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Human angiotensin-converting enzyme 2 (hACE2) is the primary receptor for cellular entry of SARS-CoV-2 into human host cells. hACE2 is heavily glycosylated and glycans on the receptor may play a role in viral binding. Thus, comprehensive characterization of hACE2 glycosylation could aid our understanding of interactions between the receptor and SARS-CoV-2 spike (S) protein, as well as provide a basis for the development of therapeutic drugs targeting this crucial interaction. Herein, 138 N-glycan compositions were identified, most of which are complex-type N-glycans, from seven N-glycosites of hACE2. Among them, 67% contain at least one sialic acid residue. At the level of glycopeptides, the overall quantification of sialylated glycan isomers observed on the sites N322 and N546 have a higher degree of NeuAc (α2-3)Gal (over 80.3%) than that of other N-glycosites (35.6-71.0%). In terms of O-glycans, 69 glycan compositions from 12 O-glycosites were identified, and especially, the C-terminus of hACE2 is heavily O-glycosylated. The terminal sialic acid linkage type of H1N1S1 and H1N1S2 are covered highly with α2,3-sialic acid. These findings could aid the investigation of the interaction between SARS-CoV-2 and human host cells.
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COVID-19 , SARS-CoV-2 , Humanos , Enzima de Conversão de Angiotensina 2/metabolismo , Glicosilação , Ácido N-Acetilneuramínico , Polissacarídeos/química , Ligação Proteica , SARS-CoV-2/metabolismoRESUMO
Clinical application of PD-1 and PD-L1 monoclonal antibodies (mAbs) is hindered by their relatively low response rates and the occurrence of drug resistance. Co-expression of B7-H3 with PD-L1 has been found in various solid tumors, and combination therapies that target both PD-1/PD-L1 and B7-H3 pathways may provide additional therapeutic benefits. Up to today, however, no bispecific antibodies targeting both PD-1 and B7-H3 have reached the clinical development stage. In this study, we generated a stable B7-H3×PD-L1 bispecific antibody (BsAb) in IgG1-VHH format by coupling a humanized IgG1 mAb against PD-L1 with a humanized camelus variable domain of the heavy-chain of heavy-chain antibody (VHH) against human B7-H3. The BsAb exhibited favorable thermostability, efficient T cell activation, IFN-γ production, and antibody-dependent cell-mediated cytotoxicity (ADCC). In a PBMC humanized A375 xenogeneic tumor model, treatment with BsAb (10 mg/kg, i.p., twice a week for 6 weeks) showed enhanced antitumor activities compared to monotherapies and, to some degree, combination therapies. Our results suggest that targeting both PD-1 and B7-H3 with BsAbs increases their specificities to B7-H3 and PD-L1 double-positive tumors and induces a synergetic effect. We conclude that B7-H3×PD-L1 BsAb is favored over mAbs and possibly combination therapies in treating B7-H3 and PD-L1 double-positive tumors.
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Antígeno B7-H1 , Receptor de Morte Celular Programada 1 , Humanos , Antígeno B7-H1/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Leucócitos Mononucleares/metabolismo , Anticorpos Monoclonais , Imunoglobulina G/metabolismoRESUMO
The energy loss inside a centrifugal pump has a significant effect on its performance characteristics. Based on the structural characteristics of the humpback pectoral fin, a new tongue was designed to improve the performance of the centrifugal pump. The influence of three sinusoidal tubercle volute tongues (STVT) and one original volute tongue (OVT) on energy dissipation using the enstrophy analysis method was investigated. To accomplish this, the pressure fluctuations and performances of four centrifugal pumps were analyzed. The results indicate that enstrophy is primarily distributed at the impeller outlet and near the tongue. The total enstrophy of the profiles of STVT was smaller than that of the profiles of OVT. This difference was more obvious near the tongue. The reductions in the total enstrophy of the pumps were 8% (STVT-1), 8.2% (STVT-2), and 9% (STVT-3). The pressure fluctuations of the STVT profiles also decreased to different degrees. The average pressure fluctuations at the monitoring points decreased by 20.6% (STVT-1), 21.7% (STVT-2), and 23.3% (STVT-3). The performances of the bionic retrofit pumps increased by 1.5% (STVT-1), 2% (STVT-2), and 2.45% (STVT-3) under the design flow rate. This study guides the structural optimization of pumps.
