Cellular Binding Research Articles

An integrated mammalian library approach for optimization and enhanced microfluidics-assisted antibody hit discovery

Recent years have seen the development of a variety of mammalian library approaches for display and secretion mode. Advantages include library approaches for engineering, preservation of precious immune repertoires and their repeated interrogation, as well as screening in final therapeutic format and host. Mammalian display approaches for antibody optimization exploit these advantages, necessitating the generation of large libraries but in turn enabling early screening for both manufacturability and target specificity. For suitable libraries, high antibody integration rates and resulting monoclonality need to be balanced – we present a solution for sufficient transmutability and acceptable monoclonality by applying an optimized ratio of coding to non-coding lentivirus. The recent advent of microfluidic-assisted hit discovery represents a perfect match to mammalian libraries in secretion mode, as the lower throughput fits well with the facile generation of libraries comprising a few million functional clones. In the presented work, Chinese Hamster Ovary cells were engineered to both express the target of interest and secrete antibodies in relevant formats, and specific clones were strongly enriched by high throughput screening for autocrine cellular binding. The powerful combination of mammalian secretion libraries and microfluidics-assisted hit discovery could reduce attrition rates and increase the probability to identify the best possible therapeutic antibody hits faster.

Exploring the impact of PEGylation on the cell-nanomicelle interactions by AFM-based single-molecule force spectroscopy and force tracing.

PEGylation has been considered the gold standard method for the modification of various drug delivery systems since the last century. However, the impact of PEGylation on the dynamic interaction between drug carriers and cell membranes has not been quantitatively clarified. Herein, the cellular binding and receptor-mediated endocytosis of a model PEGylated polypeptide nanomicelle were systematically investigated at the single-particle level using AFM-based single-molecule force spectroscopy (SMFS) and force tracing. A self-assembled elastin-like polypeptide (ELP) nanomicelle, which is capable of cross-linking, gastrin-releasing peptide (GRP) modification, and PEGylation was prepared. The cross-linked ELP-based nanomicelles exhibited outstanding stability in a broad temperature range of 4-40°C, which facilitate the drug loading, as well as our cell-nanomicelle study at the single particle level. The unbinding force between the cross-linked ELP-based nanomicelles and the GRP receptor (GRPR)-containing cell (PC-3) membranes was quantitatively measured by AFM-SMFS. It is found that the PEGylated GRP-displaying nanomicelles exhibit the highest unbinding force, indicating the enhanced specific binding effect of PEGylation. Furthermore, the receptor-mediated endocytosis of the cross-linked ELP-based nanomicelles was monitored with the help of force tracing based on AFM-SMFS. Our results show that PEGylation decreases the endocytic force, duration, and engulfment depth of the PEGylated GRP-displaying nanomicelles, but increases their endocytic velocity, which results from the elimination of non-specific interactions during endocytosis. These observations demonstrate the diverse and complex roles of PEGylation on the interaction of polypeptide nanomicelles to cell membranes and may shed light on the rational design of organic polymer-based drug delivery systems aiming for active and passive targeting strategies. STATEMENT OF SIGNIFICANCE: A self-assembled elastin-like polypeptide (ELP) nanomicelle, which can be easily cross-linked, gastrin-releasing peptide (GRP) modified, and PEGylated, is designed. The AFM-SMFS experiment shows that PEGylation can enhance specific binding of the nanomicelles to the receptors on cell membranes. The force tracing experiment indicates that PEGylation decreases the endocytic force as well as engulfment depth of the nanomicelles through the elimination of non-specific interactions. PEGylation can benefit the drug delivery systems aiming at active targeting, while might not be an ideal modification for drug carriers designed for passive targeting, whose cellular uptake mainly depends on non-specific interactions.

Activity of bovine lactoferrin in resistance to white spot syndrome virus infection in shrimp

White spot syndrome virus (WSSV) is a notorious pathogen that has plagued shrimp farming worldwide for decades. To date, there are no known treatments that are effective against this virus. Lactoferrin (LF) is a protein with many bioactivities, including antiviral properties. In this study, the activities and mechanisms of bovine LF (bLF) against WSSV were analyzed. Our results showed that bLF treatment significantly reduced shrimp mortalities caused by WSSV infection. bLF was found to have the ability to bind to surfaces of both host cells and WSSV virions. These bindings may have been a result of bLF interactions with the host cellular chitin binding protein and F1 ATP synthase β subunit protein and the WSSV structural proteins VP28, VP110, VP150 and VP160B. bLF demonstrated potential for development as an anti-WSSV agent in shrimp culture. Furthermore, these reactionary proteins may play a role in WSSV infection.

