family can interfere with vascular endothelial homeostasis by directly binding cell surface integrins. Targeting integrins that generate vascular endothelial dysfunction may contribute to γ-BGT poisoning, however, the underlying mechanisms have not been investigated in detail. In this study, the results showed that γ-BGT played a role in -promoting the permeability of the vascular endothelial barrier. Depending on its selective binding to integrin α5 in vascular endothelium (VE), γ-BGT initiated downstream events, including focal adhesion kinase dephosphorylation and cytoskeleton remodeling, resulting in the intercellular junction interruption. Those alternations facilitated paracellular permeability of VE and barrier dysfunction. Proteomics profiling identified that as a downstream effector of the integrin α5 / FAK signaling pathway cyclin D1 partially mediated the cellular structural changes and barrier dysfunction. Furthermore, VE-released plasminogen activator urokinase and platelet-derived growth factor D could serve as potential diagnostic biomarkers for γ-BGT-induced vascular endothelial dysfunction. Our results indicate the mechanisms through which γ-BGT as a novel disintegrin directly interacts with the VE, with consequences for barrier dysfunction.

Kingella kingae RtxA toxin interacts with sialylated gangliosides

The membrane-damaging RTX family cytotoxin RtxA is a key virulence factor of the emerging pediatric pathogen Kingella kingae, but little is known about the mechanism of RtxA binding to host cells. While we have previously shown that RtxA binds cell surface glycoproteins, here we demonstrate that the toxin also binds different types of gangliosides. The recognition of gangliosides by RtxA depended on sialic acid side groups of ganglioside glycans. Moreover, binding of RtxA to epithelial cells was significantly decreased in the presence of free sialylated gangliosides, which inhibited cytotoxic activity of the toxin. These results suggest that RtxA utilizes sialylated gangliosides as ubiquitous cell membrane receptor molecules on host cells to exert its cytotoxic action and support K. kingae infection.

Generation and characterization of a monoclonal antibody that binds to Galectin-1

Galectin-1 is a β-galactoside-binding lectin that has been implicated as a suppressive molecule in cancer and autoimmune diseases. Gal-1 has known immunomodulatory activity and was found to be expressed on regulatory T cells, leading to the potential for targeted immunotherapies. Anti-Gal-1 monoclonal antibodies were generated in this study using classical hybridoma techniques. MAb 6F3 was found to bind to Gal-1 by Western blot and ELISA. Flow cytometry was used to determine cell surface and intracellular binding of mAb 6F3 to Gal-1 in PBMC-derived Tregs and tumor cells, including Treg-like cell lines. These results suggest mAb 6F3 may be used to further study Gal-1 protein expression and function.

NAV-001, a high-efficacy antibody-drug conjugate targeting mesothelin with improved delivery of a potent payload by counteracting MUC16/CA125 inhibitory effects.

Subsets of tumor-produced cell surface and secreted proteins can bind to IgG1 type antibodies and suppress their immune-effector activities. As they affect antibody and complement-mediated immunity, we call these proteins humoral immuno-oncology (HIO) factors. Antibody-drug conjugates (ADCs) use antibody targeting to bind cell surface antigens, internalize into the cell, then kill target cells upon liberation of the cytotoxic payload. Binding of the ADC antibody component by a HIO factor may potentially hamper ADC efficacy due to reduced internalization. To determine the potential effects of HIO factor ADC suppression, we evaluated the efficacy of a HIO-refractory, mesothelin-directed ADC (NAV-001) and a HIO-bound, mesothelin-directed ADC (SS1). The HIO factor MUC16/CA125 binding to SS1 ADC was shown to have a negative effect on internalization and tumor cell killing. The MUC16/CA125 refractory NAV-001 ADC was shown to have robust killing of MUC16/CA125 expressing and non-expressing tumor cells in vitro and in vivo at single, sub-mg/kg dosing. Moreover, NAV-001-PNU, which contains the PNU-159682 topoisomerase II inhibitor, demonstrated good stability in vitro and in vivo as well as robust bystander activity of resident cells while maintaining a tolerable safety profile in vivo. Single-dose NAV-001-PNU demonstrated robust tumor regression of a number of patient-derived xenografts from different tumor types regardless of MUC16/CA125 expression. These findings suggest that identification of HIO-refractory antibodies to be used in ADC format may improve therapeutic efficacy as observed by NAV-001 and warrants NAV-001-PNU's advancement to human clinical trials as a monotherapy to treat mesothelin-positive cancers.

Apolipoprotein E Recognizes Alzheimer's Disease Associated 3‐O Sulfation of Heparan Sulfate

AbstractApolipoprotein E (ApoE)’s ϵ4 alle is the most important genetic risk factor for late onset Alzheimer's Disease (AD). Cell‐surface heparan sulfate (HS) is a cofactor for ApoE/LRP1 interaction and the prion‐like spread of tau pathology between cells. 3‐O‐sulfo (3‐O‐S) modification of HS has been linked to AD through its interaction with tau, and enhanced levels of 3‐O‐sulfated HS and 3‐O‐sulfotransferases in the AD brain. In this study, we characterized ApoE/HS interactions in wildtype ApoE3, AD‐linked ApoE4, and AD‐protective ApoE2 and ApoE3‐Christchurch. Glycan microarray and SPR assays revealed that all ApoE isoforms recognized 3‐O‐S. NMR titration localized ApoE/3‐O‐S binding to the vicinity of the canonical HS binding motif. In cells, the knockout of HS3ST1‐a major 3‐O sulfotransferase‐reduced cell surface binding and uptake of ApoE. 3‐O‐S is thus recognized by both tau and ApoE, suggesting that the interplay between 3‐O‐sulfated HS, tau and ApoE isoforms may modulate AD risk.