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Activation of the programmed cell death protein 1 and programmed cell death ligand 1 (PD-1/PD-L1) signaling axis plays important roles in intrinsic or acquired resistance to human epidermal growth factor receptor 2 (HER2)-directed therapies in the clinic. Therefore, therapies simultaneously targeting both HER2 and PD-1/PD-L1 signaling pathways are of great significance. Here, aiming to direct the anti-PD-L1 responses toward HER2-expressing tumor cells, we constructed a humanized bispecific IgG1 subclass antibody targeting both HER2 and PD-L1 (HER2/PD-L1; BsAb), which displayed satisfactory purity, thermostability, and serum stability. We found that BsAb showed enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity in vitro. In the late phase of peripheral blood mononuclear cell (PBMC)-humanized HER2+ tumor xenograft models, BsAb showed superior therapeutic efficacies as compared with monoclonal antibodies (mAbs) or combination treatment strategies. In cynomolgus monkeys, BsAb showed favorable pharmacokinetics and toxicity profiles when administered at a 10 mg/kg dosage. Thus, HER2/PD-L1 BsAb was demonstrated as a potentially effective option for managing HER2+ and trastuzumab-resistant tumors in the clinic. We propose that the enhanced antitumor activities of BsAb in vivo may be due to direct inhibition of HER2 signaling or activation of T cells.
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Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais Murinos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor ErbB-2/antagonistas & inibidores , Animais , Citotoxicidade Celular Dependente de Anticorpos , Linhagem Celular Tumoral , Humanos , Camundongos , Neoplasias Experimentais/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Receptor ErbB-2/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Chemotherapy-induced peripheral neuropathy (CIPN) is one of the pervasive side effects of chemotherapy, leading to poor quality of life in cancer patients. Discovery of powerful analgesics for CIPN is an urgent and substantial clinical need. Nerve growth factor (NGF), a classic neurotrophic factor, has been identified as a potential therapeutic target for pain. In this study, we generated a humanized NGF monoclonal antibody (DS002) that most effectively blocked the interaction between NGF and tropomyosin receptor kinase A (TrkA). We showed that DS002 blocked NGF binding to TrkA in a dose-dependent manner with an IC50 value of 6.6 nM; DS002 dose-dependently inhibited the proliferation of TF-1 cells by blocking the TrkA-mediated downstream signaling pathway. Furthermore, DS002 did not display noticeable species differences in its binding and blocking abilities. In three chemotherapy-induced rat models of CIPN, subcutaneous injection of DS002 produced a significant prophylactic effect against paclitaxel-, cisplatin- and vincristine-induced peripheral neuropathy. In conclusion, we demonstrate for the first time that an NGF inhibitor effectively alleviates pain in animal models of CIPN. DS002 has the potential to treat CIPN pain in the clinic.
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Antineoplásicos , Doenças do Sistema Nervoso Periférico , Ratos , Animais , Fator de Crescimento Neural , Anticorpos Monoclonais/uso terapêutico , Qualidade de Vida , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Dor , Antineoplásicos/efeitos adversos , Receptor trkA/metabolismoRESUMO
The titer of recombinant proteins is one of the key parameters in biopharmaceutical manufacturing processes. The fluorescence polarization (FP)-based assay, a homogeneous, high-throughput and real-time analytical method, had emerged as a powerful tool for biochemical analysis and environmental monitoring. In this study, an FP-based bioassay was utilized to quantify antibody fragment crystallizable (Fc)-containing proteins, such as recombinant monoclonal antibodies (mAbs) and mAb derivatives, in the cell culture supernatant, and the impacts of tracer molecular weight and FITC-coupling conditions on fluorescence polarization were methodically examined. Distinct from the fluorescence polarization potency calculated by classical formula, we for the first time proposed a new concept and calculation of fluorescence polarization intensity, based on which an analytical method with broader detection range and analysis window was established for quantifying Fc-containing proteins. This provided new ideas for the practical application of fluorescence polarization theory. The established method could detect 96 samples within 30 minutes, with dynamic titer range of 2.5-400 mg L-1, and a linear fitting R2 between the measured and actual concentration reaching 0.99. The method had great application prospects in determining the titer of recombinant proteins with Fc fragments, especially when applied to large-scale screening of high-yield and stable expression CHO cell lines commonly used in biopharmaceutical industry.