Structural basis of α-synuclein uptake by neurons.

Cell-to-cell transmission of toxic forms of α-Synuclein (αS) is thought to underlie disease progression in Parkinson's disease and other synucleinopathies. My lab identified a neuronal protein, neurexin 1β, as capable of driving selective uptake of αS; the interaction is dependent upon N-terminal aceylation of αS, as well as N-linked glycosylation of neurexin 1β. I will describe our efforts both to understand the role of N-terminal acetylation in αS binding to neurexin 1β as well as to determine whether this modification alters its interactions with other putative cellular binding partners. To do this, we use a variety of biophysical and quantitative cell biological methods. Our work provides insight into molecular details of the interaction between αS and neurexin 1β and whether this interaction may be exploited for therapeutic development.

Binding, activity and risk assessment of bisphenols toward farnesoid X receptor pathway: In vitro and in silico study

Bisphenols have been identified as emerging environmental pollutants of high concern with potential adverse effects through interactions with receptor-mediated pathways. However, their potential mechanism of action and health risks through the farnesoid X receptor (FXR) pathway remain poorly understood. In the present study, we aimed to explore the potential disruption mechanism of bisphenols through the FXR signalling pathway. Receptor binding assays showed that bisphenols bound to FXR directly, with tetrabromobisphenol A (TBBPA; 34-fold), tetrachlorobisphenol A (TCBPA; 8.7-fold), bisphenol AF (BPAF; 2.0-fold), and bisphenol B (BPB; 1.9-fold) showing a significantly stronger binding potency than bisphenol A (BPA). In receptor transcriptional activity assays, bisphenols showed agonistic activity toward FXR, with BPAF, BPB, and bisphenol F (BPF) exhibiting higher activity than BPA, but TBBPA and TCBPA showing significantly weaker activity than BPA. Molecular docking results indicated that the number of hydrogen bonds dictated their binding strength. Intracellular concentrations of bisphenols were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in receptor activity assays, and it was found that the intracellular concentrations of TBBPA and TCBPA were 40-fold lower than those of BPA. Using the bioactivity concentrations in the FXR receptor activity assay, the liver concentrations of bisphenols were estimated using physiologically-based pharmacokinetic (PBPK) models through their serum concentrations, and the hazard quotient (HQ) values were calculated. The results suggest a potentially high concern for the risk of activating the FXR pathway for some populations with high exposure. Overall, these results indicate that bisphenols can bind to and activate FXR receptors, and that the activation mechanism is dependent on cellular uptake and binding strength. This study provides important information regarding the exposure risk of bisphenols, which can promote the development of environmentally friendly bisphenols.

Gallium-68-Labeled ZPDGFRβ Affibody: A Potential PET Probe for Platelet-Derived Growth Factor Receptor β-Expressing Carcinomas.

Platelet-derived growth factor receptor β (PDGFRβ) has been demonstrated to be an effective biomarker for a variety of malignant cancers, and affibody-based PDGFRβ molecules have potential as positron emission tomography (PET) tracers for the diagnosis of cancers. Based on previous pharmacokinetics studies, short-lived positron emission radionuclides, such as fluorine-18 and gallium-68, would be more suitable for affibody-based PET imaging. Thus, in the present study, we prepared a gallium-68-labeled PDGFRβ-targeting dimeric affibody conjugate and evaluated its capability for visualizing malignant tumors by micro-PET/computed tomography (CT) imaging. The PDGFRβ-targeting ZPDGFRβ affibody was conjugated with the p-NCS-Bn-DOTA macrocyclic ligand and radiolabeled with gallium-68 to generate the 68Ga-DOTA-ZPDGFRβ PET probe . Then, several types of malignant carcinoma cells (U-87 MG, LS 174T, A549, H1688, and H446) were used to evaluate the targeted cellular binding capability of the PET probe through in vitro/in vivo cellular assays and whole-body imaging by micro-PET/CT. The 68Ga-DOTA-ZPDGFRβ was successfully prepared with a radiochemical yield of 93% and exhibited ideal stability for up to 4 h at room temperature in vitro. This radioactive conjugate demonstrated specific binding ability with PDGFRβ-expressing U-87 MG cells, which was suppressed by PDGFRβ ligands. The biodistribution of 68Ga-DOTA-ZPDGFRβ indicated fast liver clearance and a kidney-bladder excretion route. The U-87 MG xenografted tumor was clearly visualized with 68Ga-DOTA-ZPDGFRβ at 1 h postinjection using micro-PET/CT imaging. 68Ga-DOTA-ZPDGFRβ is a potential radiopharmaceutical for the diagnosis of PDGFRβ-expressing tumors.