The invariant chain CD74 protein is a cell surface binding partner of TIMP-1 in breast cancer cells.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) regulates the proteolytic activity of matrix metalloproteinases (MMPs), playing an important role in the homeostasis of the extracellular matrix. Beyond its well-known role in tissue maintenance, TIMP-1 has been associated with multiple MMP-independent cytokine-like functions. The protein structure of TIMP-1, with two distinct domains, one interacting with MMPs and another able to bind multiple partners, provides a rationale for this multi-functionality. The identification of CD63 as a cell surface receptor for TIMP-1, able to mediate intracellular signaling through the Erk/MAPK axis, provided a molecular basis for the role of TIMP-1 in cellular signaling. However, several lines of evidence suggest that TIMP-1 may be able to associate with many interaction partners, thus attaining multiple functions. To enable the identification of previously unknown interaction partners that may underpin the core cellular functions of TIMP-1, known as well as unknown, we performed a yeast two-hybrid screening using a mammary gland complementary DNA (cDNA) library. We report here the identification of multiple interactors, including MHC class II-associated invariant chain γ (CD74). We verified that CD74 interacts with TIMP-1 in breast cancer cells and that this interaction contributes to cellular internalization of TIMP-1 and mediates intracellular signaling through the Akt signaling axis in breast cancer cells. These data provide new insights into the complex nature of the functions of TIMP-1 and their potential mechanistic basis.

Apolipoprotein E Recognizes Alzheimer's Disease Associated 3-O Sulfation of Heparan Sulfate.

Apolipoprotein E (ApoE)'s ϵ4 alle is the most important genetic risk factor for late onset Alzheimer's Disease (AD). Cell-surface heparan sulfate (HS) is a cofactor for ApoE/LRP1 interaction and the prion-like spread of tau pathology between cells. 3-O-sulfo (3-O-S) modification of HS has been linked to AD through its interaction with tau, and enhanced levels of 3-O-sulfated HS and 3-O-sulfotransferases in the AD brain. In this study, we characterized ApoE/HS interactions in wildtype ApoE3, AD-linked ApoE4, and AD-protective ApoE2 and ApoE3-Christchurch. Glycan microarray and SPR assays revealed that all ApoE isoforms recognized 3-O-S. NMR titration localized ApoE/3-O-S binding to the vicinity of the canonical HS binding motif. In cells, the knockout of HS3ST1-a major 3-O sulfotransferase-reduced cell surface binding and uptake of ApoE. 3-O-S is thus recognized by both tau and ApoE, suggesting that the interplay between 3-O-sulfated HS, tau and ApoE isoforms may modulate AD risk.

Utilization of Circulating Tumor Cells in the Management of Solid Tumors.

Circulating tumor cells (CTCs) are tumor cells shed from the primary tumor into circulation, with clusters of CTCs responsible for cancer metastases. CTC detection and isolation from the bloodstream are based on properties distinguishing CTCs from normal blood cells. Current CTC detection techniques can be divided into two main categories: label dependent, which depends upon antibodies that selectively bind cell surface antigens present on CTCs, or label-independent detection, which is detection based on the size, deformability, and biophysical properties of CTCs. CTCs may play significant roles in cancer screening, diagnosis, treatment navigation, including prognostication and precision medicine, and surveillance. In cancer screening, capturing and evaluating CTCs from peripheral blood could be a strategy to detect cancer at its earliest stage. Cancer diagnosis using liquid biopsy could also have tremendous benefits. Full utilization of CTCs in the clinical management of malignancies may be feasible in the near future; however, several challenges still exist. CTC assays currently lack adequate sensitivity, especially in early-stage solid malignancies, due to low numbers of detectable CTCs. As assays improve and more trials evaluate the clinical utility of CTC detection in guiding therapies, we anticipate increased use in cancer management.

An AND‐Gate DNA Nanodevice for Dual Protein‐Mediated, Tumor‐Specific Imaging

Comprehensive SummarySensitive and specific imaging of tumor‐associated biomarkers offers an effective way for cancer diagnosis. However, most of the reported imaging probes are limited to the targeting of a single biomarker, which may impair imaging precision. Here, we report an aptamer‐based, AND‐gate DNA nanodevice that relies on the sequential processing of two tumor‐associated proteins to produce a specific fluorescent signal for precise tumor imaging. The DNA nanodevice integrates an AS1411 aptamer to bind cell surface nucleolin for tumor targeting and a DNA sensor to respond to intracellular apurinic/apyrimidinic endonuclease 1 for activatable imaging. We evaluated the performance of the system in both living cells and a tumor‐bearing mouse model, and demonstrated its capability in tumor cell‐specific imaging. We anticipate that this strategy will accelerate the design of multivariate protein‐stimulated probes for tumor diagnosis.