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Anticorpos Monoclonais , Cricetulus , Polarização de Fluorescência , Ensaios de Triagem em Larga Escala , Fragmentos Fc das Imunoglobulinas , Proteínas Recombinantes , Proteínas Recombinantes/química , Proteínas Recombinantes/análise , Células CHO , Polarização de Fluorescência/métodos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/química , Ensaios de Triagem em Larga Escala/métodos , Fragmentos Fc das Imunoglobulinas/química , Bioensaio/métodos , AnimaisRESUMO
Introduction: Humanization is typically adopted to reduce the immunogenicity of murine antibodies generated by hybridoma technology when used in humans. Methods: Two different strategies of antibody humanization are popularly employed, including "complementarity determining region (CDR) grafting" and "framework (FR) shuffling" to humanize a murine antibody against human programmed death-1 (PD-1), XM PD1. In CDR-grafting humanization, the CDRs of XM PD-1, were grafted into the human FR regions with high homology to the murine FR counterparts, and back mutations of key residues were performed to retain the antigen-binding affinities. While in FR-shuffling humanization, a combinatorial library of the six murine CDRs in-frame of XM PD-1 was constructed to a pool of human germline FRs for high-throughput screening for the most favorable variants. We evaluated many aspects which were important during antibody development of the molecules obtained by the two methods, including antibody purity, thermal stability, binding efficacy, predicted humanness, and immunogenicity, along with T cell epitope prediction for the humanized antibodies. Results: While the ideal molecule was not achieved through CDR grafting in this particular instance, FR-shuffling proved successful in identifying a suitable candidate. The study highlights FR-shuffling as an effective complementary approach that potentially increases the success rate of antibody humanization. It is particularly noted for its accessibility to those with a biological rather than a computational background. Discussion: The insights from this comparison are intended to assist other researchers in selecting appropriate humanization strategies for drug development, contributing to broader application and understanding in the field.
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Regiões Determinantes de Complementaridade , Receptor de Morte Celular Programada 1 , Animais , Humanos , Camundongos , Receptor de Morte Celular Programada 1/imunologia , Regiões Determinantes de Complementaridade/imunologia , Regiões Determinantes de Complementaridade/genética , Anticorpos Monoclonais Humanizados/imunologia , Epitopos de Linfócito T/imunologiaRESUMO
The Omicron variant is sweeping the world, which displays striking immune escape potential through mutations at key antigenic sites on the spike protein, making broad-spectrum SARS-CoV-2 prevention or therapeutical strategies urgently needed. Previously, we have reported a hACE2-targeting neutralizing antibody 3E8, which could efficiently block both prototype SARS-CoV-2 and Delta variant infections in prophylactic mouse models, having the potential of broad-spectrum to prevent SARS-CoV-2. However, preparation of monoclonal neutralizing antibodies is severely limited by the time-consuming process and the relative high cost. Here, we utilized a modified VEEV replicon with two subgenomic (sg) promoters engineered to express the light and heavy chains of the 3E8 mAb. The feasibility and protective efficacy of replicating mRNA encoding 3E8 against Omicron infection in the hamster were demonstrated through the lung targeting delivery with the help of VEEV-VRP. Overall, we developed a safe and cost-effective platform of broad-spectrum to prevent SARS-CoV-2 infection.
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COVID-19 , SARS-CoV-2 , Animais , Cricetinae , Camundongos , SARS-CoV-2/genética , COVID-19/prevenção & controle , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes , RNA Mensageiro , Glicoproteína da Espícula de Coronavírus/genética , Anticorpos AntiviraisRESUMO
Increased remissions in multiple sclerosis (MS) during pregnancy suggest that elevated levels of sex steroids exert immunoregulatory activity. Estrogen (E2=17ß-estradiol) protects against experimental autoimmune encephalomyelitis (EAE), but the cellular basis for E2-induced protection remains unclear. Studies demonstrate that depletion of B cells prior to induction of EAE exacerbates disease severity, implicating regulatory B cells. We thus evaluated pathogenic and E2-induced protective mechanisms in B-cell-deficient (µMT(-/-)) mice. EAE-protective effects of E2 were abrogated in µMT(-/-) mice, with no reduction in disease severity, cellular infiltration or pro-inflammatory factors in the central nervous system compared to untreated controls. E2 treatment of WT mice selectively upregulated expression of PD-L1 on B cells and increased the percentage of IL-10-producing CD1d(high) CD5(+) regulatory B cells. Upregulation of PD-L1 was critical for E2-mediated protection since E2 did not inhibit EAE in PD-L1(-/-) mice. Direct treatment of B cells with E2 significantly reduced proliferation of MOG(35-55)-specific T cells that required estrogen receptor-α (ERα). These results demonstrate, for the first time, a requirement for B cells in E2-mediated protection against EAE involving direct E2 effects on regulatory B cells mediated through ERα and the PD-1/PD-L1 negative co-stimulatory pathway. E2-primed B cells may represent an important regulatory mechanism in MS and have strong implications for women receiving current MS therapies that cause B-cell depletion.