Transcriptome Analysis of the Regulatory Mechanism of Exogenous Spermidine in High Temperature Stress Resistance of Tomato Seedlings

Previous studies have shown that spermidine (Spd) can improve tolerance to high temperature stress in tomato seedlings. To further understand how Spd regulates the molecular components of high temperature stress signaling pathways, we performed a genome-wide transcriptome analysis in tomato seedlings treated with high temperature and/or exogenous Spd. The results demonstrate that, under high temperature conditions, Spd significantly alleviated the inhibition of plant growth, as well as improving the net photosynthetic rate and pigment contents. The transcriptome analysis revealed thousands of differentially expressed genes (DEGs) in response to high temperature with or without Spd treatment. Half of the genes were induced by high temperature, part of the genes were induced by high temperature with exogenous Spd, and some were induced by the coordinated effect of high temperature and Spd. A GO analysis indicated that genes involved in cellular processes, metabolic processes, and nucleotide binding in the sample were subjected to high temperature. Some DEGs were also involved in plant physiological processes. These results suggest potential genes and molecular pathways were involved in the exogenous Spd-mediated tolerance to high temperature stress in tomato plants. A JA signaling test was designed, which indicated that MYC2 and JAS1 in heat-resistant materials were both increased, through quantitative RT-PCR.

Identification and characterization of a flexile G-quadruplex in the distal promoter region of stemness gene REX1

We have identified a parallel G-quadruplex (R1WT) in the distal promoter region (−821 base-pairs upstream of the TSS) of the pluripotent gene REX1. Through biophysical and biochemical approach, we have characterized the G-quadruplex (GQ) as a potential molecular switch that may control REX1 promoter activity to determine the transcriptional fate. Small- molecule interactive study of the monomeric form of R1WT (characterized as R1mut2) with TMPyP4 and BRACO-19 revealed GQ destabilization upon interaction with TMPyP4 and stabilization upon interaction with BRACO-19. This distinctive drug interactivity suggests the in cellulo R1WT to be a promising drug target. The endogenous existence of R1WT was confirmed by BG4 antibody derived chromatin immunoprecipitation experiment. Here in, we also report the endogenous interaction of GQ specific transcription factors (TFs) with R1WT region in the human chromatin of cancer cell. The wild-type G-quadruplex was found to interact with four important transcription factors, (i) specificity protein (Sp1) (ii) non-metastatic cell 2 (NM23-H2): a diphosphatase (iii) cellular nucleic acid binding protein (CNBP) and (iv) heterogenous nuclear ribonucleoprotein K (hnRNPK) in the REX1 promoter. In contrast, nucleolin protein (NCL) binding was found to be low to the said G-quadruplex. The flexibility of R1WT between folded and unfolded states, obtained from experimental and computational analysis strongly suggests R1WT to be an important gene regulatory element in the genome. It controls promoter DNA relaxation with the coordinated interaction of transcription factors, the deregulation of which seeds stemness characteristic in cancer cells for further metastatic progression.

Transcriptomic study of Suaeda salsa in response to salt and drought stress.

Drought and salinity are the main factors limiting agricultural production. Improving crop resistance to relieve land stress is a major challenge in agriculture. The salt-tolerant species Suaeda salsa is a typical indicator of saline soil. It has a strong drought tolerance and can be used as a model plant to study salt and drought tolerance in plants. In this study, transcriptome sequencing and bioinformatic analysis were performed to study gene expression changes in S. salsa under salt and drought stresses, and to screen out differentially expressed genes. The genetic changes were most abundant in cellular processes, metabolic processes, ion binding, signalling, post-translational modifications, protein conversion, and molecular chaperones, suggesting that the above methods may play a significant role in the response of S. salsa to external salt and drought stress. Enrichment analysis showed that carbohydrate metabolic processes, oxidoreductase activity, transmembrane transport, kinase activity, cellular protein modification processes, and ion-binding pathways are involved in the stress response of S. salsa .