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Linfócitos B/imunologia , Encefalomielite Autoimune Experimental/imunologia , Estrogênios/imunologia , Animais , Antígenos CD1d/imunologia , Antígeno B7-1/imunologia , Antígeno B7-H1 , Antígenos CD5/imunologia , Células Cultivadas , Estradiol/imunologia , Feminino , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptídeos/deficiência , Peptídeos/imunologia , Baço/imunologiaRESUMO
Bispecific antibodies (BsAbs) are typically monoclonal antibody (mAb)-derived molecular entities engineered to bind to two distinct targets, including two antigens or two epitopes on the same antigen. When compared to parental monoclonal antibodies or combinational therapies, the generated BsAbs have the ability to bridge the two targets and thus may offer additional clinical benefits. Characterizing BsAbs' ability to bind to both targets simultaneously is critical for their biotherapeutic development. A range of bi-functional quantitative bridging assays to enable target-specific capture and detection of binding properties include enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and cell-based flow cytometry. Developing suitable and robust cell-based bioassays is more challenging than non-cell-based binding assays because cell-based assays with complex matrices can be inherently variable and often lack precision. Compared to SPR, ELISA has a rapid setup and readily available method, being widely and extensively applied in almost every laboratory. Here, we describe a dual-target bridging ELISA assay that characterizes the ability of a HER2(human epidermal growth factor receptor 2)/PD-L1(programmed cell death ligand 1) BsAb in binding to both HER2 and PD-L1 simultaneously, a prerequisite for its envisioned mode of action. Graphical abstract.
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Purpose: Trophoblast cell surface antigen 2 (TROP2) has emerged as a promising target of antibody-drug conjugates (ADCs) for triple-negative breast cancer (TNBC), as well as other breast cancers (BCs). This study aims to investigate the biomarker value of TROP2 for patient-tailoring and prognostic for BC patients, including TNBC. Methods: The levels of TROP2 expression in 404 Chinese BC tissues on tissue microarrays (TMAs) were quantified by immunohistochemistry and their correlations to the clinicopathological factors and the overall survival rate were analyzed. Also, BC cell lines and patient-derived organoids (PDOs) with different TROP2 expression levels were employed to investigate the correlation between TROP2 expression levels and the therapeutic responses to DS001, a TROP2-directed ADC molecule with stable linker and potent payload. Results: TROP2 overexpression was identified in significantly more (P = 0.046) tumor tissues (41.08%, 99/241) than normal adjacent tissues (31.29%, 51/163) from Chinese BC patients, and in significantly more (P = 0.024) TNBC patients (59.38%, 19/32) than in other BC types (38.28%, 80/209). BC cell line with the lowest TROP2 expression level failed to respond to DS001 treatment. The levels of TROP2 expression were determined to be significantly correlated with the potencies of DS001 treatment, but not with the overall survival rates of the patients. Conclusion: Our results demonstrated that TROP2 could serve as a patient-tailoring and predictive biomarker for ADC therapeutics but not as a general prognostic biomarker to predicate patient survival.
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Current treatment options for diabetic neuralgia are limited and unsatisfactory. Tanezumab, a monoclonal antibody that blocks nerve growth factor (NGF) signaling, has been shown to be effective in relieving the clinical symptoms of osteoarthritis pain, chronic low back pain, cancer pain induced by bone metastasis, and diabetic neuralgia. However, the clinical development of tanezumab has been terminated due to the risk of induction of rapidly progressive osteoarthritis (RPOA), and no other NGF antibodies have been examined for their ability to treat diabetic neuralgia in either animal models or clinical trials. In this study, a humanized high-affinity NGF monoclonal antibody (mAb), huAb45 that could neutralize the interaction between NGF and its high-affinity receptor TrkA. In a mouse diabetic neuralgia model, it effectively relieved neuropathic pain. This study may serve as the necessary foundation for future studies of huAb45 to potentially treat diabetic neuralgia.
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Diabetes Mellitus , Neuropatias Diabéticas , Osteoartrite , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Animais , Anticorpos Monoclonais/farmacologia , Diabetes Mellitus/tratamento farmacológico , Neuropatias Diabéticas/tratamento farmacológico , Modelos Animais de Doenças , Camundongos , Fator de Crescimento Neural/metabolismoRESUMO
Purpose: To evaluate the safety, tolerability, pharmacokinetics and immunogenicity of DS002 injection, an anti-nerve growth factor (anti-NGF) monoclonal antibody for treating pain conditions, in healthy Chinese subjects. Methods: This study was a single-center, randomized, double-blind, single-dose escalation, placebo-controlled design (CTR20210155). A total of 53 healthy subjects, 27 male and 26 female, were enrolled in this study, and one subject withdrew from the study before administration. Seven dose groups were set up, which were 0.5 mg, 1.0 mg, 2.0 mg, 4.0 mg, 7.0 mg, 12.0 mg and 20.0 mg, respectively. The drug was administered by single subcutaneous injection. Four subjects were enrolled in the first dose group (0.5 mg) received DS002. Other dose groups enrolled eight subjects each, six of whom received DS002 while the other two received a placebo. Safety, tolerability, pharmacokinetic parameters and immunogenicity of DS002 were assessed. Results: DS002 was well tolerated; all adverse events were Grade 1-2, and did not reach the termination standard of dose increment within the range of 0.5-20.0 mg. Adverse event rates were generally similar across treatments. After a single subcutaneous injection, the median Tmax in different dose groups ranged 167.77-337.38 h; mean t1/2 ranged 176.80-294.23 h, the volume of distribution (Vz) ranged 5265.42-7212.00 ml, and the clearance rate (CL) ranged 12.69-24.75 ml/h. In the dose range of 0.5-20.0 mg, Cmax ranged from 51.83 ± 22.74 ng/ml to 2048.86 ± 564.78 ng/ml, AUC0-t ranged from 20615.16 ± 5698.28 h·ng/mL to 1669608.11 ± 387246.36 h·ng/mL, and AUC0-inf ranged from 21852.45 ± 5920.21 h·ng/mL to 1673504.66 ± 389106.13 h·ng/mL. They all increased with dose escalation, and Cmax and AUC0-t did not have a significant dose-linear relationship, whilst AUC0-t was not dose-dependent at all. anti-drug antibody test results of each group of all subjects in this trial were negative. Conclusion: DS002 showed satisfactory safety within the dose range of 0.5 mg-20.0 mg. The absorption and metabolism of DS002 were slow, it exhibited a low volume of distribution and the clearance rate was low. These data suggest that DS002, by blocking nerve growth factor, is expected to become a novel, safe and non-addictive treatment for pain conditions.
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Dinutuximab (ch14.18) was the first approved monoclonal antibody against the tumor-associated antigen disialoganglioside GD2. Despite its success in treating neuroblastoma (NB), it triggers a significant amount of neuropathic pain in patients, possibly through complement-dependent cytotoxicity (CDC). We hypothesized that modifying ch14.18 using antibody engineering techniques, such as humanization, affinity maturation, and Fc engineering, may enable the development of next-generation GD2-specific antibodies with reduced neuropathic pain and enhanced antitumor activity. In this study we developed the H3-16 IgG1m4 antibody from ch14.18 IgG1. H3-16 IgG1m4 exhibited enhanced binding activity to GD2 molecules and GD2-positive cell lines as revealed by ELISA, and its cross-binding activity to other gangliosides was not altered. The CDC activity of H3-16 IgG1m4 was decreased, and the antibody-dependent cellular cytotoxicity (ADCC) activity was enhanced. The pain response after H3-16 IgG1m4 antibody administration was also reduced, as demonstrated using the von Frey test in Sprague-Dawley (SD) rats. In summary, H3-16 IgG1m4 may have potential as a monoclonal antibody with reduced side effects.