Light controlled reversible Michael addition of cysteine: a new tool for dynamic site-specific labeling of proteins.

Cysteine-based Michael addition is a widely employed strategy for covalent conjugation of proteins, peptides, and drugs. The covalent reaction is irreversible in most cases, leading to a lack of control over the process. Utilizing spectroscopic analyses along with X-ray crystallographic studies, we demonstrate Michael addition of an engineered cysteine residue in human Cellular Retinol Binding Protein II (hCRBPII) with a coumarin analog that creates a non-fluorescent complex. UV-illumination reverses the conjugation, yielding a fluorescent species, presumably through a retro-Michael process. This series of events can be repeated between a bound and non-bound form of the cysteine reversibly, resulting in the ON-OFF control of fluorescence. The details of the mechanism of photoswitching was illuminated by recapitulation of the process in light irradiated single crystals, confirming the mechanism at atomic resolution.

M6A modifications of circular RNAs in ischemia-induced retinal neovascularization.

Ischemia-induced pathological neovascularization in the retina is a leading cause of blindness in various age groups. The purpose of the current study was to identify the involvement of circular RNAs (circRNAs) methylated by N6-methyladenosine (m6A), and predict their potential roles in oxygen-induced retinopathy (OIR) in mice. Methylation assessment via microarray analysis indicated that 88 circRNAs were differentially modified by m6A methylation, including 56 hyper-methylated circRNAs and 32 hypo-methylated circRNAs. Gene ontology enrichment analysis predicted that the enriched host genes of the hyper-methylated circRNAs were involved in cellular process, cellular anatomical entity, and protein binding. Host genes of the hypo-methylated circRNAs were enriched in the regulation of cellular biosynthetic process, the nucleus, and binding. According to the Kyoto Encyclopedia of Genes and Genomes analysis, those host genes were involved in the pathways of selenocompound metabolism, salivary secretion, and lysine degradation. MeRIP-qPCR verified significant alterations in m6A methylation levels of mmu_circRNA_33363, mmu_circRNA_002816, and mmu_circRNA_009692. In conclusion, the study revealed the m6A modification alterations in OIR retinas, and the findings above shed light on the potential roles of m6A methylation in circRNA regulatory functions in the pathogenesis of ischemia-induced pathological retinal neovascularization.

Tumor Targeting of 211At-Labeled Antibody under Sodium Ascorbate Protection against Radiolysis.

Astatine-211 (211At) is an alpha emitter applicable to radioimmunotherapy (RIT), a cancer treatment that utilizes radioactive antibodies to target tumors. In the preparation of 211At-labeled monoclonal antibodies (211At-mAbs), the possibility of radionuclide-induced antibody denaturation (radiolysis) is of concern. Our previous study showed that this 211At-induced radiochemical reaction disrupts the cellular binding activity of an astatinated mAb, resulting in attenuation of in vivo antitumor effects, whereas sodium ascorbate (SA), a free radical scavenger, prevents antibody denaturation, contributing to the maintenance of binding and antitumor activity. However, the influence of antibody denaturation on the pharmacokinetics of 211At-mAbs relating to tumor accumulation, blood circulation time, and distribution to normal organs remains unclear. In this study, we use a radioactive anti-human epidermal growth factor receptor 2 (anti-HER2) mAb to demonstrate that an 211At-induced radiochemical reaction disrupts active targeting via an antigen-antibody interaction, whereas SA helps to maintain targeting. In contrast, there was no difference in blood circulation time as well as distribution to normal organs between the stabilized and denatured immunoconjugates, indicating that antibody denaturation may not affect tumor accumulation via passive targeting based on the enhanced permeability and retention effect. In a high-HER2-expressing xenograft model treated with 1 MBq of 211At-anti-HER2 mAbs, SA-dependent maintenance of active targeting contributed to a significantly better response. In treatment with 0.5 or 0.2 MBq, the stabilized radioactive mAb significantly reduced tumor growth compared to the denatured immunoconjugate. Additionally, through a comparison between a stabilized 211At-anti-HER2 mAb and radioactive nontargeted control mAb, we demonstrate that active targeting significantly enhances tumor accumulation of radioactivity and in vivo antitumor effect. In RIT with 211At, active targeting contributes to efficient tumor accumulation of radioactivity, resulting in a potent antitumor effect. SA-dependent protection that successfully maintains tumor targeting will facilitate the clinical application of alpha-RIT.