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Anticorpos Monoclonais , Neuralgia , Animais , Anticorpos Monoclonais/farmacologia , Gangliosídeos , Neuralgia/tratamento farmacológico , Ratos , Ratos Sprague-DawleyRESUMO
AIMS: Small molecule compound tyrphostin A9 (A9), an inhibitor of platelet-derived growth factor (PDGF) receptor, was previously reported by our group to stimulate extracellular signal-regulated kinase 1 (ERK1) and 2 (ERK2) in neuronal cells in a PDGF receptor-irrelevant manner. The study aimed to investigate whether A9 could protect axons in experimental autoimmune encephalomyelitis through activation of ERKs. MAIN METHODS: A9 treatment on the protection on neurite outgrowth in SH-SY5Y neuroblastoma cells and primary substantia nigra neuron cultures from the neurotoxin MPP+ were analyzed. Then, clinical symptoms as well as ERK1/2 activation, axonal protection induction, and the abundance increases of the regeneration biomarker GAP-43 in the CNS in the relapsing-remitting experimental autoimmune encephalomyelitis (EAE) model were verified. KEY FINDINGS: A9 treatment could stimulate neurite outgrowth in SH-SY5Y neuroblastoma cells and protect primary substantia nigra neuron cultures from the neurotoxin MPP+. In the relapsing-remitting EAE model, oral administration of A9 successfully ameliorated clinical symptoms, activated ERK1/2, induced axonal protection, and increased the abundance of the regeneration biomarker GAP-43 in the CNS. Interestingly, gene deficiency of ERK1 or ERK2 disrupted the beneficial effects of A9 in MOG-35-55-induced EAE. SIGNIFICANCE: These results demonstrated that small molecule compounds that stimulate persistent ERK activation in vitro and in vivo may be useful in protective or restorative treatment for neurodegenerative diseases.
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Axônios/efeitos dos fármacos , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/prevenção & controle , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Neuroblastoma/tratamento farmacológico , Tirfostinas/farmacologia , Animais , Encefalomielite Autoimune Experimental/etiologia , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , MAP Quinases Reguladas por Sinal Extracelular/genética , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Ratos , Ratos Sprague-DawleyRESUMO
Antibody-drug conjugates (ADCs) are commonly heterogeneous and require extensive assessment of exposure-efficacy and exposure-safety relationships in preclinical and clinical studies. In this study, we report the generation of a monoclonal antibody against monomethyl auristatin E (MMAE) and the development, validation, and application of sensitive and high-throughput enzyme-linked immunosorbent assays (ELISA) to measure the concentrations of MMAE-conjugated ADCs and total antibodies (tAb, antibodies in ADC plus unconjugated antibodies) in cynomolgus monkey sera. These assays were successfully applied to in vitro plasma stability and pharmacokinetic (PK) studies of SMADC001, an MMAE-conjugated ADC against trophoblast cell surface antigen 2 (TROP-2). The plasma stability of SMADC001 was better than that of similar ADCs coupled with PEG4-Val-Cit, Lys (m-dPEG24)-Cit, and Val-Cit linkers. The developed ELISA methods for the calibration standards of ADC and tAb revealed a correlation between serum concentrations and the OD450 values, with R 2 at 1.000, and the dynamic range was 0.3-35.0 ng/mL and 0.2-22.0 ng/mL, respectively; the intra- and inter-assay accuracy bias% ranged from -12.2% to -5.2%, precision ranged from -12.4% to -1.4%, and the relative standard deviation (RSD) was less than 6.6% and 8.7%, respectively. The total error was less than 20.4%. The development and validation steps of these two assays met the acceptance criteria for all addressed validation parameters, which suggested that these can be applied to quantify MMAE-conjugated ADCs, as well as in PK studies. Furthermore, these assays can be easily adopted for development of other similar immunoassays.
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Although estrogens exert a pronounced protective effect on multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), their therapeutic application has been limited by undesirable side effects thought to be mediated primarily through estradiol binding to intracellular estrogen receptor alpha. In this study, we found that signaling through the putative membrane estrogen receptor, G protein-coupled receptor 30 (GPR30), was sufficient to mediate protection against EAE, which was significantly impaired in GPR30 gene-deficient mice. Treatment with G-1, an agonist that selectively activates GPR30 without engagement of the intracellular estrogen receptors, retained the ability of estradiol to protect against clinical and histological EAE without estradiol-associated side effects, deviated cytokine profiles, and enhanced suppressive activity of CD4(+)Foxp3(+) T regulatory cells through a GPR30- and programmed death 1-dependent mechanism. This study is the first to evaluate the protective effect of GPR30 activation on EAE, and provides a strong foundation for the clinical application of GPR30 agonists such as G-1 in multiple sclerosis.