Cellular retinol-binding protein 1: a therapeutic and diagnostic tumor marker.

Cellular Retinol Binding Protein 1 (CRBP1) gene is a protein coding gene located on human chromosome 3q21, which codifies a protein named CRBP1. CRBP1 is widely expressed in many tissues as a chaperone protein to regulate the uptake, subsequent esterification and bioavailability of retinol. CRBP1 combines retinol and retinaldehyde with high affinity to protect retinoids from non-specific oxidation, and transports retinoids to specific enzymes to promote the biosynthesis of retinoic acid. The vital role of CRBP1 in retinoids metabolism has been gradually discovered, which has been implicated in tumorigenesis. However, the precise functions of CRBP1 in different diseases are still poorly understood. The purpose of this review is to provide an overview of the role of CRBP1 in various diseases, especially in both the promotion and inhibition of cancers, which may also offer a novel biomarker and potential therapeutic target for human diseases.

Lectin-based phototherapy targeting cell surface glycans for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is resistant to current treatments but lectin-based therapy targeting cell surface glycans could be a promising new horizon. Here, we report a novel lectin-based phototherapy (Lec-PT) that combines the PDAC targeting ability of rBC2LCN lectin to a photoabsorber, IRDye700DX (rBC2-IR700), resulting in a novel and highly specific near-infrared, light-activated, anti-PDAC therapy. Lec-PT cytotoxicity was first verified in vitro with a human PDAC cell line, Capan-1, indicating that rBC2-IR700 is only cytotoxic upon cellular binding and exposure to near-infrared light. The therapeutic efficacy of Lec-PT was subsequently verified in vivo using cell lines and patient-derived, subcutaneous xenografting into nude mice. Significant accumulation of rBC2-IR700 occurs as early as 2hours postintravenous administration while cytotoxicity is only achieved upon exposure to near-infrared light. Repeated treatments further slowed tumor growth. Lec-PT was also assessed for off-target toxicity in the orthotopic xenograft model. Shielding of intraperitoneal organs from near-infrared light minimized off-target toxicity. Using readily available components, Lec-PT specifically targeted pancreatic cancer with high reproducibility and on-target, inducible toxicity. Rapid clinical development of this method is promising as a new modality for treatment of pancreatic cancer.

Gastrodin ameliorates exercise-induced fatigue via modulating Nrf2 pathway and inhibiting inflammation in mice

Gastrodia elata Bl. is traditional Chinese medicine used to alleviate fatigue, but its underlying mechanism is still unclear. This study explored the anti-fatigue mechanism of gastrodin by exercise-induced fatigue model and network pharmacology. This study found that gastrodin (200 mg/kg/day) had significant anti-fatigue effects in C57BL/6J mice based on mouse energy and endurance measurements. Gastrodin could effectively ameliorate biochemical indexes in the fatigue mice. The putative targets of “Gastrodin” and “Fatigue” were obtained by integrating multiple databases, and a virtual network containing 220 interactive targets was constructed by STRING and Cytoscape. Functional annotation analysis of these targets by g:Profiler showed that they mainly contribute to the cellular processes, protein binding, and other functions and participate in metabolic pathways, cancer pathways, PI3K-Akt signaling pathway, etc. We found that oxidation and inflammatory factors played an important role in the virtual network of gastrodin anti-fatigue, which was supported by microarray dataset analysis and a molecular docking prediction. Additionally, real time-quantitative PCR and Western blot analysis indicated that gastrodin could promote the activation of the Nrf2 signal pathway, which could activate HO-1 and NQO1; gastrodin also could down-regulate the expressions of IL-1β, TNF-α, and IL-6. In summary, gastrodin can ameliorate exercise-induced fatigue by modulating the Nrf2 pathway and inhibiting expressions of inflammatory factors, this provides a new clue for the development of gastrodin-functional foods or anti-fatigue drugs.

Microfluidically Partitioned Dual Channels for Accurate Background Subtraction in Cellular Binding Studies by Surface Plasmon Resonance Microscopy.

Unlike conventional surface plasmon resonance (SPR) using an antifouling film to anchor biomolecules and a reference channel for background subtraction, SPR microscopy for single-cell analysis uses a protein- or polypeptide-modified gold substrate to immobilize cells and a cell-free area as the reference. In this work, we show that such a substrate is prone to nonspecific adsorption (NSA) of species from the cell culture media, resulting in false background signals that cannot be correctly subtracted. To obtain accurate kinetic results, we patterned a dual-channel substrate using a microfluidic device, with one channel having poly-l-lysine deposited in situ onto a preformed polyethylene glycol (PEG) self-assembled monolayer for cell immobilization and the other channel remaining as PEG-covered for reference. The two 2.0 mm-wide channels are separated by a 75 μm barrier, and parts of the channels can be readily positioned into the field of view of an SPR microscope. The use of this dual-channel substrate for background subtraction is contrasted with the conventional approach through the following binding studies: (1) wheat germ agglutinin (WGA) attachment to the N-acetyl glucosamine and N-acetyl-neuraminic acid sites of glycans on HFF cells, and (2) the S1 protein of the COVID-19 virus conjugation with angiotensin-converting enzyme 2 (ACE2) on the HEK293 cells. Both studies revealed that interferences by NSA and the surface plasmon polariton wave diffracted by cells can be excluded with the dual-channel substrate, and the much smaller refractive index changes caused by the injected solutions can be correctly subtracted. Consequently, sensorgrams with higher signal-to-noise ratios and shapes predicted by the correct binding model can be obtained with accurate kinetic and affinity parameters that are more biologically relevant. The affinity between S1 protein and ACE2 is comparable to that measured with recombinant ACE2, yet the binding kinetics is different, suggesting that the cell membrane does impose a kinetic barrier to their interaction.

Study of FA12 peptide-modified PEGylated liposomal doxorubicin (PLD) as an effective ligand to target Muc1 in mice bearing C26 colon carcinoma: in silico, in vitro, and in vivo study

ABSTRACT Objectives This study tried to achieve active targeting of Muc1 in cancer; the surface of PEGylated liposomal doxorubicin (PLD/Doxil®) was decorated with FA12 peptide. Methods According to docking results, FA12 was selected for this study, among four different peptides. MD simulation was also conducted as an additional confirmation of the binding interaction between FA12 and Muc1. Liposomal formulations were prepared; 1HNMR and HPLC techniques were used to verify peptide conjugation to DSPE-PEG2000-COOH. Afterward, DSPE-PEG2000-FA12 was post-inserted into the PLD at 50, 100, 200, and 400 peptides per liposome. The size, zeta potential, release profile, cytotoxicity (IC50), and cell uptake (using fluorescence microscopy and flow cytometry) were evaluated. In vivo biodistribution and antitumor activities were studied on mice bearing C-26 colon carcinoma. Results Cell uptake and cytotoxicity results revealed that PLD-100 (targeted PLD with 100 FA12 per liposome) could significantly enhance cellular binding. Furthermore, PLD-100 demonstrated higher antitumor efficacy, indicating more remarkable survival compared to PLD and other targeted PLDs. PLD-100 exhibited higher doxorubicin tumor accumulation compared to PLD. Conclusions FA12 peptide is a promising targeting ligand for PLD to treat cancers with a high level of Muc1 expression and merits further investigations.

Peptide-Based [68Ga]Ga Labeled PET Tracer for Tumor Imaging by Targeting Tumor-Associated Macrophages.

Tumor-associated macrophages (TAMs) are known to promote cancer development and metastasis. In this study, a TAMs-targeting peptide named M2pep was selected to investigate the feasibility of [68Ga]Ga-labeled M2pep as a noninvasive probe in targeted TAMs imaging. The peptide M2pep was conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and radiolabeled with 68Ga. The cellular uptake and binding assay were assessed in M2 macrophages and in the B16F10 cell line. Micro-PET imaging and a biodistribution study were performed on B16F10 tumor-bearing mice. High radiochemical purity [68Ga]Ga-DOTA-M2pep (>95%) was prepared and was stabilized in saline and bovine serum at 37 °C for 2 h. In vitro studies demonstrated high uptake of [68Ga]Ga-DOTA-M2pep in M2 macrophages, which was effectively blocked by the "cold" M2pep (free peptide). The micro-PET imaging and biodistribution study revealed that [68Ga]Ga-DOTA-M2pep reached the tumor site rapidly and showed high accumulation in the tumor at 1 h post-injection. In addition, the probe was rapidly cleared from the blood and mainly excreted via the kidneys, resulting in a high tumor/background ratio. Preclinical studies have shown that [68Ga]Ga-DOTA-M2pep specifically targets TAMs and might be a promising molecular probe for the noninvasive visualization of TAMs expression.