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Antígenos de Superfície/biossíntese , Proteínas Reguladoras de Apoptose/biossíntese , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/prevenção & controle , Proteínas de Membrana/fisiologia , Receptores de Estrogênio/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Regulação para Cima/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Superfície/fisiologia , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/fisiologia , Células Cultivadas , Ciclopentanos/farmacologia , Encefalomielite Autoimune Experimental/metabolismo , Estradiol/fisiologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Receptor de Morte Celular Programada 1 , Quinolinas/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Regulação para Cima/genéticaRESUMO
The evolution of coronaviruses, such as SARS-CoV-2, makes broad-spectrum coronavirus preventional or therapeutical strategies highly sought after. Here we report a human angiotensin-converting enzyme 2 (ACE2)-targeting monoclonal antibody, 3E8, blocked the S1-subunits and pseudo-typed virus constructs from multiple coronaviruses including SARS-CoV-2, SARS-CoV-2 mutant variants (SARS-CoV-2-D614G, B.1.1.7, B.1.351, B.1.617.1, and P.1), SARS-CoV and HCoV-NL63, without markedly affecting the physiological activities of ACE2 or causing severe toxicity in ACE2 "knock-in" mice. 3E8 also blocked live SARS-CoV-2 infection in vitro and in a prophylactic mouse model of COVID-19. Cryo-EM and "alanine walk" studies revealed the key binding residues on ACE2 interacting with the CDR3 domain of 3E8 heavy chain. Although full evaluation of safety in non-human primates is necessary before clinical development of 3E8, we provided a potentially potent and "broad-spectrum" management strategy against all coronaviruses that utilize ACE2 as entry receptors and disclosed an anti-coronavirus epitope on human ACE2.
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Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Anticorpos Monoclonais Murinos/farmacologia , Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , SARS-CoV-2/imunologia , Enzima de Conversão de Angiotensina 2/imunologia , Animais , Anticorpos Monoclonais Murinos/imunologia , Antivirais/imunologia , Chlorocebus aethiops , Modelos Animais de Doenças , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Células VeroRESUMO
The regulatory role of programmed death 1 (PD-1) was investigated in the development of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Typical EAE could be induced by immunization without pertussis toxin (PTX) in PD-1-null but not in wild-type (WT) mice. However, both strains developed a similar EAE phenotype when immunized with PTX or by adoptive transfer of pathogenic T cells. In WT mice that did not develop EAE after immunization without PTX, the frequency of CD4(+)FoxP3(+) Treg cells was boosted in the periphery but not in the thymus. This increase in Treg frequency was abrogated by PD-1 deficiency or inclusion of PTX. In addition, PD-1 expression was critical to in vitro conversion of naïve myelin-specific CD4 T cells into Treg cells and was directly related to Treg suppressive activity. Finally, PD-1 was markedly down-modulated in the periphery of WT mice after administration of PTX. Therefore, down-modulation of PD-1 in Treg cells may abrogate Treg-mediated immune suppression, permitting the activation of myelin-reactive T cells and induction of EAE.
Assuntos
Antígenos de Superfície/genética , Proteínas Reguladoras de Apoptose/genética , Encefalomielite Autoimune Experimental/genética , Linfócitos T Reguladores/imunologia , Animais , Antígenos de Superfície/imunologia , Proteínas Reguladoras de Apoptose/imunologia , Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Regulação para Baixo , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Citometria de Fluxo , Adjuvante de Freund/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Toxina Pertussis/imunologia , Receptor de Morte Celular Programada 1 , Medula Espinal/patologia , Coloração e RotulagemRESUMO
The mechanism by which oestrogens suppress experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, is only partially understood. We here demonstrate that treatment with 17beta-oestradiol (E(2)) in C57BL/6 mice boosted the expression of programmed death 1 (PD-1), a negative regulator of immune responses, in the CD4(+) FoxP3(+) regulatory T (Treg) cell compartment in a dose-dependent manner that correlated with the efficiency of EAE protection. Administration of E(2) at pregnancy levels but not lower concentrations also enhanced the frequency of Treg cells. Additionally, E(2) treatment drastically reduced the production of interleukin-17 (IL-17) in the periphery of immunized mice. However, E(2) treatment did not protect against EAE or suppress IL-17 production in PD-1 gene-deficient mice. Finally, E(2) failed to prevent Treg-deficient mice from developing spontaneous EAE. Taken together, our results suggest that E(2)-induced protection against EAE is mediated by upregulation of PD-1 expression within the Treg-cell compartment.