Complement Shields Fc Regions on HIT Immune Complexes and Prevents Recognition By Cellular Fcγ Receptors

Heparin-induced thrombocytopenia (HIT) is a life-threatening immune-mediated thrombotic disorder caused by immune complexes (ICs) containing IgG antibodies to platelet factor 4 (PF4)/heparin complexes. In previous studies, we established that complement opsonization of HIT ICs is essential for cellular binding of ICs and subsequent engagement of Fcγ receptors (FcγRs). We additionally showed that complement inhibition alone prevented FcγR-mediated effector functions, such as neutrophil degranulation, monocyte tissue factor expression, and procoagulant activity (Khandelwal, Blood 2021). As KKO and HIT ICs can also bind FcγRs in media without complement, we undertook these studies to examine the direct effects of complement on IC-FcγR interactions. For these studies, we used THP-1 cells or U937 cells, monocytic cell lines expressing FcγRI and FcγRII, but not complement receptors (CRs). Cells were incubated with AF-647 labeled KKO (50 mg/ml), a HIT-like monoclonal antibody, either alone or with PF4 (25 mg/ml) and heparin (1U/ml) to form in situ KKO ICs. Labeled KKO or KKO ICs were added to RPMI-containing media (lacking complement) or undiluted citrated plasma (source of complement) in the presence or absence of anti-FcγRIIA. Binding of labeled IgG or anti-C3c was examined by flow cytometry. Consistent with prior observations, THP-1 cells incubated in media show robust binding of KKO ICs in a FcγRII dependent manner (70% reduction in binding with anti-FcγRII; Figure 1A). Surprisingly, in undiluted plasma, there was minimal KKO IC binding to THP-1 cells, despite adequate complement generation (Figure 1B). Similar effects of plasma vs. media were seen in confocal experiments using KKO ICs with THP-1 cells (Figure 1C & 1D), HIT ICs incubated with THP-1 cells (containing HIT patient IgG, PF4/heparin; data not shown), or KKO ICs incubated with U937 cells (data not shown). In other studies, we note that binding of KKO ICs was dependent on the source of complement, with undiluted human plasma showing maximal inhibitory effects (100%), while undiluted normal human serum (NHS) and fetal bovine serum (FBS) reduced KKO IC binding by 70% and 50%, respectively (data not shown). To demonstrate that the inhibitory effects of undiluted plasma on KKO IC binding was complement-mediated, THP-1 cells were incubated in plasma containing the complement inhibitors BBK32 (C1r inhibitor) or CP40 (C3 inhibitor) prior to addition of labeled KKO ICs. In other experiments, THP-1 cells were incubated with labeled KKO ICs in media containing human complement proteins (C1+C3+C4). As shown, complement inhibition in plasma restored KKO IC binding to cellular FcγR (Figure 2A). Conversely, addition of purified complement components to media resulted in reduced KKO IC binding (Figure 2B). Similar effects of complement proteins and inhibitors were noted in ELISA-based assays using immobilized FcγRIIA incubated with KKO ICs in plasma or media (data not shown). These studies demonstrate striking inhibitory effects of complement on IC-FcγR interactions. Specifically, complement shields Fc regions on ICs from recognition and binding to cellular FcγRs and this masking effect is reversed by complement inhibition. We additionally show that these masking effects of complement are less apparent in media containing FBS, the most commonly used source of complement for cell-based studies. Taken together with our recent observations showing requirements for complement receptors in binding complement-opsonized KKO ICs, these studies suggest that the masking effects of complement on ICs prevent FcγR activation on cells lacking CRs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Protein Microarrays and their Fabrication.

Protein microarrays are an important tool when analyzing multiple analytes simultaneously. As the human genome contains approximately 20,000 genes, examining the interactions of even just one representative protein for each gene requires a high-throughput technique. For instance, the interaction between glycosaminoglycans (GAGs), a form of polysaccharide, and chemokines, small chemoattractant proteins, is critical for local inflammation. GAGs present in the glycocalyx on the surface of the cell bind to chemokines, which are released in response to injury. These chemokines can then form concentration gradients that direct the migration and recruitment of leucocytes via leukocyte receptors which in turn leads to immune cell responses, inflammation, or innate immunity and cell or antibody-mediated immune responses. Discovering the novel interactions between the GAGs and chemokines can help in designing drugs which can alter cellular binding to organ tissues, thereby potentially reducing damaging innate immune (inflammation) or acquired immune (antibody-mediated)responses.